The genomic and genetic basis of mammalian sexual reproduction:
Sequence of the mouse Y chromosome, and
a gene regulatory program for meiotic prophase
by
MASSACHUSElTS INSTITIJTE
OF rECHNOLOLGY
Ying Qi Shirleen Soh
MAY 27 2015
A.B. Biology
Harvard College (2008)
LIBRARIES
SUBMITTED TO THE DEPARTMENT OF BIOLOGY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY IN BIOLOGY
AT THE
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
JUNE 2015
C 2015 Massachusetts Institute of Technology. All rights reserved
3ignature redacted
Signature of Author ....
Department of Biology
C
Certified by ...............
Accepted by..................
ignature redacted
Signature redacted
David C. Page
Professor of Biology
Thesis Supervisor
Amy E. Keating
Associate Professor of Biology
Co-Chair, Biology Graduate Committee
The genomic and genetic basis of mammalian sexual reproduction:
Sequence of the mouse Y chromosome, and
a gene regulatory program for meiotic prophase
by
Ying Qi Shirleen Soh
Submitted to the Department of Biology
on May 22, 2015 in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy in
Biology
ABSTRACT
Mammalian sexual reproduction requires sexual determination, sexual differentiation, and the
production of haploid gametes. In this thesis, I examined the genomic evolution of the mouse Y
chromosome, which instructs sexual determination, and genetic regulation of a program of gene
expression for meiosis, a specialized cell cycle which gives rise to haploid gametes.
Chapter 2 describes the study of the mouse Y chromosome. Contrary to popular theory
that Y chromosomes should be degenerate and gene poor, we find that the mouse male-specific
region of the Y chromosome (MSY) is almost entirely euchromatic and contains about 700
protein-coding genes. Almost all of these genes belong to three acquired, massively amplified
gene families that have no homologs on primate MSYs but do have acquired, amplified
homologs on the mouse X chromosome. We propose that lineage-specific convergent acquisition
and amplification of X-Y gene families is a result of sex-linked meiotic drive.
Chapter 3 describes the gene regulatory program of meiotic prophase. Meiotic prophase
comprises a complex chromosomal program results in the production of haploid gametes. This
must be supported by a program of gene expression via which the required genes are induced.
We interrogated gene expression in fetal ovaries over time and space, and in mutants of Dazl and
Stra8 - key genes required for meiotic initiation. We determined that genes are regulated in three
classes. Class 1 genes are expressed independently of Stra8, class 2 genes are expressed partially
independently of Stra8, and Class 3 genes are dependent on Stra8 to be expressed. All genes
require Dazl to be expressed. We propose that the Stra8-independent genes may represent genes
required to be expressed prior to or early during meiotic initiation. Following initiation of
meiosis, we found that Stra8 is required to induce down-regulation of its own expression. We
propose that induction of down-regulation of the initiating signal by itself serves to ensure timely
cessation of and one-time activation of the chromosomal program of meiotic prophase.
Thesis Supervisor: David C. Page
Title: Professor of Biology
3
ACKNOWLEDGEMENTS
I would like to thank David Page for guidance, support, motivation and encouragement
throughout my scientific training. I would also like to thank Helen Skaletsky for guidance
especially on the mouse Y project, and Dirk de Rooij for expertise on testis histology. Jana
Koubova and Jessica Alfdldi laid the foundation for work described in this thesis. I especially
thank my fellow inhabitants of room 405 - Laura Brown, Katherine Romer, and Winston Bellott
- for advice, criticism, and support. My co-authors made work possible - Jennifer Hughes, Jacob
Mueller, Mark Gill, Tatyana Pyntikova, Natalia Koutseva, Steve Rozen, and Yueh-Chiang Hu. I
thank Gregoriy Dokshin, Kyomi Igarashi, Mina Kojima, Peter Nicholls, Mary Goodheart, Sidaly
Phou, Tsutomu Endo, and Michelle Carmell for generous help and advice. I had help from a
talented undergraduate, Marsha Wibowo. Our administrative staff - Jorge Adarme and Susan
Tocio - make the impossible possible. I thank members of my committee - Richard Hynes and
Peter Reddien - for advice and criticism. I thank my undergraduate advisors - Richard Losick
and Andrew Berry - for their continued interest in me long after I have graduated college. I
thank my family for their support, patience, understanding, and encouragement. I thank my
husband Guo-Liang for endless optimism.
5
CONTENTS
A bstract..........................................................................................................................................3
A cknow ledgem ents .......................................................................................................................
5
C ontents .........................................................................................................................................
7
C hapter 1. Introduction .......................................................................................................
9
A . Features of m am m alian sexual reproduction .....................................................................................
11
B. Evolution of the m am malian sex chromosom es ............................................................................
13
References (sections A , B) .....................................................................................................................
31
C . M aking a gam ete ................................................................................................................................
41
D . M eiotic prophase and meiotic initiation .......................................................................................
47
References (sections C, D) .....................................................................................................................
63
Chapter 2. Sequencing the Mouse Y Chromosome Reveals Convergent Gene Acquisition
and Amplification on Both Sex Chromosomes.....................................................................
75
A bstract...................................................................................................................................................77
Introduction.............................................................................................................................................79
Results.....................................................................................................................................................83
Discussion.............................................................................................................................................
119
Supplem entary tables ............................................................................................................................
139
Supplementary data ..............................................................................................................................
141
Chapter 3. A Gene Regulatory Program for Meiotic Initiation in the Fetal Ovary..........157
A bstract.................................................................................................................................................
159
Introduction...........................................................................................................................................
160
Results...................................................................................................................................................
163
7
Discussion .............................................................................................................................................
199
Experimental Procedures ......................................................................................................................
207
References.............................................................................................................................................
211
Supplementary information ..................................................................................................................
215
C hapter 4. Conclusions............................................................................................................229
Sequence of the m ouse Y chromosome ................................................................................................
231
The gene regulatory program of meiotic prophase in the fetal ovary...................................................
239
A ppendix....................................................................................................................................251
A . Retinoic acid activates two pathways required for meiosis in mice ...............................................
253
B. Licensing of primordial germ cells to gametogenesis is dependent upon the genital ridge ........... 265
8
CHAPTER 1.
INTRODUCTION
9
t
A. FEATURES OF MAMMALIAN SEXUAL REPRODUCTION
In sexually reproducing organisms, the propagation of species depends ultimately on the
production of gametes. Gametes deliver segregated and assorted genetic material to form a
unique zygote of the next generation. This thesis examines the evolutionary forces and
developmental processes that shape gamete production in a mammalian model, the mouse.
Mammalian sexual reproduction is characterized by these key features: meiosis, sexually
-
dimorphic gametes, sexually dimorphic somatic differentiation, and sex chromosomes. Meiosis
the reassortment and halving of parental genetic material into haploid gametes, which then
combine to create a new individual - is the defining feature of all sexually reproducing
eukaryotes. In mammals, sexual reproduction additionally encompasses sexual differentiation of
both the gamete and the organism. Sexually dimorphic gametes are termed sperm and egg, and
are produced by males and females respectively. Sexual differentiation in mammals is
determined by sex chromosomes- XX or XY sex chromosomes direct female and male
differentiation respectively.
This thesis presents work relating to two of these features of mammalian sexual
reproduction: (1) Sex chromosome evolution; and (2) meiotic initiation and prophase. In this
introduction, I review: (1) theoretical and empirical models of mammalian sex chromosome
evolution, and pre-genomic sequencing studies of the mouse Y chromosome; and (2) germ cell
development, particularly the regulation of meiotic initiation. In Chapter 2, 1 present the
sequence and analysis of the mouse Y chromosome, and the insights it lends to mammalian sex
chromosome evolution. In Chapter 3, I characterize the gene regulatory network of meiotic
11
prophase in mouse fetal ovarian germ cells. In the final chapter I discuss implications and future
directions.
12
B. EVOLUTION OF THE MAMMALIAN SEX CHROMOSOMES
In mammals, sexual differentiation is directed by sex chromosomes. XX individuals, or the
homogametic sex, develop as females, whereas XY individuals, the heterogametic sex, develop
as males (Painter, 1921). Male differentiation is determined by a single gene on the Y
chromosome - Sex determining region Y (SRY) (Berta et al., 1990; Gubbay et al., 1990; Sinclair
et al., 1990).
The sex chromosomes are distinct from autosomes. Unlike each pair of autosomes, which
are identical throughout (having only allelic differences), the X and the Y chromosomes are
identical only in the pseudoautosomal region (PAR). Consequently, during meiosis in the male,
while each pair of autosomes may synapse and undergo crossing over throughout the entire
length of the chromosome, the X and Y chromosomes synapse and cross over only at the PAR
(Burgoyne, 1982). During female meiosis, the X homologs synapse and may cross over
throughout the entire length, like in the autosomes. The male-specific region of the Y
chromosome (MSY) thus does not cross over with a homolog.
These heteromorphic chromosomes nevertheless have a common origin - they evolved
from an ordinary pair of ancestral autosomes in a mammalian ancestor (Ohno, 1967). The
trajectory of sex chromosome evolution has been the subject of much theorizing. However,
accumulating empirical genomic data demonstrates that the sex chromosomes are evolving in
ways that are unanticipated by prior theoretical work. Complete, high-quality genomic data had
existed only for three primates (Hughes et al., 2010, 2012; Skaletsky et al., 2003). We therefore
sequenced the mouse Y chromosome, both to enable expanded comparative analysis across
mammals, and to serve as a resource for this mammalian genetic model organism. In the
following sections, I summarize the theory and empirical data underlying present models of sex
13
chromosome evolution, and the state of mapping and sequencing of the mouse Y chromosome
prior to our sequencing efforts (previously extensively reviewed in Alfdldi, 2008; Bellott and
Page, 2009; Hughes and Rozen, 2012).
PRE-GENOMIC THEORIES OF SEX CHROMOSOME EVOLUTION: Y CHROMOSOME DECA Y
The first theories of sex chromosome evolution arose out of genetic mapping studies in
Drosophila.As the first genetic maps of the Drosophila chromosomes were generated, Muller
(1914), noting that all chromosomes except the Y chromosome had at least one known gene,
postulated the first theory of sex chromosome evolution: The X and Y chromosomes evolved
from a pair of autosomes. However, because recombination does not occur in Drosophila male
gametes, the male-specific Y chromosome accumulated deleterious mutations, thus losing all its
genes. The mammalian Y chromosome had a similar apparent paucity of genes. By the late
1950s, several traits had been mapped to the X chromosome, but the only gene mapped to the Y
chromosome was for male-sex determination (Ford et al., 1959; Jacobs and Strong, 1959; Lee et
al., 2004; Stern and American, 1957; Welshons and Russell, 1959).
Ohno (1967) proposed two major steps in the evolution of vertebrate sex chromosomes
(Figure 1.1). He speculated that the vertebrate common ancestor had no sex chromosomes. The
first event in sex chromosome evolution was a mutation arising on an ordinary autosome causing
the emergence of a male sex-determining gene. This turned the pair of autosomes carrying the
sex-determining gene into incipient sex chromosomes that are initially homomorphic. In
vertebrates, unlike in Drosophila, crossing over occurs in male gametes - thus, the incipient sex
chromosomes would continue to cross over. Ohno thus proposed that the second event in sex
chromosome evolution is the suppression of crossing over between the sex chromosomes by an
inversion on the sex-specific chromosome.
14
A A
X Y
X Y
X Y
Inversion
X Y
Ydegeneation
)
ex-delenning
genes
Figure 1.1
Theoretical model of sex chromosome evolution
Ohno (1967) proposed that sex chromosomes arose from an ordinary pair of autosomes (AA).
Acquisition of a male sex-determining gene on one of the pair of autosomes results in the
formation of an incipient pair of sex chromosomes: the male-specific chromosome is referred to
as the Y chromosome, and the homolog shared between the sexes is referred to as the X
chromosome. Subsequently, recombination between the sex chromosomes would be suppressed
by an inversion. Fisher (1931) and Rice (1987) suggest a reason for why suppression of
recombination might be selected for. They predict that male-benefit sexually antagonistic genes
would preferentially accumulate near the sex-determining locus, so that their expression would
more likely be restricted to males only. This would result in selection for suppression of
recombination between the X and Y chromosomes. Isolation of the Y chromosome from
recombination renders natural selection inefficient on the Y, ultimately resulting in its
degeneration (Bachtrog, 2008). The X, on the contrary, remains largely unchanged.
15
Suppression of crossing over is thought to be selected for on the incipient Y chromosome
because it restricted expression of Y-linked sexually antagonistic genes to males (Fisher, 1931;
Rice et al., 1987). Sexually antagonistic genes, which are beneficial to males but deleterious to
females, would accumulate on the male-specific chromosome in tight linkage to the sexdetermining locus so that they would be preferentially transmitted to male offspring. Suppression
of recombination between the sex chromosomes would permanently restrict these sexually
antagonistic genes to male-only expression.
Isolation of the sex-specific chromosome from crossing over results in permanent linkage
of loci on the sex-specific chromosome. This renders natural selection inefficient on the sexspecific chromosome, resulting in its eventual degeneration via processes such as Muller's
ratchet, genetic hitchhiking, and background selection (Bachtrog, 2008). Muller's ratchet
describes the accumulation of deleterious mutations in the absence of sexual recombination. In
an asexual population of chromosomes that is accumulating mutations, one without deleterious
mutations cannot be created by recombination. As the fittest chromosome is stochastically lost to
genetic drift, the number of deleterious mutations on the remaining chromosomes of the
population accumulate (Felsenstein, 1974; Muller, 1964). Permanent linkage between loci
additionally enables selection at one site to impede selection at linked sites. Genetic hitchhiking
describes how weakly deleterious mutations may "hitchhike" along when strongly beneficial
mutations are selected for (Maynard Smith and Haigh, 1974; Rice, 1987). Background selection
describes how selection against strongly deleterious alleles increases the odds that weakly
deleterious alleles can become fixed by drift (Charlesworth, 1994).
In counterpoint to the dramatically degenerating Y chromosome, the X chromosome is
predicted to be unchanging. This idea, that genes on the X chromosome should be conserved, is
16
codified as "Ohno's law" (Ohno, 1967). Ohno noted that degeneration of the sex-specific
chromosome would require a mechanism to correct gene dosage on the sex chromosomes
between the sexes. Once dosage compensation was in place, genes would not be able to move
between autosomes and X chromosomes, because it would result in mis-regulation of dosage
levels.
GENOMIC ERA OF MAMMALIAN SEX CHROMOSOME EVOLUTION: SELECTED SURVIVORS,
AND GENE ACQUISITION AND AMPLIFICA TION
In the last couple of decades, sequencing of mammalian sex chromosomes has provided
empirical evidence for these theoretical models of sex chromosome evolution, but also revealed
aspects of sex chromosome evolution that were unanticipated by prior theoretical work.
Accumulating sequence data of the sex chromosomes support the idea that the sex
chromosomes are a "mosaic of sequence classes" (Skaletsky et al., 2003) (Figure 1.2). Sequences
on the sex chromosomes may be classified by their origin - ancestral or acquired, or by copy
number - single-copy or multicopy/ampliconic. Ancestral sequences derive from the ancestral
autosomes from which the sex chromosomes arose. Ancestral sequences on the Y chromosome
had previously been referred to as "X-degenerate" sequences, until a reconstruction of the
ancestral autosomal gene content made it possible to identify genes as originating from the
ancestral autosomes from which the sex chromosomes arose (Bellott et al., 2010, 2014).
Acquired sequences arrived on the sex chromosomes after the evolution of the sex chromosomes.
It was observed that while ancestral sequences exist in both single and multi-copy, acquired
sequences are most often highly amplified (Lahn and Page, 1999a; Saxena et al., 1996).
17
Human Y
Arcesa Wng.-ccp
*X-ranhpossd
H othir
0 Psudoautosonm
R Hetsrochrcmabc
10 Mb
Chimpanzee Y
con
Rhesus Y
-u
--
I-
ul~I
-
El
- a
I
*
Mouse X
con
-
b
U I
1I --1
I3
-
~lIl
~lfl
-
a
_
10Mb
Figure 1.2
Mammalian sex chromosomes for which complete, high-quality genomic
sequences exist
(a) Human, chimpanzee, and rhesus Y chromosomes.
(b) Mouse X chromosome.
The primate sex chromosomes are a mosaic of sequence classes: ancestral (often existing in
single-copy), ampliconic (which are often acquired), pseudoautosomal, and heterochromatic. The
human Y additionally contains sequences transposed from the X chromosome after the
divergence of human and chimpanzee.
Figure adapted from Hughes et al., 2012, Mueller et al., 2008.
18
Despite a common origin, the mammalian X and Y chromosome differ dramatically in
their composition of ancestral sequences. As theory predicts, following suppression of X-Y
crossing over, the human X and Y chromosomes have become highly differentiated - whereas
the ancestral genes of the X chromosome are largely intact (Ross et al., 2005), few ancestral
genes remain on the Y chromosome (Skaletsky et al., 2003). The persisting Y ancestral genes,
and their X homologs, have also diverged in sequence. Examining the synonymous nucleotide
divergence of the persisting X-Y gene pairs reveals a likely evolutionary path of the human sex
chromosomes (Lahn and Page, 1999b). Lahn and Page (1999b) observed that divergences
between X-Y gene pairs grouped into four clusters, or "strata", along the X chromosome. They
proposed that each stratum represents a single inversion event that isolates the genes within the
inversion from crossing over. Thus, consistent with Ohno's model of sex chromosome evolution,
the human Y chromosome has been subject to suppression of crossing over by at least four
inversion events (Bellott et al., 2014; Lahn and Page, 1999b; Ross et al., 2005).
Speculation abounds about the looming extinction of the human Y chromosome (Aitken
and Marshall Graves, 2002; Graves, 2006). However, comparative analysis across mammals has
repeatedly demonstrated that such fears are unfounded. Comparisons of the ancestral Y genes
across a diverse panel of men revealed remarkably little variation in protein-coding sequences,
which implied that natural selection has acted to preserve the coding sequences of the ancestral
Y genes during human migration and population expansion (Rozen et al., 2009). The ancestral Y
genes are evolutionary stable across even longer time scales. Comparison of human and
chimpanzee, which diverged about 6 million years ago, demonstrated that all ancestral Y genes
present in the common ancestor of human and chimpanzee have been preserved in the human
lineage (Hughes et al., 2005). Comparison of human and rhesus showed that only one gene in the
19
youngest stratum has been lost since human and rhesus diverged (Hughes et al., 2012). Thus,
gene loss on the human MSY ceased at least 25 million year ago. Across multiple mammalian
species, the retained Y ancestral genes are a non-random set: a conserved set of genes have been
preserved across multiple mammalian lineages (Bellott et al., 2010). Two of these genes were in
fact independently retained in the placental and marsupial lineages. These ancestral Y genes and
their X homologs were found to be enriched for dosage-sensitive, broadly expressed regulators
of transcription, translation and protein stability. Thus, survival of the Y ancestral genes may
have been driven by selection to maintain the ancestral dosage of homologous X-Y gene pairs
that have regulatory functions in many tissues.
While the Y chromosome has lost the vast majority of its ancestral genes, it has also
acquired genes. Acquired genes are often amplified; they are frequently located within large
tracts of highly identical sequences, termed amplicons. These exceptional amplicons (often > 100
kb in length, > 99.9% in identity) were first noticed on the human Y chromosome, the first
mammalian Y chromosome to be completely sequenced (Skaletsky et al., 2003). 30% of the
human Y euchromatic sequence consists of amplicons. The majority of ampliconic sequences on
the human Y chromosome occur as palindromes, or inverted repeats separated by a short
spacerthe largest of which span 3 Mb. Complete Y chromosome sequence from the chimpanzee
and rhesus show that these amplicons are particularly labile. The chimpanzee Y contains twice as
many amplicons as human, which are massively rearranged. In contrast, the rhesus Y has little
ampliconic sequence (Hughes et al., 2010, 2012). Ampliconic Y sequences have also been found
in the accruing sequence data of other mammalian species (Murphy et al., 2006; Paria et al.,
2011). In human and other mammals, ampliconic Y genes are enriched for testis-specific
expression. Acquisition of genes with testicular function on the mammalian Y chromosomes
20
may have been foreshadowed by theoretical predictions that male-benefit genes would
accumulate in linkage to the sex-determining locus. However, their rampant amplification is
unanticipated. Growth and innovation of the mammalian Y chromosome by gene acquisition and
amplification stands counter to theories of Y chromosome decay.
The remarkable similarity between amplicons of the Y chromosome led to the hypothesis
that intrachromosomal homologous recombination might occur between amplicons. Indeed,
comparison of the palindromes between human and chimpanzee, and between humans yields
evidence of gene conversion between palindrome arms (Rozen et al., 2003). Such gene
conversion might enable the Y chromosomes to rescue deleterious mutations occurring within
ampliconic genes. However, intrachromosomal recombination between ampliconic repeats also
makes the Y chromosome prone to rearrangements. Extensive structural polymorphism has been
documented among human Y chromosomes (Repping et al., 2006). These structural
polymorphisms have significant medical relevance. Recurrent interstitial and terminal deletions
mediated by these amplified sequences cause spermatogenic failure (Balaresque et al., 2008;
Blanco et al., 2000; Giachini et al., 2009; Kamp et al., 2000; Kuroda-Kawaguchi et al., 2001;
Reijo et al., 1995, 1996; Repping et al., 2002, 2003; Stouffs et al.; Sun et al., 2000; Visser et al.,
2009; Vogt et al., 1996). Recombination between palindromes on the long arm results in
isodicentric Y chromosomes (idicYp): mirror-imaged chromosomes with an axis of symmetry
through the center of the involved palindrome. Such chromosomes can result in spermatogenic
failure as well as sex reversal or Turner syndrome (Lange et al., 2009).
Similarly unanticipated observations have been made of the mammalian X chromosome.
Ohno's law predicts that X chromosome gene content should be invariable amongst placental
mammals. Indeed, X chromosomal content is broadly constant across diverse mammals (Band et
21
al., 2000; Delgado et al., 2009; Kuroiwa et al., 2001; Murphy et al., 1999; Prakash et al., 1996;
Raudsepp et al., 2004; Ross et al., 2005; Spriggs et al., 2003; Watanabe et al., 1999). However,
the X chromosome is not perfectly unchanging. On both human and mouse X chromosomes,
genes have been acquired (Emerson et al., 2004; Palmer et al., 1995; Rugarli et al., 1995).
Additionally, X chromosomes also appear enriched for ampliconic sequences and gene families
expressed specifically in the testis (Mueller et al., 2008; Warburton et al., 2004). A systematic
comparison of gene content of human and mouse X chromosomes demonstrated that although
the majority of X-linked single-copy genes abide by Ohno's law, being shared by both human
and mice, most X-ampliconic genes are exceptions to Ohno's law - over two-thirds of human
and mouse X-ampliconic genes have no orthologs in the other species (Mueller et al., 2013).
Thus, many X genes have been independently acquired and amplified since divergence from the
common ancestor of humans and mice. The mammalian X chromosome, like the Y chromosome,
has experienced growth and innovation via gene acquisition and amplification.
MAPPING AND SEQUENCING OF THE MOUSE
Y CHROMOSOME
Genomic analysis of mammalian sex chromosomes has revealed unanticipated
characteristics of the sex chromosomes, and highlighted their relevance to human health and
disease. To date, complete genomic sequences exist only for the human, chimpanzee, and rhesus
male-specific region of the Y chromosomes (MSYs). Further study of mammalian sex
chromosomes would benefit from high-quality reference sequence across a broad range of
mammals, especially in a tractable model organism. The mouse MSY would be an ideal
candidate for sequencing.
Prior to our sequencing efforts, there was limited knowledge of content of the mouse Y
chromosome. It was estimated by flow cytometry to be 94.7 Mb in size (Bergstrom et al., 1998),
22
but only a mere two megabases from the putative short arm had been mapped, and 750 kb
sequenced (Figure 1.3a). Conventional genetic mapping methods cannot be applied to the MSY,
as it does not undergo crossing over. Early mapping on the mouse Y chromosome relied on gross
chromosomal translocations and deletions (Figure 1.3b-i). These mapping efforts were aided by
parallel mapping efforts of human orthologs.
Mapping and sequencing of the mouse MSY began with and was most complete for the
putative short arm. The first phenotypes mapped to the mouse Y were the Testis determining
factor, Y-linked (Tdy) (Welshons and Russell, 1959) and Male-specific antigens (Hya)
(Eichwald and Silmser, 1955; Eichwald et al., 1958). Mapping of these and subsequently
discovered loci relied heavily on a translocation of the Y chromosome to the X chromosome, the
Sxra translocation, and its derivatives. Tdy and Hya map to the Sxra region (Cattanach et al.,
1971; Evans et al., 1982; Simpson et al., 1981). Hya and Tdy are genetically separable - only
Hya maps to ASxrb, a region deleted in a variant of Sxra (McLaren et al., 1984; Roberts et al.,
1988; Simpson and Page, 1991). Subsequently, a spermatogonial proliferation factor, Y-linked,
Spy, was proposed to also map to ASxrb (Burgoyne et al., 1986).
23
Figure 1.3
Map of mouse MSY, and Y chromosome-related translocations and deletions
(a) Map of mouse MSY, as of 2002. The mouse MSY was thought to have a short arm, which the
Sxra region mapped to. Most genes identified are located within the Sxra region. The long arm of
the chromosome was thought to contain highly-repetitive sequences, including a testis-specific
transcript, Ssty. The pseudoautosomal region (PAR) lies on the distal end of the long arm.
(b) XSxra carrying translocation from the Y chromosome. The translocation contains Sry, thus
XXSxra mice develop as males.
(c) XSxrb is a variant of XSxra, where an interstitial deletion has occurred between ZfyJ and Zfy2.
(d) YSxra carries the Sxra translocation on the distal end of the pseudoautosomal region. It is
thought to result from crossing over at the PAR between XSxra and a wild-type Y chromosome.
(e)
YTdY"
has a deletion of Sry. X YTl"J mice develop as females.
(f) Yd-1 has a deletion of sequences outside of Sry, but X Yd-~ mice also develop as females. It is
also deleted for some copies of Rbmy.
(g) B 10.BR-Ydel is deleted for sequences in the long arm; it is estimated to retain only about
25% of long arm sequences.
(h) RIII-Ydel is deleted for sequences in the long arm; it is estimated to retain only about 33% of
long arm sequences.
(i) YTdy"-qdel is deleted for sequences in the long arm; it is estimated to retain only about 10%
of long arm sequences. The deletion arose on the
YTy"
Figure adapted from Alftldi, 2008; Tour6 et al., 2004.
24
chromosome.
a
b
Mouse MSY map (-2002)
XSx*
C XSxr
zfy-
short am
3Ltely
Jandid
E2s3y
ULy
~gwm
Sm* ASxi
reginon
ZPy2
Dd3y
x
Rbwy(5-30) J.
affn
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chrmosonm
dvomosome
IJarild
(1OD+)
Rmy (2-10)
Sey
Zfy2
Uspey
Ddx3y
Uzy
PAR
PAR I
r
d
e
YSxra
short arm
suA
f
__
lya
Yd4
short arm
Zy1
*Ubely
short arm
Zy1
Sia* [
zjyl
YT*'"l
I Ibmy (2-10)
PAR E
Mtely
Zy1
*tbely
Jadd
JaRbdmdy
Ei2a3y
E0283y
(Ny
Uly
D&x3y
Uly
Ddx3y
Ddxy
uDW3y
Rbmy
S
(100+)
lorv am
SSIY
(100+)
+
R&Wy (5-,W)
Mw2-10)
long+m
Zfy2
Sty
RWmy (2-10)
(5-w)
(100+)
D"x~
iAy
EM2V3
PAR E
___
PAR r
zld
travaxaton
g
BIO.BR-Ydel
short armI
h
Rill-Ydel
short arm
Yrd--qdeI
shoda
mm
Zy
Uftly
-25%
"a
PARm
25
(Z0)
Molecular identification of genes followed. Sex determining region, Y-linked, (Sry), was
first identified and proposed to be a candidate for Tdy (also known as TDF) in human (Sinclair et
al., 1990). In parallel, Sry was cloned in mouse (Gubbay et al., 1990). A Y-chromosome deletion
removing Sry, YTd", results in XYT" female mice (Lovell-Badge and Robertson, 1990).
Conversely, transgenic addition of Sry is sufficient for male-determinination in the mouse
(Koopman et al., 1991). A repeat sequence, Sx1, located outside of Sry and deleted on a Y dchromosome, was also found to result in sex-reversal (Capel et al., 1993). Also identified in the
course of the search for Tdy were Zfy and Zfy2 (Mardon and Page, 1989; Mardon et al., 1989;
Nagamine et al., 1989; Page et al., 1987).
Mapping and sequencing of Sxra and its derivatives led to identification of an additional
handful of genes: Ubaly (Ubely), Kdm5d (Jaridld/Smcy),Rbmy, Ddx3y (Dby), Eia2s3y, Usp9y
(Dffry), and Uty (Agulnik et al., 1994; Brown et al., 1998; Capel et al., 1993; Laval et al., 1995;
Mazeyrat et al., 1998; McLaren et al., 1992; Mitchell and Bishop, 1992; Mitchell et al., 1991).
The Hya and Spy phenotypes could now be accounted for. Hya was shown to be encoded for by
two genes, Jarid1dand Uty (Greenfield et al., 1996; King et al., 1994; Scott et al., 1995). Spy is
encoded for Eij2s3y, but not Ubely, Ddx3y, or Jarid1d(Agulnik et al., 2001; Mazeyrat et al.,
2001).
Earlier cytogenetic studies suggested that the mouse Y had a small short arm (Ford,
1966). Sxra appeared to be located on this short arm by DNA FISH (Roberts et al., 1988), though
it was difficult to distinguish if it indeed resided on the short arm, or simply close to the
centromere on the long arm. Analyses of the structure of one Sxrb variant led investigators to
infer that Sxrb had undergone recombination with the Y chromosome. For this to be possible, the
Sxra region must normally be located on the short arm (McLaren et al., 1988). Together, these
26
observations suggest that the mouse Y has a short arm, unlike all the other telocentric mouse
chromosomes. The mouse Y is also distinct from all other mouse chromosomes in having
centromeric sequence that is dissimilar from all other mouse centromeres (Broccoli et al., 1991;
Matsuda and Chapman, 1991; Pardue and Gall, 1970).
Outside of Sxra, the mouse Y was thought to contain highly repetitive sequences on the
long arm (Bergstrom et al., 1997; Bishop et al., 1985; Eicher et al., 1989; Hutchison et al., 1989;
Navin et al., 1996; Nishioka, 1988; Nishioka and Lamothe, 1986; Nishioka et al., 1993, 1994;
Phillips et al., 1982). The repetitive sequence is highly variable amongst the Mus genus, and is
thus thought to be rapidly evolving. The sequences were known to contain at least one testis
transcript, Ssty (Spermiogenesis-specific transcript, Y-linked) (Bishop and Hatat, 1987; Prado et
al., 1992; Toure et al., 2004).
Two sets of evidence led to the hypothesis that these long arm repetitive sequences play
roles in spermiogenesis. XO mice carrying the Sxr" translocation (containing most of the genes
on the short arm, including Sry, but none of the long arm) are phenotypically male but sterile, as
their germ cells arrest during meiotic metaphase. This meiotic metaphase arrest can be rescued
by providing a meiotic pairing partner. However, this does not restore fertility because the sperm
produced have abnormal sperm heads. This led to the conclusion that the mouse Y long arm is
required for normal sperm head development. A series of mice with partial deletions of the
mouse Y long arm (Figure 1.3g-i), B 1 O.BR-Ydel (~25% of normal Yq present), RIII-Ydel
(-33%), and
yTdyml -qdel
(-10%), also have fertility and sperm head abnormalities (Conway et
al., 1994; Styrna et al., 1991, 2002; Tourd et al., 2004; Xian et al., 1992). The severity of
phenotype correlates with the extent of deletion.
27
Some studies have suggested that the mouse Y long arm sequences are engaged in a
battle with the X chromosome to be preferentially transmitted to the next generation. Such
conflict, whereby a locus within a genome favors preferential transmission of itself over another
allele, is referred to as meiotic drive. This speculation arises out of observations that RIII-Ydel
and BR10.BR-Ydel mice, whose Y chromosomes carry fewer copies of the repetitive long arm
sequences, produce more female than male offspring (Conway et al., 1994; Moriwaki et al.,
1988). Antagonism between the X and Y chromosomes can also be observed at the level of gene
expression - mice lacking some or part of the Y long arm exhibit higher transcription of several
X-linked spermatid expressed genes (Ellis et al., 2005). Thus, it was suggested that a possible
function of the mouse Y long arm sequences may be to repress transcription from the X
chromosome in post-meiotic germ cells. The exact mechanism by which transmission distortion
occurs remains unknown. For example, whether mice with deletions of the Y long arm make
more X than Y-bearing sperm has not yet been directly tested. However, if fertilization is
performed by intracytoplasmic sperm injection, sex-ratio balance is restored, suggesting that sexratio distortion results from a fertilization defect of Y-bearing sperm, rather than an imbalance in
production of X- versus Y-bearing sperm (Ward and Burgoyne, 2006).
SINGLE-HAPLOTYPE ITERATIVE MAPPING AND SEQUENCING
Sequencing the mouse Y chromosome requires special attention to its repetitive structure. In
addition, we have found that all previously sequenced mammalian Y chromosomes, of human,
chimp, and rhesus, contained ampliconic sequences - long stretches of duplicated sequences that
share high nucleotide identity. To resolve such repetitive ampliconic sequences requires a
method with an extraordinary level of accuracy and precision.
28
Approaches that are presently popular, such as whole-genome shotgun, would fail to
accurately assemble ampliconic sequences with long, highly similar repeats. In a whole-genome
shotgun approach, the entire genome is sequenced at once in short reads (typically < 1 kb in
length). Such short reads could not be used to distinguish between two repeat units spanning
kilobases, and that only differ by one basepair per thousands. Ampliconic repeat units would thus
be collapsed into a single unit, not representative of actual sequences.
Our laboratory previously developed and used single-haplotype iterative mapping and
sequencing (SHIMS) to assemble ampliconic sequences (Hughes and Rozen, 2012; Hughes et
al., 2010, 2012; Mueller et al., 2013; Skaletsky et al., 2003). SHIMS is a clone-based sequencing
strategy whereby the genome is first mapped with large insert clones which are then individually
sequenced. The large-insert clones used in these instances were bacterial artificial chromosomes
(BACs), which can contain up to 200 kb of insert sequence, thus enabling the assembly of large
contiguous regions within long, highly-identical repeats. SHIMS iterates between mapping and
sequencing, and requires close attention to identify rare mismatches between highly similar
sequences, termed sequence family variants (SFVs). First, BACs are identified for sequencing.
BACs with SFVs matching assembled sequence (sequence contigs) are considered true
overlapping neighbors and therefore are sequenced to extend existing contigs. New SFVs are
considered to represent a new repeat unit, and new neighbors would need to be identified. This
process is iterated until no new SFVs between BACs are found.
In Chapter 2, I describe our SHIMS efforts of the mouse Y chromosome. We expect the
sequence to both enable expanded comparative analysis across mammals, and also to serve as a
29
resource for genetic studies of the Y chromosome in a mammalian genetic model organism.
Analysis of the mouse MSY reveals a potential role of sex chromosome meiotic drive in the
evolution of the mammalian sex chromosomes.
30
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40
C. MAKING A GAMETE
During the development of multicellular organisms, cells differentiate into two fundamental
lineages. Somatic cells, which form the bulk of the organism, ultimately perish. Germ cells, by
contrast, deliver genetic material from the parental generation to future generations, and
therefore may be considered a privileged, in vivo immortal cell type. During their life span, germ
cells differentiate from being unipotent precursors to highly specialized and sexually dimorphic
haploid gametes, and yet retain the capacity to form a totipotent zygote. Importantly, they
undergo meiosis, a specialized cell division to halve and generate new combinations of parental
genetic material. I will briefly summarize the differentiation of mouse germ cells from initial
specification to the time of initial sexual differentiation (reviewed in Lesch and Page, 2012), and
then focus on the processes of meiotic initiation and meiotic prophase.
GERM CELL SPECIFICATIONAND MIGRATION
In metazoans, germ cells may be specified in two ways: pre-formation, or induction (reviewed in
Extavour and Akam, 2003). In pre-formation, germ cell fate results from inheritance of
maternally-provided determinants, called the germ plasm. In induction, germ cell fate is induced
by extracellular signals.
Mammalian germ cells are specified by induction (Tam and Zhou, 1996). Primordial
germ cells (PGCs) can be identified in the posterior proximal epiblast by as early as E6.25, by
markers specific to or enriched in primordial germ cells (PGCs), including Tnap, Dppa3 (Stella),
fragilis, Prdml (Blimp]), Prdm14, and Tcfap2c (Bortvin, 2003; Chiquoine, 1954; Ginsburg et
al., 1990; Ohinata et al., 2005; Saitou et al., 2002; Sato et al., 2002; Weber et al., 2009; Yabuta et
al., 2006). PGCs are induced primarily by BMP4 from the extra-embryonic ectoderm, and
WNT3 from the epiblast (Lawson et al., 1999; Ohinata et al., 2009) (Figure 1.4). WNT3 induces
41
T (Brachyury), and together with BMP4, enables robust activation of PGC markers Prdml and
PrdmJ4 (Aramaki et al., 2013). Additional BMP8b and BMP2 signals from the extraembryonic
ectoderm oppose yet unidentified signals that antagonize germ cell formation in the anterior
epiblast (de Sousa Lopes et al., 2004; Ying and Zhao, 2001; Ying et al., 2000).
Despite many genes identified as expressed in early PGCs, the specifier of germ cell
identity remains unknown. None of these genes appear to be required for germ cell specification
per se - embryos deficient for these genes still specify germ cells, but these germ cells fail to
progress and die (Bortvin et al., 2004; Lange et al., 2008; Ohinata et al., 2005; Payer et al., 2003;
Weber et al., 2009; Yamaji et al., 2008). Additionally, several of these markers are not specific to
PGCs (Mikedis and Downs, 2012, 2013; Rosner et al., 1990; Yamaguchi et al., 2005).
Nevertheless, these genes do play important roles in PGC development. Prdml appears to
prevent somatic differentiation via suppression of somatic markers such as Hoxbl (Ohinata et al.,
2005). Prdml is then thought to induce Prdm14, and together they are required to re-active
expression of pluripotency markers, such as Sox2 (Yamaji et al., 2008). In culture, Prdml,
Prdm14, and Tcfap2c, appear to cooperate in PGC induction (Magnnisd6ttir et al., 2013; Nakaki
et al., 2013; Schemmer et al., 2013). Studies in PGC culture systems suggest additional potential
roles for Lin28 as a positive regulator of Prdml expression and PGC numbers (West et al.,
2009).
42
4
extraembryonic
ectoderm
genital ridge
somatic
testis
IBMP4
prospermatogonia
G1/GO
WNT3
proximal
specification
epibla
primordial
germ cell migration
arrest,
initiate meiosis
Dazi
gametogenesis
licensing competent cell
after birth
t
E6.25 -E7.25
Figure 1.4
E8.5-E11.5
E10.0- 11.5
oocyte
initiate
somatic
ovary
meiosis
E12.5
EI&5
Model for germ cell induction, licensing, and subsequent differentiation.
Primordial germ cells are induced from the posterior proximal epiblast by signals from the
extraembryonic ectoderm. They migrate to the genital ridge, where unidentified signals from the
genital ridge induce Dazi expression. Dazl is required for germ cells to be licensed as
gametogenesis-competent cells. Gametogenesis-competent cells then sexually differentiate
depending on whether they find themselves in a testis or ovary. Testicular germ cells develop as
prospermatogonia. They enter cell cycle arrest and delay meiosis until after birth. Ovarian germ
cells develop as oocytes. They initiate meiosis immediately. Below: Approximate timings of key
transitions. E = embryonic day.
Adapted from Hu et al., 2015.
43
PGCs must migrate from their site of specification, the proximal epiblast, through the
developing hindgut, and eventually into the genital ridges (Anderson et al., 2000; Molyneaux and
Wylie, 2004; Molyneaux et al., 2001). During migration, PGCs maintain a pluripotent fate
(McLaren, 2003; Rosner et al., 1990; Yabuta et al., 2006; Yamaguchi et al., 2005), mitotically
divide (Tam and Snow, 1981), and are thought to reset their epigenetic landscape to that of an
early undifferentiated embryo (Hajkova et al., 2008; Seki et al., 2005; Surani et al., 2008)
ARRIVAL AT THE GONAD AND GERM CELL LICENSING
PGCs arrive at the gonad between E 10.5 and E 11.5. PGC entry into the gonad is
associated with DNA demethylation (Hajkova et al., 2002), and with transcriptional changes
(Molyneaux et al., 2004), including the induction of a set of genes, including Dazl, Mvh, Gcna,
and Sycp3 (Di Carlo et al., 2000; Enders and May, 1994; Fujiwara et al., 1994; Seligman and
Page, 1998; Tanaka et al., 2000; Toyooka et al., 2000). Soon after entry into the gonad, germ
cells down-regulate pluripotency genes (Bullejos and Koopman, 2004; Menke et al., 2003;
Western et al., 2005) and sexually differentiate according to cues from the somatic ovary or
testis. The transition from PGC to differentiating germ cell, termed germ cell licensing, requires
the RNA-binding protein, DazL. On a C57BL6 background, Dazl-deficient germ cells fail to
differentiate, and instead retain expression of pluripotency genes (Gill et al., 2011; Lin et al.,
2008). Induction of Dazl and germ cell licensing is dependent on signaling by the genital ridge
(Hu et al., 2015), but the soma-dependent factors that induce licensing remain unidentified.
Upon germ cell licensing, ovarian and testicular germ cells embark on divergent paths of
differentiation. Ovarian germ cells immediately initiate meiosis and an oogenesis differentiation
program, whereas testicular germ cells delay meiosis until after birth. Instead, they mitotically
arrest and begin a spermatogenesis differentiation program (Adams and Mclaren, 2002;
44
McLaren, 2003). The testicular germ cells develop into spermatogonial stem cells which
differentiate and initiate meiosis in cyclical waves every 8.6 days (Oakberg, 1956). One of many
important decisions that ovarian and testicular germ cells make upon germ cell licensing is
therefore whether and when to initiate meiosis.
45
I
D. MEIOTIC PROPHASE AND MEIOTIC INITIATION
Meiosis is a specialized cell division program that results in the halving of parental genetic
material and the production of haploid gametes. During meiosis, a single round of DNA
replication is followed by two consecutive cell divisions without an intervening S-phase. The
first meiotic division is reductive - homologous chromosomes segregate, thereby halving ploidy.
During the second meiotic division, sister chromatids segregate, as in a mitotic division.
The reductive division of meiosis depends on a set of complex chromosomal processes
that occur meiotic prophase, termed here the chromosomal program of meiotic prophase.
Meiosis-specific cohesins must be loaded, homologs must pair and synapse, and homologs must
recombine. This chromosomal program must be supported by expression of a large suite of
genes, termed here the gene expression program. While the intricacies of the chromosomal
program are well-studied, how its initiation is regulated has only been discovered in the past
decade. How the prerequisite gene expression is regulated has also received little attention. In
Chapter 3, 1 describe our efforts to characterize the genetic regulation of the gene expression
program of meiotic prophase, which I term the gene regulatory program. In the following
sections, I first summarize the key events of the chromosomal program (extensively reviewed in
Cohen et al., 2006; Handel and Schimenti, 2010; Marston and Amon, 2004), with a focus on
mouse meiosis. Next, I review what is known about the regulation of meiotic initiation and gene
expression program.
THE CHROMOSOMAL PROGRAM OF MEIOTIC PROPHASE
The chromosomal program of meiotic prophase is the subject of intense study (Cohen et al.,
-
2006; Handel and Schimenti, 2010; Marston and Amon, 2004). It comprises three major events
47
loading of meiotic cohesins, synapsis, and recombination - which occur starting from premeiotic S-phase through the four stages of meiotic prophase, leptotene, zygotene, pachytene, and
diplotene (Figure 1.5a).. Proteins involved in the chromosomal program are largely conserved
and well-studied from yeast to mouse. I summarize here results pertaining only to mouse.
Meiotic cohesins are loaded onto chromosomes during the S-phase preceding meiotic
division. The cohesin complex is thought to form a ring that holds together sister chromatids
(Nasmyth and Haering, 2009; Peters et al., 2008; Uhlmann et al., 1999). Four meiosis-specific
cohesin proteins have thus far been identified in mouse: SMCIB (Revenkova et al., 2004), REC8
(Bannister et al., 2004; Xu et al., 2005), RAD21L (Gutidrrez-caballero et al., 2011; Herrin et al.,
2011; Ishiguro et al., 2011; Lee and Hirano, 2011), and STAG3 (Pezzi et al., 2000; Prieto et al.,
2001). Meiotic cohesins must replace the mitotic cohesins to ensure sister chromatid cohesion
throughout the first meiotic division. During mitotic anaphase, the mitotic cohesin RAD21 is
cleaved to allow segregation of sister chromatids to opposite poles. During pre-meiotic S-phase,
however, REC8, replaces RAD21. During anaphase I of meiosis, REC8 is cleaved along the arm
regions but remains protected at centromeres until metaphase II (Buonomo et al., 2000; Moore
and Off-Weaver, 1998; Tachibana-Konwalski et al., 2010). Thus, sister chromatids remain
connected at the centromeres throughout the first meiotic division, and segregate to the same
pole. Cohesins loaded during pre-meiotic S-phase form the initial core of the axial elements
along the sister chromatids, which eventually form part of the synaptonemal complex responsible
for synapsis.
48
SYCP3
SYCp1
SYCE1
SYCE2
SYCP2
TEX12
Formation of
axial elements
DNA replication
Formation of
central elements
Synapsis
Do-synapsis
0
Loading of
cohesins
REC8
SMC1B
STAG3
RAD21L
DSB formation
spoil
Recombination
DSB repair
DMC1
MSH4
MSH5
.
a
Cross-over
stabilization
MLHI
MLH3
b
Female
Male
Figure 1.5
E13.5
E14.5 - E15.5
E15.5 - E16.5
E17.5 - E18.5
E1S.5 -PO
P9
PI
P12
P14
P20
Stages and events of meiotic prophase
a. Meiotic prophase is preceded by pre-meiotic DNA replication. Prophase itself may be divided
into leptotene, zygotene, pachytene, and diplotene. Three major chromosomal events occur
during prophase. (1) Meiotic cohesins are loaded during pre-meiotic DNA replication. (2)
Chromosomes synapse via formation of axial/lateral and central elements. (3) Recombination
occurs via formation and repair of double-strand breaks (DSBs). A subset of the initial DSBs are
stabilized to form cross-overs. Genes known to play roles in in these processes are listed at the
approximate stage at which they are thought to function. b. Approximate timings of stages
occurring in female germ cells (from Speed, 1982). E = embryonic day. Approximate timings of
the first appearance of stages during the first wave of spermatogenesis in male germ cells (from
Bellve et al., 1977). Subsequent to the first wave, all stages are continually present in the testis. P
= postnatal day.
49
Synapsis of homologous chromosomes is mediated by the synaptonemal complex (SC), a
protein scaffold that forms along the length of chromosomes. During leptotene, chromosomes
begin to condense and homologs begin to align but are not paired. Axial elements, comprising
proteins such as SC proteins SYCP3 (Yuan et al., 2000, 2002) and SYCP2 (Yang et al., 2006), in
addition to the cohesins, assemble on each chromosome homolog. During zygotene, synapsis
between homologs is initiated: central elements, comprising proteins such as SYCP1, SYCE1,
SYCE2, TEX12, form between homologs (Bolcun-Filas et al., 2007, 2009; Hamer et al., 2008;
de Vries et al., 2005), resulting in pairing along the length of homologs. At pachytene, the
homologs are fully synapsed. At diplotene, homologs desynapse.
The chromosomal synapsis orchestrated by the SC is accompanied by homologous
recombination. Homologous recombination is initiated during leptotene by formation of doublestrand breaks (DSBs). DSBs are catalyzed by SPO 1 (Baudat et al., 2000; Keeney et al., 1999;
Romanienko and Camerini-Otero, 2000), and also require Meil, Mei4 and Recl14 (Kumar et al.,
2010; Libby et al., 2002, 2003; Panizza et al., 2011). DSBs can be detected by phosphorylated
H2A.X (gH2A.X) (Rogakou et al., 1998). DSBs are processed and repaired by DMC1, a
meiosis-specific homolog of RecA (Bishop et al., 1992; Pittman et al., 1998; Yoshida et al.,
1998). Repair and maturation of DSBs into sites of crossovers additionally require the mismatch
repair machinery (Baker et al., 1996; Edelmann et al., 1996, 1999; Kneitz et al., 2000; Kolas et
al., 2005; Lipkin et al., 2002; Vries et al., 1999). MSH4 and MSH5 localize to DSBs at leptotene.
MLH 1 and MLH3 localize to DSB at pachytene and are required to stabilize crossovers. A
subset of DSBs mature into chiasmata that hold together homologous chromosomes, so as to
ensure their accurate segregation during the first meiotic division.
50
REGULA TION OF MEJOTIC INITIA TION
The timing of meiotic prophase differs in males and females (Figure 1.5b). In females, meiotic
prophase begins in the fetal ovary. The first ovarian germ cells enter leptotene beginning E 14.5
of gestation, with the entire cohort of germ cells initiating and progressing through meiotic
prophase relatively synchronously (Borum, 1961; Speed, 1982). There is an anterior-to-posterior
wave of progression, which is reflected by an anterior-to-posterior wave of expression of Stra8,
DmcJ, and Sycp3 (Bullejos and Koopman, 2004; Menke et al., 2003). Ovarian germ cells reach
the end of meiotic prophase, or diplotene, by around birth, upon which they arrest. They resume
the first meiotic division only upon ovulation, and then arrest at metaphase II until fertilization.
In males, the earliest wave of meiotic prophase begins postnatally around P10. Thereafter,
continually throughout life and in cyclical waves in each tubule of the testis, differentiated
spermatogonia initiate meiosis every 8.6 days (Oakberg, 1956).
Several mechanisms had been postulated for this sexual dimorphism in timing of meiotic
entry. One theory was that the fetal testis produces a meiosis preventing substance. This was
supported by observations that germ cells that mis-migrate to the adrenal glands, or that are cocultured with lung tissue, nevertheless initiate meiosis (McLaren and Southee, 1997; Upadhyay
and Zamboni, 1982; Zamboni and Upadhyay, 1983); only testicular germ cells do not initiate
meiosis. In the absence of a testicular meiosis preventing substance, it was suggested that germ
cells would enter meiosis on a cell-autonomous clock. An alternate theory was that meiosis is
induced by a meiosis-inducing substance, and some evidence suggested as candidate a steroidlike molecule from the mesonephros which lies adjacent to the ovary (Andersen et al., 1981;
Grinsted and Byskov, 1981). Such a substance would have to exist in tissues where mis-migrated
germ cells initiated meiosis.
51
The molecular identities of both a meiosis inducing substance and a meiosis preventing
substance were identified in the past decade (Figure 1.6). In mouse fetal ovarian germ cells,
meiosis is initiated by a meiosis inducing substance, retinoic acid (RA) (Bowles et al., 2006;
Koubova et al., 2006). RA induces expression of Stra8, a putative helix-loop-helix-containing
transcription factor which had originally been identified as being induced by retinoic acid (RA)
in P19 embryonal carcinoma cells (Oulad-Abdelghani et al., 1996). Stra8 is required for the
chromosomal events of meiotic prophase: in the absence of Stra8, germ cells fail to undergo premeiotic DNA replication, make DSBs, load meiotic cohesins, and synapse (Baltus et al., 2006;
Bowles et al., 2006; Koubova et al., 2006). In mouse fetal ovarian germ cells, RA induction of
Stra8 and meiotic initiation requires the germ cell-expressed competence factor Dazl (Lin et al.,
2008). In the postnatal testis, Stra8 and meiotic initiation is also induced by RA (Ghyselinck et
al., 2006; Koubova et al., 2006; Zhou et al., 2008). Thus, even though the timing of meiosis is
sexually dimorphic, the signals and regulators that initiate meiosis are similar. In the fetal testis,
meiosis does not initiate because of expression of the RA metabolizing enzyme Cyp26bJ
degrades RA (Bowles et al., 2006; Koubova et al., 2006; MacLean et al., 2007). One can now
interpret earlier observations in light of these recent findings. For example, the observation that
mis-migrated or cultured germ cells initiate meiosis does not necessitate invoking a cellautonomous clock. Instead, meiotic initiation in mis-migrated germ cells or tissue culture could
be induced by RA, which is a ubiquitous signaling molecule in developing tissues.
52
........
................
- - .......................
b
a
Dade
Dart
Cyp26bl-I Z A
RA
Fgffi
W8
Chromosomalprogram
of melatic prophase
Figure 1.6
Model for regulation of meiotic initiation in fetal gonads.
a. In the fetal ovary, the extrinsic signal, retinoic acid (RA), in the presence of competence
factor, Dazl, induces Stra8. Stra8 in turn is required for initiation of meiotic prophase, as assayed
by the chromosomal events of meiotic prophase. How Dazl, RA, and Stra8 regulate the gene
expression program of meiotic prophase has not yet been systematically examined. However, it
has been observed that at least two genes, Rec8 and Sycp3, are expressed in the absence of Stra8,
even though they do not appear to functionally load or assemble on the chromosomes.
Expression of one gene, DmcJ, in contrast, requires Stra8.
b. In the fetal testis, meiosis is prevented primarily via the presence of the RA-metabolizing
enzyme Cyp26bJ. Cyp26bJ degrades RA, thus preventing Stra8 induction and meiotic initiation.
Additional mechanisms involving Fg9 and Nanos2 are thought to aid in suppressing Stra8
expression.
53
Additional factors modulate the expression of Stra8 and meiotic initiation in the fetal
ovary. Polycomb repressive complex 1 (PRC1) suppresses premature Stra8 induction by
maintaining the chromatin at the Stra8 promoter in a repressed but poised state, until a critical
threshold of RA is reached (Yokobayashi et al., 2013). In a germ cell-specific knockout of PRCI
component RnJ2, fetal ovarian germ cells prematurely express Stra8, other meiotic genes, and
initiate meiosis. The Doublesex-relatedtranscription factor Dmrtl (Krentz et al., 2011), and
homeodomain transcription factors MsxJ and Msx2 (Le Bouffant et al., 2011) are required for
full expression of Stra8. Full body knockouts of these genes result in fewer germ cells with
STRA8 expression, and fewer germ cells successfully initiating meiosis. Nevertheless, a reduced
number of germ cells do progress through meiosis and mature into follicles. In the Dmrtl
knockout, these remaining germ cells are sufficient for fertility. A caveat to these studies is that it
is unclear if the effects of Dmrtl and Msx]/Msx2 on Stra8 expression are germ cell-autonomous:
these transcription factors are expressed in both germ and somatic cells of the ovary. Thus, the
full-body knockouts of these genes would not be able to distinguish germ cell-autonomous from
somatic effects.
In the testis, Stra8 is primarily prevented from being expressed by the RA-metabolizing
enzyme Cyp26bl. Fgf9 is also thought to act directly on germ cells to additionally suppress Stra8
expression and meiotic initiation, amongst other roles in promoting male germ cell fate (Bowles
et al., 2010). After Cyp26bJ expression decreases, Nanos2, an RNA-binding protein expressed in
testicular germ cells, maintains the suppression of meiosis by preventing Stra8 expression
(Suzuki and Saga, 2008). In the postnatal testis, Dmrtl acts in spermatogonia to restrict RA
responsiveness and directly repress Stra8 transcription, thereby preventing meiosis. Germ-cell
specific loss of Dmrtl causes spermatogonia to precociously exit the spermatogonial program
54
and enter meiosis. Dmrt] thus appears to have opposing effects on Stra8 expression in ovarian
versus testicular germ cells (Matson et al., 2010).
The decision to initiate meiosis had historically been conflated with the decision of germ
cell sexual differentiation (Kocer et al., 2009; McLaren, 2003), but recent evidence suggests
these are genetically distinct decisions. The initial confusion resulted partly because these two
processes are temporally intertwined, and partly due to lack of distinguishing characteristics of
early oogenesis versus spermatogenesis beyond meiotic initiation. Close examination of the
Stra8 mutant demonstrated that oocyte-like cells can differentiate even in the absence of the
meiotic chromosomal program (Dokshin et al., 2013). Reciprocal analyses of oogenesis mutants
might demonstrate that meiosis can proceed in the absence of oogenesis.
Studies of meiotic initiation in diverse model organisms have demonstrated that although
conceptual parallels exist between species, the molecular mechanisms regulating meiotic
initiation are poorly conserved (reviewed in Kimble, 2011). Meiosis in both mouse and yeast
require both a cell-intrinsic and extrinsic signal, converging upon a key regulator of meiotic
initiation. In mouse, the extrinsic signal, RA, and the regulator of meiotic initiation, Stra8, are
found only in vertebrates. In yeast, extrinsic nutrient signals converge with the cell-intrinsic
requirement to express the a/a heterodimer to induce IME 1, which is required for meiotic
initiation (reviewed in Marston and Amon, 2004; van Werven and Amon, 2011). In both worm
and fly, entry into meiosis is determined by loss of repressive signals from the somatic niche as
germ cells move away from the niche. In C. elegans the distal tip cell, the somatic niche,
produces the Delta/LAG-2 signal that promotes mitotic division and inhibits meiosis. Germ cells
initiate meiosis when they reach a critical distance from the niche. Initiation is primarily
regulated in parallel by GLD-1 and GLD-2, which are hypothesized to repress translation of
55
mitosis-promoting mRNAs and activate translation meiosis-promoting mRNAs respectively
(reviewed in Kimble and Crittenden, 2007). In Drosophila,BMP signals from the cap cells in
females, and JAK/STAT signaling from the hub cells in males suppress differentiation (reviewed
in Morrison and Spradling, 2008). As germline stem cells move away from the niche, they
differentiate into cystoblasts and embark on meiosis. Differentiation and the switch to meiosis
require the translational regulators bag-of-marbles(bam) and benign gonialcell neoplasm (bgcn)
(reviewed in Kimble, 2011). This lack of conservation means that while we can look to other
models for conceptual parallels, understanding vertebrate regulators like Stra8 and RA requires
unique studies in a mouse model.
THE GENE EXPRESSION PROGRAM OF MEIOTIC PROPHASE
In most studies of meiosis and of initiation of meiosis, "meiosis" most often refers to the
chromosomal program of meiotic prophase - the events that directly result in the tethering of
homologs to each other, thus enabling meiotic cell division. The Stra8 mutant, for instance, was
evaluated primarily for, and showed absence of, pre-meiotic DNA replication, REC8 (a cohesin)
and SYCP3 (a synaptonemal complex protein) loading along meiotic condensed chromosomes,
and double-strand break formation as marked by gH2A.X (Anderson et al., 2008; Baltus et al.,
2006).
The chromosomal program of meiotic prophase must be preceded and enabled by
expression of a large suite of proteins required to directly mediate the chromosomal events. I
refer to the suite of such genes as a gene expression program, and the regulation of this
expression program as a gene regulatory program.
56
A genome-wide view of the gene regulatory program of meiotic prophase has not yet
been determined for mouse, nor of any other eukaryote with a segregated germline. In many
model organisms, a genome-wide view of gene expression has been determined for germ cells
and/or for cells undergoing meiotic initiation, including in budding yeast (Chu et al., 1998;
Primig et al., 2000), fission yeast (Mata et al., 2002), worm (Ortiz et al., 2014; Reinke et al.,
2000), and Arabidopsis (Yang et al., 2011). However, it is unknown in most cases how this gene
expression is regulated.
The best existing model for a meiotic gene regulatory program is that of budding yeast
(Chu et al., 1998; Kassir et al., 2003; Primig et al., 2000; Smith and Mitchell, 1989). It had been
shown that meiotic genes are induced in multiple waves, often referred to as early, middle, and
late genes. The timing of gene induction correlates with timing of gene function. Genome-wide
transcriptome studies refined these patterns of induction to seven classes (Chu et al., 1998).
Regulating this gene expression program are two transcriptional activators, IME 1 and NDT80.
The predominant view is that IME 1 is a transcriptional activator for early genes, and NDT80 is a
transcriptional activator for middle genes. Transcriptome analysis of the Ndt8O-mutant revealed
nuances to this simplified preliminary view (Chu et al., 1998). In the Ndt8O mutant, the
magnitude of induction of most middle genes was reduced compared to that in wild-type strains,
indicating the importance of Ndt80 for their induction. There remained, however, significant
Ndt80-independent induction of many of the middle genes, comparable in magnitude and timing
to that seen in wild-type cells. Thus, induction of several middle genes appeared to be in fact
independent of Ndt8O. Thus, a genome-wide approach to determining regulation of meiotic gene
expression revealed that an additional factor in addition to Ndt80 likely regulates the induction of
middle genes.
57
I propose to characterize the gene regulatory program of mammalian meiotic prophase.
This comprises identifying first what genes are expressed and when - "the gene expression
program", then determining how it is genetically regulated by mutant analyses - "the gene
regulatory program". I expect this to be useful in the following ways. First, characterization of
the gene expression program of meiotic prophase would benefit identification of additional genes
with roles in meiotic prophase. Most genes known to be involved in meiosis in mouse thus far
have been identified as well-conserved orthologs of proteins with demonstrated meiotic
functions in lower eukaryotes (Schurko and Logsdon, 2008; Villeneuve and Hillers, 2001).
However, for many other meiotic proteins identified in lower eukaryotes, their evolutionary
conservation is uncertain. One recent study identified a poorly conserved mouse ortholog of
yeast Mei4, and found that despite poor sequence conservation of 8% identity, the divergent
proteins nevertheless had functionally conserved roles in DSB formation (Kumar et al., 2010).
Identifying a gene expression program thus provides an orthogonal means of identifying
mammalian meiotic proteins that have poor sequence conservation but are functionally
conserved. Such a gene list would also enable identification of novel, mammalian-specific
meiotic proteins that do not have counterparts in lower eukaryotes. Second, characterizing the
gene regulatory program can reveal yet unidentified pathways or factors required to account for
the network architecture of meiotic gene expression.
To date, several transcriptomic studies have been performed on germ cells or whole
gonads at the time of meiotic initiation in both ovarian and testicular germ cells in the mouse,
either in an attempt to define the meiotic gene expression program, or more generally of gonad
development. In the paragraphs that follow, I summarize the existing transcriptomic studies, as
58
categorized by their main approach used to sort and dissect the stages of germ cell differentiation
and meiotic progression:
(1) Profiling of sorted germ cells from whole adult testes. In the adult testis, germ cells in
all stages of meiosis are present. Therefore, sorting must not only separate germ from
soma, but also germ cells at various stages of meiosis. Germ cells can be sorted from
somatic cells and separated into spermatogonial (pre-meiotic), pachytene (mid-meiosis),
and round spermatid (post-meiotic) populations by gravity sedimentation. Two
transcriptomic studies have been performed on these sorted germ cell populations
(Chalmel et al., 2007; Soumillon et al., 2013). However, the limited resolution provided
by gravity sedimentation precludes finer dissection of the stages of meiotic prophase.
Another possible approach for sorting germ cells from somatic cells is fluorescenceactivated cell sorting of genetically fluorescently marked germ cells. However, there is
not yet a way of further sorting the purified germ cells into various meiotic stages. It is
also possible to isolate the stages of spermatogenesis by transillumination-assisted
microdissection of the seminiferous tubule. Spermatogenesis proceeds in synchronized
waves along the seminiferous tubules. A given cross-section of the tubule will contain
only a specific combination of cell types, which gives rise to specific light absorption
patterns. One can therefore use the transillumination properties of a given region of the
seminiferous tubule to isolate specific stages of spermatogenesis. However, this method
generates small numbers of cells from each tubule, making it challenging to further
separate the mitotic, meiotic, and post-meiotic cell types at each tubule cross-section.
(2) Profiling of whole postnatal testes during the first wave of spermatogenesis. Several
transcriptomic studies of the first wave of spermatogenesis have been performed
59
(Margolin et al., 2014; Schultz et al., 2003; Shima et al., 2004, and Griswold et al.,
unpublished: GSE12769 and GSE926). This approach enables finer resolution of meiotic
progression than sorting of germ cells by gravity sedimentation. However, most studies
examine expression from whole testis, which does not enable identification of germ cellspecific or enriched expression. One study combined the first wave approach with
enriching for germ cells by fractionation on a Percoll gradient (Kogo et al., 2010).
However, even after sorting germ cells from somatic cells, the germ cell population will
still be a mixed population consisting of the most advanced meiotic stage, as well as all
stages preceding it. A recent developed method to chemically manipulate RA levels in
the testis using the retinaldehyde inhibitor WIN 18446 (Hogarth et al., 2015) might enable
synchronization of meiotic progression, which would improve yields of a homogeneous
cell population.
(3) Profiling of whole fetal ovaries and testes at timepoints corresponding to meiotic
entry in ovarian germ cells (Harding et al., 2011; Small et al., 2005). Fetal ovarian germ
cells progress through meiotic prophase relatively synchronously. The comparative
synchrony of meiotic progression in the female makes it easier to finely isolate specific
stages of meiotic prophase compared to testes. The downside to performing
transcriptomic analyses in the fetal ovary is that there are many fewer germ cells than in
the adult or postnatal testis. We chose to characterize the gene expression program from
whole fetal ovaries. We undertook a subtractive approach - comparing mutants lacking
germ cells with wildtype gonads - to identify both germ-cell-enriched as well as ovarian
versus testicular germ cell-enriched genes. A similar study has since been reported
(Rolland et al., 2011). Germ cells can also be sorted by FACS, but the small amounts of
60
material yielded would have challenged sequencing technologies available to us at the
time.
These transcriptomic studies provided a description of the gene expression program of
meiotic prophase, but not of how it is regulated. Studies of regulators of meiotic initiation so far
have assayed expression of only a limited set of genes. A global view of gene regulation is thus
still lacking. Studies of a limited gene set suggested that expression of some meiotic genes might
be independent of the signal required for initiation of the chromosomal program, Stra8. The
cohesin, REC8, and synaptonemal complex protein, SYCP3, are expressed even in the absence
of Stra8, even though their loading onto chromosomes remains dependent on Stra8 (Baltus et al.,
2006). In fact, Rec8 expression can be induced in the fetal testis in the absence of Stra8 by
ectopic RA (Koubova et al., 2014). On the other hand, expression of Dmcl, a meiotic
recombinase, is dependent on Stra8. These three observations suggest opposing models for how
the meiotic gene expression program may be regulated. Meiotic genes may be regulated
independently of Stra8, and therefore have the potential to be induced prior to initiation of the
chromosomal program. On the other hand, meiotic genes may be regulated by Stra8, so that the
decision to induce meiotic gene expression and initiate the chromosomal program are not
genetically separable by Stra8. One question is which of these principles, or perhaps both, might
be generalizable across the entire gene expression program. In Chapter 3, I describe a genomewide approach to characterizing the gene regulatory program of meiotic prophase in the fetal
ovary.
61
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CHAPTER 2.
SEQUENCING THE MOUSE Y CHROMOSOME REVEALS
CONVERGENT GENE ACQUISITION AND AMPLIFICATION ON
BOTH SEX CHROMOSOMES
Y.Q. Shirleen Soh, Jessica Alfdldi, Tatyana Pyntikova, Laura G. Brown, Tina Graves, Patrick J.
Minx, Robert S. Fulton, Colin Kremitzki, Natalia Koutseva, Jacob L. Mueller, Steve Rozen,
Jennifer F. Hughes, Elaine Owens, James E. Womack, William J. Murphy, Qing Cao, Pieter de
Jong, Wesley C. Warren, Richard K. Wilson, Helen Skaletsky, David C. Page
AUTHOR CONTRIBUTIONS
J.A., S.R., W.C.W., R.K.W., H.S. and D.C.P. planned the project. H.S. designed the mapping
strategy. J.A., T.P., L.G.B., N.K. and H.S. performed BAC mapping. T.G., P.R.M., R.S.F., and
C.K. performed BAC sequencing. T.P. performed FISH analyses. E.O., J.W., and W.J.M.
generated the mouse RH panel. Q.C. and P.d.J. selected BACs from CHORI-33, CHORI-26 and
CHORI-35 libraries. Y.Q.S.S. and H.S. performed RH mapping. Y.Q.S.S., J.A., J.L.M., S.R.,
J.F.H., and H.S. performed sequence analyses. Y.Q.S.S., H.S., and D.C.P. wrote the paper.
ACKNOWLEDGEMENTS
We thank M. Goodheart for assistance with mice; D.W. Bellott, J.F. Hughes, B.L. Lesch, K.
Romer, and L. Teitz for comments on the manuscript.
Adapted from Soh, Y.Q.S.*, Alfdldi, J.*, Pyntikova, T., Brown, L.G., Graves, T., Minx, P.J.,
Fulton, R.S., Kremitzki, C., Koutseva, N., Mueller, J.L., et al. (2014). Sequencing the mouse Y
chromosome reveals convergent gene acquisition and amplification on both sex chromosomes.
Cell 159, 800-813. *Equal contributions
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ABSTRACT
We sequenced the MSY (Male-Specific region of the Y chromosome) of the C57BL/6J strain of
the laboratory mouse Mus musculus. In contrast to theories that Y chromosomes are
heterochromatic and gene poor, the mouse MSY is 99.9% euchromatic and contains about 700
protein-coding genes. Only two percent of the MSY derives from the ancestral autosomes that
gave rise to the mammalian sex chromosomes. Instead, all but 50 of the MSY's genes belong to
three acquired, massively amplified gene families that have no homologs on primate MSYs, but
do have acquired, amplified homologs on the mouse X chromosome. The complete mouse MSY
sequence brings to light dramatic forces in sex chromosome evolution: lineage-specific
convergent acquisition and amplification of X-Y gene families, possibly fueled by antagonism
between acquired X-Y homologs. The mouse MSY sequence presents opportunities for
experimental studies of a sex-specific chromosome in its entirety, in a genetically tractable
model organism.
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INTRODUCTION
The mammalian sex chromosomes arose from an ordinary pair of autosomes (Lahn and Page,
1999; Ohno, 1967). Following the emergence of the sex-determining locus on the Y
chromosome, a series of inversions on the Y chromosome suppressed X-Y crossing over.
Suppression of X-Y crossing over liberated the X and Y chromosomes to radically differentiate.
They remain identical only in the pseudoautosomal region, where X-Y crossing over still occurs.
Reconstruction of mammalian sex chromosome evolution has identified two major
developments, and highlighted their implications for human health and disease. First, the Y
chromosome lost most of its ancestral genes, whereas the X chromosome retained them (Hughes
et al., 2005, 2010, 2012; Ross et al., 2005; Skaletsky et al., 2003). Surviving ancestral Y genes
are enriched for widely expressed, dosage-sensitive regulators of gene expression that may play
crucial roles in Turner syndrome and sexual dimorphism in disease (Bellott et al., 2014). Second,
both X and Y chromosomes have acquired and amplified genes independently of each other.
These gene families are often expressed specifically in the testis, suggestive of functions in male
gametogenesis (Hughes et al., 2010; Mueller et al., 2008, 2013; Murphy et al., 2006; Paria et al.,
2011; Ross et al., 2005; Skaletsky et al., 2003; Warburton et al., 2004). The ampliconic structure
of these amplified gene families makes them prone to recurrent rearrangements that can cause
spermatogenic failure, sex reversal and Turner syndrome (Kuroda-Kawaguchi et al., 2001; Lange
et al., 2009; Reijo et al., 1995; Repping et al., 2002, 2003).
Study of mammalian sex chromosomes and their relevance to human biology is hindered
by the lack of high-quality reference sequences of X and Y chromosomes across a broad range of
mammals. Our understanding of mammalian sex chromosomes and their evolution is presently
79
supported by a limited set of high-quality single-haplotype sequences: complete X chromosome
sequences from human and mouse, complete MSY sequences from human, rhesus, and
chimpanzee, and the ancestrally-derived sequences of the MSY in marmoset, mouse, rat, bull,
and opossum (Bellott et al., 2014; Church et al., 2009; Hughes et al., 2005, 2010, 2012; Mueller
et al., 2013; Ross et al., 2005; Skaletsky et al., 2003). Other analyses rely on cDNA sequences,
physical maps, and partial genomic sequences (Li et al., 2013; Paria et al., 2011). None of the
fully sequenced Y chromosomes is from a genetically tractable model organism.
The mouse could potentially serve as such a model, offering rich opportunities to study
mammalian Y chromosome biology. We thus embarked on this sequencing effort, whose results
we now report, in 2002. Prior to our efforts, only limited sequences from the presumptive short
arm were available to facilitate genetic studies of the mouse Y chromosome. The mouse MSY,
which does not undergo crossing over with a homolog, is impervious to conventional genetic
mapping methods, requiring instead approaches such as deletion mapping. When this study
began, most identified genes, including the sex-determining gene Sry, mapped to the short arm
(Burgoyne, 1998; Mazeyrat et al., 1998). The long arm was comparatively impenetrable due to
its highly repetitive nature (Eicher et al., 1989; Nishioka and Lamothe, 1986; Phillips et al.,
1982). Its repetitive sequences were known to produce at least one testis-specific transcript, and
were thought to play roles in spermatogenesis and fertility (Bishop and Hatat, 1987; Burgoyne et
al., 1992; Conway et al., 1994; Moriwaki et al., 1988; Prado et al., 1992; Styrna et al., 1991;
Tourd et al., 2004a).
We recognized that mapping and sequencing the mouse MSY, given the repetitive longarm sequences, would require a methodology with an extraordinary level of accuracy and
precision. Our laboratory previously developed and used single-haplotype iterative mapping and
80
sequencing (SHIMS) to assemble ampliconic sequences, long stretches of duplicated sequences
that share high nucleotide identity, which are a common feature of primate sex chromosomes
(Hughes et al., 2010, 2012; Mueller et al., 2013; Skaletsky et al., 2003). Based on our previous
success, we employed the same approach with the mouse, obtaining high-quality, almostcomplete sequence of the short arm, centromere, and long arm. These sequences, made
immediately available in GenBank, have already enabled design and interpretation of numerous
studies (Cocquet et al., 2009, 2010, 2012; Ellis et al., 2005, 2007, 2011; Ferguson et al., 2009;
Pertile et al., 2009; Reynard et al., 2009; Tourd et al., 2005; Wang et al., 2013a, 2013b).
Here we report the sequence of the mouse MSY and its implications for mammalian sex
chromosome evolution and biology. Like previously sequenced primate MSYs, the mouse MSY
has lost most of its ancestral genes and acquired and amplified other genes during the past 200300 million years. These processes of gene decay and acquisition are especially prominent in the
mouse compared to the primate MSYs. The mouse MSY retains only nine of 639 ancestral
genes, far fewer than in primates. Remarkably, the overwhelming majority of the mouse MSY
consists instead of newly acquired, massively amplified, rodent-specific sequence, which
contains three major Y gene families. These Y gene families have X homologs that are products
of convergent acquisition and amplification. The work reported here sheds light on the
paradoxical combination of divergence and convergence between the X and Y chromosomes in
mouse sex chromosome evolution and offers a genetically tractable model for experimentation in
Y chromosome biology.
81
RESULTS
SEQUENCING, MAPPING, AND ASSEMBLY OF THE MOUSE
Y CHROMOSOME
The mouse MSY presented a technical challenge insurmountable by typical sequencing
strategies. Prior evidence suggested that the mouse MSY contains highly repetitive sequences.
We confirmed this by analyzing fingerprint contigs from C57BL/6J XY and XX bacterial
artificial chromosome (BAC) libraries (RPCI-24 and RPCI-23, British Columbia Cancer Agency
and Michael Smith Genome Science Center): male-specific fingerprint contigs displayed clone
depths as great as 162-fold, whereas expected depth for single-copy Y sequence was 5-fold.
Despite this unprecedented clone depth, we surmised from fingerprint analysis that individual
repeat units were sufficiently large and complex to be amenable to BAC-based sequencing
(Experimental Procedures).
We sequenced the C57BL/6J mouse MSY using SHIMS (Extended Experimental
Procedures). We started by identifying 170 mouse MSY BACs containing unique base-pair
substitutions termed Sequence Family Variants (SFVs) (Saxena et al., 2000) (Data S2.1). These
BACs were used to seed 121 contigs, which were expanded by iteratively selecting and
sequencing BAC clones with SFV matches to existing contig ends. To ensure accurate SFV
identification and BAC assembly, we aimed to sequence BACs with large overlaps of about 5060 kb. We obtained a tiling path of 88.8 Mb in 19 contigs, of which 60.0 Mb was sequenced in at
least two independent BACs (Table S2.1). This redundancy enabled a high degree of accuracy,
an error rate of about 1 in 143,000 nucleotides (Extended Experimental Procedures), which
allowed us to resolve large, almost perfect repeats. We were able to resolve and assemble the
massively ampliconic mouse MSY sequences only by SHIMS; whole-genome shotgun methods
83
would have produced a collapsed assembly, not representative of actual sequences. SHIMS, as
demonstrated here, will enable sequencing and assembly of complex ampliconic genomic
structures, variants of which are often associated with human disease (Bailey and Eichler, 2006;
Freeman et al., 2006).
To confirm and validate the sequence map, and order and orient the 19 sequence contigs,
we constructed a radiation hybrid (RH) map spanning the entire length of the chromosome. We
generated a panel of 93 high-resolution RH clones, which we genotyped for 215 markers
designed to distinguish sequence differences between mouse MSY amplicons (Data S2.1). RH
mapping independently confirmed our sequence assembly in each of the 19 sequence contigs,
and enabled us to order and orient the contigs (Table S2.2).
We created a model Y chromosome assembly of 89.6 Mb, which comprises the 19
ordered and oriented contigs connected by estimated gaps (Data S2.1, and S2.2, Experimental
Procedures). We used this model assembly for all subsequent analyses. Based on the total
sequence we obtained (88.8 Mb) and estimated gap sizes, we estimate our sequence to be 99.1%
complete. This total sequence is within 6.2% of previous size estimates of the mouse Y
chromosome as measured by flow cytometry (94.7 Mb) (Bergstrom et al., 1998).
THE MOUSE Y CHROMOSOME IS EUCHROMA TIC AND MASSIVELYAMPLICONIC
The mouse MSY dwarfs the human, chimp, and rhesus MSYs in both the absolute and relative
amounts of euchromatic and ampliconic sequence: 89.5 Mb, or 99.9%, of the mouse MSY is
euchromatic, and 87.7 Mb, or 98.0%, of this euchromatin is ampliconic (Figure 2.1 a, b). The
MSY's ampliconic sequences are distinct from what are typically referred to as repetitive
sequences, which are often thought of as gene poor, high in interspersed repeats, or even
84
heterochromatic. In contrast, the mouse MSY ampliconic sequence (and consequently the entire
mouse MSY) is gene dense and is similar in interspersed-repeat content to the sequenced primate
MSYs or to mouse autosomes (Figure 2. ic, d). Thus, the mouse MSY stands in contrast to
theories that Y chromosomes should be degenerate, small, gene poor, and heterochromatic
(Bachtrog, 2013; Charlesworth and Charlesworth, 2000; Graves, 2006).
A
COMPLETE SEQUENCE OFA MAMMALIAN CENTROMERE
We obtained the complete sequence of the mouse Y centromere (Figure 2.2). Consisting of 90 kb
of satellite repeats, the centromere is the only heterochromatic sequence (defined as satellite
sequence) that we identified in the entire mouse MSY. This centromeric sequence has been
shown to associate with kinetochore-specific histone H3 CENPA (Pertile et al., 2009). It is
located between 3.5 Mb of short-arm and 86.0 Mb of long-arm sequence, confirming that the
mouse Y is the only acrocentric chromosome amongst all the other telocentric mouse
chromosomes (Ford, 1966; McLaren et al., 1988; Roberts et al., 1988).
85
Figure 2.1
Comparison of mouse, human, chimpanzee, and rhesus Y chromosomes
(A) Schematic representations of mouse, human, chimpanzee, and rhesus Y chromosomes, to
scale. Ancestral single-copy corresponds to what was previously referred to as X-degenerate
(Hughes et al., 2010, 2012; Skaletsky et al., 2003). (B) Sizes (in Mb) of euchromatic sequence
classes in MSYs. (C, D) Gene and interspersed repeat content of mouse MSY euchromatic
sequence compared to human, chimpanzee, and rhesus Y chromosomes, and breakdown by
euchromatic sequence classes compared to mouse X chromosome and autosomes. (C) Gene
densities (numbers per Mb) of coding genes. (D) Percentages of nucleotides contained in SINEs,
retroviral, LINEs, and total interspersed repeats. Human, chimpanzee, and rhesus data are
previously described (Hughes et al., 2010, 2012; Skaletsky et al., 2003). See also Data S1 for
mouse MSY sequence assembly.
86
A
Mouse Y
con
10 Mb
Human Y
* Ampiloic
con
E] Ancestral single-copy
N X-transposed
* other
Chimpanzee Y
E P* udoaubsonW
Rhesus Y
E Hterochromatic
cem
B
Human
Mouse
Rhesus
Chimpanzee
0.24 1.6
0.5 1.0
1.5
25.8
22.8
89.6
SINEs
D
C
ii
12-
E
80-
Si
l0o
40-
I
.9x
20-
Id
Ii I
I1Ii
>
3
E>.
UNEs
ENAll tersperse repeat
60-
z~.
Z 0-
11.0
Moa
>
E.
E
87
II1
]II
|II
> xi
cc
(
Total size of
"I
euchromatic
portion of MSY (Mb):
.
...
. ..........
- .. .....
......
A
I
M
10 kb
B
Centromere (BAC RP24-110P17)
Short arm (BAC RP24-291 C04)
PAR (BAC RP24-500104)
Figure 2.2
Structure of the mouse Y centromere
(A) Triangular dot plot of the mouse Y chromosome centromere. Each dot represents 100%
intra-chromosomal identity within a 100-bp window. (B) DNA FISH on cell spreads from male
C57BL/6Tac mouse. Chromosomes were labeled with probes detecting the centromere (red),
short arm (green), and pseudo-autosomal region (green). Centromere probe: RP24-1 lOP 17; short
arm probe: RP24-291C04; pseudo-autosomal region probe: RP24-500104.
88
THE MOUSE
MSY
RETAINS LITTLE ANCESTRAL SEQUENCE, BUT NEWLY ACQUIRED
SEQUENCE IS MASSIVELY AMPLIFIED
The mouse MSY euchromatin contains two sequence classes with distinct origins: ancestral and
acquired. Ancestral sequence, which originates from the autosomal ancestors of the mammalian
sex chromosomes, occupies only 2.2%, or 2 Mb, of the total euchromatin and is located entirely
within the short arm (Figures 2.1, 2.3). Of this 2 Mb of ancestral sequence, 1.6 Mb is nonampliconic and contains seven single-copy genes and one duplicated gene (Bellott et al., 2014);
the remaining 0.4 Mb is ampliconic and contains one ampliconic gene family (Table 2.1, Data
S2. 1). Relative to the primate MSYs, the mouse MSY retains fewer distinct ancestral genes,
within a much smaller region. (Human, rhesus, and chimp retain 17, 18, and 13 ancestral genes
respectively.) Thus, it appears to have experienced greater degeneration than the primate MSYs,
and correspondingly has diverged more from its homolog, the mouse X chromosome.
The remaining 97.5%, or 87.4 Mb, of euchromatin consists of acquired sequence not
originally on the ancestral autosomes, and not found on other sequenced mammalian MSYs.
Almost all of this acquired sequence is ampliconic. One Mb of acquired ampliconic sequence is
located on the short arm, interspersed among ancestral sequence (Figure 2.3); it includes one
amplified gene pair, H2al2y, and one amplified testis-expressed transcript (Table S2.3,
Experimental Procedures). Taken in its entirety, the acquired sequence contains only two genes
that exist in single copy, Prssly (Protease, serine-like, Chr Y) and Teyorfl (Testis-expressed Y
open readingframe 1); these genes map to the distal tip of the short arm, adjacent to telomeric
sequence (Experimental Procedures).
89
A
*Amphoonic
Arce*aI singI.-copy
Htrochromat
Jftl4
-o" arm
cn
B
500kb
Q.2~
Z III
+
Protein-coding
U
"lingo I
-
genes
a C
Figure 2.3
Gene content and structure of mouse Y short arm
(A) Triangular dot plot of DNA sequence identities within the mouse Y short arm. Each dot
represents 100% intrachromosomal identity within a 100-bp window. Direct repeats appear as
horizontal lines, inverted repeats as vertical lines. The centromere (cen) is visible as a dense
triangle representing highly identical heterochromatic sequence. (B) 12 families of proteincoding genes on the short arm.
90
Sequence
Class
Y gene
Copy
number
Ancestral
Zfy1, Zfy2
Ubely]
Kdm5d
Ei2s3y
Uty
2
1
1
I
1
Ddx3y
1
Usp9y
Sry
1
1
Rbmy
35a
H2al2y
Rbm3iy
Sly
2
2
126
Sstyl
Ssty2
Srsy
85
221
197
Acquired
a
X-homolog
Copy
number
Zfx
1
Ubeix
Kdm5c
Ei2s3x
Utx
Ddx3x
1
1
1
1
1
1
1
1
Usp9x
Sox3
Rbmx
H2aiJ
Rbm3Jx
Six
Six/-i
Ssx
Srsx
1
25 b
14b
14 b
la
Prssly
la
Teyorf]
Estimate, see Extended Experimental Procedures.
b Estimates
Table 2.1
(Mueller et al., 2008)
Genes and gene families on the mouse Y chromosome, and their X homologs
See Data SI for sequences of individual members of amplified gene families. See Figure S3 for
phylogenetic analyses of origins of acquired ampliconic genes. See Table S3 for accession
numbers, and non-coding transcripts identified on the mouse Y chromosome.
91
The remaining 86.4 Mb of acquired sequence is located on the long arm and is massively
amplified (Figure 2.4a). The long-arm ampliconic sequence consists of a half-megabase unit
amplified about 200 times. Each half-megabase unit is comprised of three core blocks defined by
the boundaries of an internal duplication: internally unique sequences (depicted as red and blue)
are bounded by the duplication (yellow) (Figure 2.4b). Amplification of the half-megabase unit
results in > 96% of the mouse MSY sharing at least 98% intrachromosomal identity (Figure 2.5).
This amplified sequence makes up 3% of the haploid male mouse genome. Within the long-arm
amplicons are two regions of extended and outstanding identity: one pair of 7-Mb direct repeats
of 99.999% identity, and a second pair of 4.5-Mb tandem repeats of 99.995% identity (Figure
2.4a, 2.5). In comparison, the next largest pair of sequenced repeats from any organism is the P1
palindrome on the human MSY, which has a span of 1.45 Mb for each arm and arm-to-arm
identity of 99.97% (Kuroda-Kawaguchi et al., 2001). The mouse MSY long-arm amplicons exist
in two varieties: more regular and prevalent 515-kb tandem repeats (Figure 2.4c), and less
regular, less prevalent 400-kb palindromes (Figure 2.4d). The two varieties occur in clusters
along the length of the long arm (Figure 2.4a, e, Table S2.4).
Each long-arm ampliconic unit contains three protein-coding gene families, which are
consequently massively amplified (Figure 2.4b-d). The Sly and Srsy gene families were
discovered through our sequencing efforts, and Ssty was previously described (Bishop and Hatat,
1987; Prado et al., 1992). Members of the Ssty family fall into two subfamilies, Sstyl and Ssty2.
We find 132, 197, and 317 copies of Sly, Srsy, and Ssty, respectively, with intact ORFs
(Experimental Procedures, Table 2.1, Data S2. 1). Two of the three Y gene families have
identifiable homology to autosomal genes whose products associate with chromosomes and have
functions in meiotic chromosome synapsis and segregation. Sly is related to Sycp3 (chromosome
92
10), a component of the synaptonemal complex (Moens and Spyropoulos, 1995), and Ssty is
related to Spin] (chromosome 13), which has been shown to associate with the meiotic spindle in
the mouse oocyte (Oh et al., 1997) (Figure 2.6). In addition to the three gene families, several
non-coding transcripts also map to the long-arm amplicons (Table S2.3).
The 86.4 Mb of long-arm amplicons are interrupted in only eleven locations by a total of
0.76 Mb of sequence (Figure 2.4a, Data S2.1): nine copies of a 40-kb segment originating from
chromosome 3, and two copies of a 200-kb segment that contains the acquired and amplified
gene Rbm3Jy (Figure 2.6).
93
Figure 2.4
Gene content and structure of mouse Y long arm
(A) Triangular dot plot of DNA sequence identities of the entire mouse Y chromosome. Each
black or blue dot represents 100% intrachromosomal identity within a 500-bp or 200-bp window,
respectively. Underneath the dot plot: gray arrows represent regions of extended and high
identity; pink and green bars represent locations of Rbm3Jy and chromosome 3 transposition
respectively. Gray italicized small letters label boundaries between clusters of different amplicon
organization. (B) Triangular dot plot of DNA sequence identity and gene content of a consensus
tandem amplicon unit. (C, D) Triangular dot plots of DNA sequence identity of two major
amplicons type and organization: tandem (C), and palindromic (D). For (B), (C), and (D), each
black dot represents 100% intrachromosomal identity within a 1 00-bp window. Underneath each
dot plot is a representation of the substructure of each ampliconic unit, consisting of red, yellow,
and blue core blocks. Directionality of the amplicons is indicated by gray arrows. Locations of
protein coding genes within the amplicons are indicated by black bars. (E) Detailed long-arm
amplicon substructure. Green and pink correspond to chromosome 3 transposition and Rbm3ly
respectively. Gray corresponds to other sequence, including the short arm and PAR. Gray
italicized small letters label boundaries between different organizations and directions of
amplicons, and correspond to labels in Figure 2.4a. Gray arrows indicate directionality of
amplicons.
94
U Ampioick
SAncssal inle-copy
*Other
* Pseudoautosomal
* Hetrochromatic
* ELong armnamplicon core *)cks
i, 01a
yf-
4;j
PM-keen.I
-a
$|d
Ci
b
C
B
1
. 10
g
0
h
i
k
10
E
i-
C
a m as m a s a a
as u
I
I
I
I
,
W
I1
8fy'
--
0
o'I
bb
D
s8y1
Sy
SMY
Im1
95
a
Figure 2.5
Ampliconic sequences of the mouse Y chromosome
(A) Electronic fraction of mouse, human, chimpanzee, and rhesus MSY sequences by
intrachromosomal similarity. Intrachromosomal similarity, or percent identity, is plotted on a
logarithmic scale. (B) Cumulative electronic fraction of mouse, human, chimpanzee, and rhesus
MSY sequences by intrachromosomal similarity. Intrachromosomal similarity, or percent
identity, is plotted on a logarithmic scale. Only percent identities > 97% were considered in the
cumulative fraction. (C, D) Annotation of two extended regions of high identity: 4.5 Mb of
99.995% identity (C), and 7 Mb of 99.999% identity (D). For each region, both repeat units (Unit
1 and Unit 2) are shown and annotated. The precise region of high identity is represented by light
purple background. Sequence family variants (SFVs) that distinguish Unit I from Unit 2 are
represented by gray lines. BAC clones are represented by black or gray bars labeled with the
library identifier. BAC clones with no prefix are from the RPCI-24 library. BAC clones with
prefix "E" are from the CHORI-36 library. RH markers are represented by ticks along the scale
axis, labeled with the RH marker number. Local divergence between Unit 1 and Unit 2
represents divergence in a 10 kb window with step of 1 kb.
96
..,.....
....
..
I
UnIt 2
UnR I
0
Unit 2
Unit I
C,
Lbff
a.
8io
E.-
L
L
Ii
3-
C
Cumulative fraction of
MSY euchromatic sequence
w
1
S
3- I
0
P
p P
:C
0
I
0 0
Fraction of
MSY euchronatc sequence
Figure 2.6
Phylogenetic analysis of origins of ampliconic and multicopy genes
Phylogenetic analysis of origins of ampliconic and multicopy gene families on the mouse X and
Y chromosomes. Phylogenies reconstructed by DNAML. (A) Sly, Slx, and SxlI originated from
Sycp3 in the mouse lineage. (B) Sstyl, Ssty2, and Sstx originated from Spin2 in the mouse
lineage, which in turn originated from Spin]. (C) H2al2y and H2all originated from H2aJbJ in
the mouse lineage. (D) Rbm3Jx was identified only in the murid rodents, and Rbm3Jy was
identified only in mouse. Thus, Rbm3lx likely arose in the murid rodent lineage, and Rbm31y
arose after the mouse and rat lineages diverged. Rbm3Jx and Rbm3Jy are most similar to
Hnrnpdl, but are too diverged from Hnrnpdl to determine the timing of their evolutionary origin
from Hnrnpdl.
Abbreviations: MMU - Mus musculus, HSA - Homo sapiens, GGA - Gallus gallus, BTA - Bos
taurus, CFA - Canisfamiliaris,MDO - Monodelphis domestica, RNO - Rattus novegicus, CGR
--
Cricetulus griseus, M AU - Mesocricetus auratus,MOC - Microtus ochrogaster.
Accession numbers: SYCP3 HSA: NM_001177949, Six MMU: NM_001136476, Sixli MMU:
BC108389, Sly MMU: NM_201530, Sycp3 MMU: NM_011517, Sycp3 GGA: XM_416330;
Sstx MMU: XM_006536339, Spin2 MMU: NM_001005370, Sstyl MMU: XM_006542637
(consensus sequence), Ssty2 MMU: NM_001017394 (consensus sequence), Spin2 BTA:
NM_001079770, Spin2B CFA: XM_005641406, LOC100126054 BTA: NM_001105656, Spin2b
BTA: NM_001101120, SPIN2B HSA: NM_001006683, SPIN2A HSA: NM_019003, Spin4
MMU: NM_178753, SPIN4 HSA: NM_001012968, Spin4 BTA: XM_005228274, Spin4 CFA:
XM_005641408, SPIN3 HSA: NM_001010862, Spin] CFA: XM_005615963, Spin] BTA:
98
NM_001192821, SPIN1 HSA: NM_006717, Spin] MMU: NM_146043, Spin] MDO:
XM_001376210;
H2ab] RNO: XM_003754739, H2ab] MMU: NM_026627, H2alJ_2 MMU NM_001034100,
H2all_8 MMU: NM_029588, H2al]_3 MMU: NM_001085537, H2all_5 MMU:
NM_001242953, H2all_6 MMU: NM_001242953, H2all_7 MMU: NM_001242947, H2all_4
MMU: NM_001025260, H2alJ_1 MMU: NM_001085517, H2al2y MMU: EU315689, H2ab]
CGR: XM_003508159;
Rbm3Jx RNO: AC_000089 (92037228-92038868), Rbm3lx MMU: EU315690, Rbm3Jy MMU:
NM_028970, Rbm3Jx MAU: XM_005072584, Rbm3Jx CGR: XM_003509646, Rbm3Jx MOC:
NC_022026 (31921134-31922744)
99
--------------------------- -------------------------------------Sycp3 HSA
............................. six mm u
SIX11 m m u
----...............
. ............................................................ Sly M M U
.............................. ................. ...... - Sycp3 M M U
....... ............................................................................... Sycp3 G G A
0.2
......
SStK MMU
d
9
Spin2 MMU
sspyl MMU
Ssty2 MMU
...........
.............. ................. Spin2 BTA
............................ ........................... Spin2b C FA
LOC100126054 BTA
................................................... Spin2b BTA
................................................................. Spin2b HSA
Spin2a HSA
--------- - Spin4 MMU
Spin4 HSA
BTA
.................... Spin4
..............................
..............
Spin4 C FA
Spin3 HSA
Spinl CFA
Spinl BTA
........... ......... ........................................... Spinl HSA
................. *"*'............
Spinl MMU
Spinl MDO
0.06
..................................................................
.............................................................................
............................................
........ ........ ...............................
...................................................
................................................
................................................
H2afbl RNO
H2afbl M M U
H2all 2 M M U
H2all-8 M M U
H2all 3 M M U
H2all 5 M M U
H2all 6 MM U
U
H2all 7 M M U
H2all 4 M M
................................
.........
..................................................
.......
.................................................
H2all 1 M M U
....................... .................... .............. H2al2y M M U
H2afbl CGR
0.04
............................................
Rbm3l x RNO
Rbm3lx MMU
-- --------------------------------------------- Rbm3ly MMU
........................................................... Rbm 3l x M AU
.................................................................. Rbm 3l x C G R
Rbm3l x MOC
E
0.03
100
THE MOUSE MSYLONG-ARMAMPLICONS
EVOLVED AT LEAST 3 MILLION YEARS AGO
The mouse MSY long-arm amplicons are present not only in C57BL/6J, but also in other Mus
musculus strains and even other Mus species, demonstrating that the amplicons are at least 3
million years old. Nevertheless, the sequence structure and size are highly variable. We surveyed
Y chromosomes from M musculus domesticus (from AKR/J), M musculus castaneus
(CAST/EiJ), and M spretus (SPRET/EiJ), which are estimated to have diverged from the
C57BL/6 Mus musculus musculus Y chromosome approximately 1 mya, 1 mya, and 3 mya,
respectively (Silver, 1995). For each Y chromosome, we identified and sequenced three BACs
containing sequence similar to the C57BL/6 long-arm amplicon (Table S2.5). Dot-plot analysis
demonstrated that sequences from all three additional Y chromosomes align to the C57BL/6
long-arm amplicon, albeit with rearrangement (Figure 2.7a, 2.8a). In comparison, dot-plot
analysis of a region of autosomal sequence shows no rearrangements between C57BL/6 and
SPRET/EiJ (Figure 2.8b). From the sequenced BACs, we identified intact open reading frames
(ORFs) for Sly and Ssty in all three additional Y chromosomes. We identified intact ORFs for
Srsy in AKR/J and CAST/EiJ Y chromosomes, and found sequence alignment but no intact ORF
within the three SPRET/EiJ Y chromosome BACs sequenced (Data S2.1). To determine whether
these sequences are amplified in the three additional Mus Y chromosomes as in C57BL/6, we
used fluorescence in situ hybridization to probe each Y chromosome with a BAC that contains
long-arm ampliconic sequence (Figure 2.7b). We observed that the long-arm amplicons are
amplified to different degrees in the four Y chromosomes. We conclude that the ampliconic
structure is at least three million years old, but rapidly evolving.
101
Figure 2.7
Comparison of long-arm ampliconic sequence in related Mus species
(A) Dot plots of DNA sequence identity between the C57BL/6J Y chromosome long-arm
consensus amplicon and BACs from AKR/J (CH33-213P12), CAST/EiJ (CH26-73N1), and
SPRET/EiJ (CH35-73N05). Each dot represents 100% identity within a 25-bp window. (B) DNA
FISH on cell spreads containing the Y chromosome of C57BL/6Tac (Mus musculus musculus),
AKR/J (Mus musculus domesticus), CAST/EiJ (Mus musculus castaneus), and SPRET/EiJ (Mus
spretus). Chromosomes were labeled with a single probe from C57BL/6J from the short arm
sequence (green), and a single probe deriving from each respective strain from the long-arm
sequence (red). Short arm C57BL/6J probe: RP24-084F20; long-arm C57BL/6J probe: RP24088120; long-arm AKR/J probe: CH33-204D 11; long-arm CAST/EiJ probe: CH26-073N11;
long-arm SPRET/EiJ probe: CH35-062A13.
102
A
(-
-
C57BL/6J long arm amplicon consensus unit
103
Figure 2.8
Long arm ampliconic sequences in related Mus species
(A) Dot plots of DNA sequence identity between the C57BL/6J Y chromosome long arm
consensus ampliconic unit and BACs from AKR/J, CAST/EiJ, and SPRET/EiJ. (B) Dot plots of
DNA sequence identity between C57BL/6J chromosome 7 142835222-143038175, and
SPRET/EiJ chromosome 7 BAC (CH35-123K8; Wellcome Trust Sanger Institute; accession no.
CU074422). Each dot represents 100% identity within a 25-bp window.
104
...........
0
a
CH35-123K8 ~
SPRET/EIJ
CR
-C-
0
U
CH35-74005
-
CH35-62A13
SPRET/EJ
*/
/
CH26-87M02
CH26-75A04
CAST/EiJ
-
//
/ -
CH33-226F23
-/
/
-
CH33-204D11
AKR/J
co
QJ
CONVERGENTLYACQUIRED AND AMPLIFIEDHOMOLOGS ON THE MOUSE
X CHROMOSOME
All acquired and amplified MSY genes have convergently acquired counterparts on the X
chromosome (Table 2.1, Figure 2.9, Data S2.1) (Mueller et al., 2008; Reynard et al., 2007), and
in all but one case, the X homolog is also amplified. All acquired and amplified X and Y genes
are specific to the rodent or mouse lineage (Figure 2.6).
We considered the two ways by which X-Y gene families, in particular the three most
massively amplified X-Y gene families, could have been co-amplified: by recombination
between the X and Y chromosomes, or by recombination within each chromosome. Three pieces
of evidence served to rule out X-Y recombination. First, the global physical distributions of the
X-Y homologs make X-Y pairing and recombination unlikely: the three Y gene families are
physically intermingled throughout the mouse Y long arm, whereas each X ampliconic gene
family is found in distinct clusters on the X chromosome (Figure 2.9a). Second, local X-Y
homology is limited: we find that for Sly/Six/Sixl1 and Ssty/Sstx, homology is limited to the
genes themselves. For Srsy/Srsx, X-Y sequence similarity is limited to 30 kb of sequence
surrounding the gene (Figure 2.10). Finally, each pair of X and Y homologs, and their
surrounding sequences, display sequence divergence that is inconsistent with on-going
interchromosomal recombination (Table 2.2, Figure 2.9b). We conclude that intrachromosomal
recombination both within the X chromosome and within the Y chromosome is the likely
mechanism of co-amplification of X and Y gene families.
106
A
*Ampilon.*
MEAnwestral sing-copy
y
Sly
Ssy1
MCther
*PSwudoautosonaJ
IMH.tslrlll~chrmaltc
'
P:II
II
Ssty2
Srsy
U
x
10 Mb
Six
Sixl1
Ssb
Srsx
li i
||
"I
B
Sly
Ssty2
97
Srsy
94
100
00
Ssfyl
Srsy
75
1
s
SI.
82
Sbr
100
100i
SMk
0.1
Figure 2.9
Comparison of X and Y ampliconic genes
(A) Location of ampliconic genes on the mouse Y long arm, and their homologs on the mouse X
chromosome, to scale. (Note that the location of the Sstx cluster at 30 Mb is likely misplaced in
this genome assembly: based on BAC-end sequence matches, it should be located together with
the Sstx cluster at the proximal tip.) (B) Phylogenetic analysis of mouse Y long-arm ampliconic
genes and their X homologs. All trees are drawn on same scale. Unit length represents expected
substitutions per site. Branch labels indicate bootstrap confidence values. See also Figure S5 for
dotplot analyses of X and Y ampliconic regions.
107
Figure 2.10
Dot plot analysis of sequences surrounding Y and X acquired genes
(A - D) Dot plots of DNA sequence identity between the C57BL/6J Y chromosome long arm
consensus ampliconic unit (repeat masked), and X chromosome ampliconic units containing X
homologs of Y long arm genes. Each dot represents 100% identity within a 50-bp window. Red,
yellow, and blue represent the substructure of the long arm ampliconic unit. Representative X
ampliconic units containing (A) Six, (B) Sixi], (C, D) Sstx (from 3 Mb and 73 Mb location), and
(E) Srsx. X amplicons containing Six, Six/i, and Sstx do not bear homology to the Y long arm
outside of the gene. X amplicon containing Srsx shares 93% identity with the Y long arm
ampliconic unit in 2 regions: in a 30 kb region surrounding Srsy within the yellow subunit, and a
20 kb region within the blue subunit. Dot plots of DNA sequence identity between sequences
flanking (F) H2al2y and H2al2x, and (G) Rbm3ly and Rbm3ix. H2al2x/y reside in 5 kb of
sequence sharing 85% identity between X and Y chromosomes. Rbm3Jx/y reside in 6 kb of
sequence sharing 85% identity between X and Y chromosomes. Gene locations are indicated by
black bars.
108
B
-
8
E
C Sbcx -.
E sftx
-
I,,
x
*1
"I
/,
k
30kb
Y Chromosome long arm amplicon consensus unit
G
F
I
JI
42aL2X
-
Rbm3lx 0
I
10
'I-..
U
10 kb
109
10 kb
Nucleotide percent identity within
Y homologs
Max
Min
Avg
Sly
98.6
93.7
100.0
Sstyl
98.1
91.4
100.0
Ssty2
98.2
92.4
100.0
Srsy
96.8
92.6
100.0
Table 2.2
Nucleotide percent identity within
X homologs
Max
Min
Avg
Six
98.6
96.5
100.0
SxI
99.6
98.9
100.0
Sstx
98.4
96.9
100.0
Srsx
97.4
94.8
99.7
Nucleotide percent identity between
X-Y homologs
Max
Min
Avg
Sly vs
Six
Sly vs
Sixl1
Sstyl vs
Sstx
Ssty2 vs
Sstx
Srsy vs
Srsx
77.2
64.4
81.2
75.7
70.8
80.8
62.7
61.0
64.2
62.4
60.5
63.6
94.6
92.8
96.5
Nucleotide identity between X and Y amplified gene families
110
This hypothesis is supported by abundant evidence of rearrangements within the mouse Y
long-arm amplicons. Earlier in this manuscript, we described 7 Mb and 4.5 Mb segments of
99.999% identity and 99.995% identity, which most likely resulted from intrachromosomal
duplications that either occurred recently (within the last few thousand years), or have been
maintained by extremely efficient gene conversion. We found several long-arm amplicons that
deviate from the canonical red-yellow-blue-yellow arrangement, probably resulting from
recombination between ampliconic units (Figure 2.4e, 2.11). Finally, we observed a deletion of
several megabases in the C57BL/6J strain when compared to the C57BL/6JTac strain, from
which the RH panel was constructed (Experimental Procedures). These two C57BL/6 strains
have been reproductively isolated only since 1951. Several naturally-occurring deletions of
significant portions of the long arm have also been documented (Conway et al., 1994; Styrna et
al., 1991; Tourd et al., 2004a). Thus, the massively ampliconic sequences of the long arm likely
act as extensive substrates for intrachromosomal recombination and facilitate rearrangements.
111
A
~0
Sa
Figure 2.11
l Ia I
_t$
Evidence of recombination between long arm ampliconic units
(A) Detailed long arm amplicon substructure, represented by red, yellow, and blue. Deviations
from canonical substructure of red-yellow-blue-yellow are indicated by gray lines. (B) Model of
how recombination between ampliconic units can yield deviations from canonical substructure.
112
X-YACQUIRED AND AMPLIFIED GENES ARE EXPRESSED SPECIFICALLY IN THE MALE
GERMLINE
We next considered pressures that may have selected for co-acquisition and amplification of X-Y
gene families. We assessed likely functions of X-Y gene families by measuring their expression
across a panel of adult mouse tissues (ENCODE Project Consortium, 2012; Merkin et al., 2012).
To determine if expression within the testis is in germ cells, we also examined wildtype
(Kit/Kitw') and germ-cell-deficient (Kitw/Kitwv) testes (Mueller et al., 2013). From this and
previous analyses (Mueller et al., 2008; Reynard et al., 2007, 2009; Tourd et al., 2004b), we
conclude that Sly, Ssty, and Srsy, and their X homologs, Six and Slxli, Sstx, and Srsx, are
expressed predominantly in the male germline (Figure 2.12). By examining SFVs that
distinguish individual members of each gene family, we found evidence of transcription of at
least one third of the members of the Ssty and Sly gene families (Extended Experimental
Procedures). Male germline expression of these genes is consistent with observations that mice
bearing deletions of the mouse Y long arm - and therefore possessing reduced numbers of Sly,
Ssty, and Srsy genes - suffer sperm abnormalities and subfertility (Burgoyne et al., 1992;
Conway et al., 1994; Moriwaki et al., 1988; Reynard et al., 2009; Styrna et al., 1991; Tourd et al.,
2004a). Other acquired Y genes and their X counterparts -- H2ai2y/H2a2x,Rbm31y/Rbm31x,
Prssly and Teyorfl -- are also expressed predominantly in testicular germ cells, again supporting
the idea that acquired genes on the sex chromosomes have functions in male gametogenesis
(Bellott et al., 2010; Hughes et al., 2010, 2012; Lahn and Page, 1997; Skaletsky et al., 2003). As
a control, we analyzed expression of the ancestral single-copy Y genes and their X homologs.
Many of these ancestral genes are ubiquitously expressed, as previously noted, consistent with
113
the idea that surviving ancestral genes are widely expressed, dosage-sensitive regulators of gene
expression (Bellott et al., 2014; Lahn and Page, 1997).
114
Figure 2.12
Expression of mouse Y chromosome genes and their X homologs
Expression of Y genes and their X homologs in various adult tissues, as measured by RNA-seq.
We also measured expression in germ-cell-deficient (Kitw/Kitwv) and wildtype control
(Kit/Kitw') testes. Expression is measured by the total number of reads aligning to each gene,
normalized by the length of the gene (or the average length of the gene for a multi-copy or
ampliconic gene family), and the total number of reads mapped to the transcriptome.
116
1000
Sly
Six
10100
Six/I
Acquired
ampliconic
Ssty1l
II-
Ssty2
I'
Srsy
I
Ssx
Srsx
--
H2al2x
H2ai2y
Rbm31y
Acquired
Prsy
single-copy
]..
Teyorfl
Ddx3y
Eif2s3y
Kdm5d
Ancestral
single-copy
Sty
U-
UEHEENE
Uty
Ancestral
multi-copy
Zfy2
Ancestral
ampliconic
Ddx3x
~.E
ENf2s3x
Emlumlul U..
I
].....
U--.m.m..E
Ubely
Usp9y
Rbm31x
U.
Kdm5c
U
Sox3
IuumuuuhmmuIj Ubal
Eu.
I
]
]
I
-
I
mmm~.m
Uba1
EUUUEEUUIUsp~x
U
Zfx
.U.....
Rbmx
Rbmy
~jjj4~1~,i
I ~
I
117
H
DISCUSSION
We have assembled, using SHIMS, the sequence of the mouse MSY. Despite the shared
evolutionary origin of placental mammalian Y chromosomes, the mouse MSY is spectacularly
different from the human, rhesus, and chimpanzee MSYs. Only 2 Mb, or 2.2 % of its sequence,
has shared ancestry with the primate MSYs. Instead, the mouse MSY is dominated by a single
family of acquired amplicons not found in the primate MSYs. These amplicons, each of which
spans 500 kb and contains three gene families, comprise 86.4 Mb, or 96.5% of the chromosome.
How has the mouse MSY chromosome evolved to be so different from the primate
MSYs? The same processes underlie the evolution of both mouse and primate MSYs; the
difference lies in the extent to which they have played out. Mouse and primate MSYs alike have
lost most genes from the ancestral autosomes, with the mouse retaining fewer genes than the
primates. Both mouse and primate MSYs have acquired and amplified testis-specific genes.
However, the relative and absolute scale of amplification in the mouse is unmatched among
sequenced sex chromosomes.
AMPLIFICATION OF ACQUIRED SEQUENCE IS CONSISTENT WITH SEX-LINKED MEIOTIC
DRIVE
We suggest that massive amplification of acquired sequence on the mouse MSY resulted from
sex-linked meiotic drive. Sex-linked meiotic drive occurs when a driver arises on a sex
chromosome, causing it to be transmitted to offspring more often than its counterpart. The
resulting skew in sex ratio exerts strong countervailing selection for a suppressor, on the
autosomes or the sex chromosome counterpart, to restore sex ratio balance. If both driver and
suppressor are dosage sensitive, they would undergo iterated cycles of expansion, resulting in
119
rapid co-amplification of both driver and suppressor (Jaenike, 2001; Partridge and Hurst, 1998).
In Drosophilamelanogaster,the X- and Y-linked multicopy genes Stellate and Suppressorof
Stellate are hypothesized to be such a pair of meiotic driver and suppressor (Hurst, 1992, 1996;
Palumbo et al., 1994). The mouse MSY's three acquired and massively amplified gene families
and their X homologs are reminiscent of a meiotic driver and suppressor pair: in all three cases,
both the X and Y genes are highly amplified, they are expressed specifically in testicular germ
cells, and perturbation of gene family copy number results in sex ratio distortion. Mice that have
fewer members of the Y gene families, due to partial deletions of the Y long arm, produce more
female than male offspring (Conway et al., 1994; Moriwaki et al., 1988). Knock-down of Sly or
Slx, one of the three X-Y gene pairs, also distorts sex ratio in favor of females or males,
respectively (Cocquet et al., 2009, 2012).
While we presently lack sufficient information to reconstruct the evolutionary history of
the X-Y acquired and amplified gene families with certainty, we speculate that in mouse, one or
more meiotic drivers were initially acquired by the X chromosome, and then countered by one or
more suppressors acquired by the Y chromosome. We observe that the X chromosome has many
more acquired genes than the Y chromosome (Mueller et al., 2013), whereas all Y-acquired and
amplified genes have X homologs, consistent with their having been acquired in response to a
subset of X-acquired genes. Subsequently, intrachromosomal recombination within acquired
ampliconic regions would facilitate rapid evolution and expansion of both drivers and
suppressors. We note that the amplification of X-acquired genes is restricted to discrete,
localized clusters, whereas Y-acquired genes are intermingled and massively amplified, likely
reflecting different constraints with respect to crossing over: amplification on the X chromosome
120
is constrained by the need to maintain crossing-over between X homologs in the female, whereas
amplification on the MSY has no such limitations.
Strong selective pressure to acquire and amplify Y-linked suppressors of meiotic drive
may account not only for the massively amplified Y-acquired genes, but also for the muchdecayed Y ancestral genes, thereby shaping the character of the entire mouse MSY. Specifically,
we speculate that ancestral gene decay may be a by-product of strong positive selection for
meiotic drive suppressors on the mouse MSY. Since the MSY does not cross over with a
homolog, it is inherited and selected as a unit. Strong selection for beneficial mutations in
acquired, amplified MSY genes could propel deleterious mutations in ancestral MSY genes to
fixation in a population (Charlesworth and Charlesworth, 2000; Rice, 1987). Indeed, our
laboratory previously drew attention to such a correlation between enhanced gene
acquisition/amplification and increased ancestral gene decay within the primates: the chimpanzee
MSY, which has acquired twice as many palindromes as the human MSY, has sustained more
inactivating mutations in ancestral genes than human (Hughes et al., 2005, 2010). Thus, in both
mouse and chimpanzee, single-copy, ancestral MSY genes may have been casualties of selective
forces directed at the ampliconic, acquired genes.
X-Y interchromosomal conflict, and its consequent impact on gene acquisition and
-
amplification on sex chromosomes, may be widespread in mammals. In both human and mouse
the only two species with high-quality reference sequences for both sex chromosomes - the X
and Y have co-acquired and amplified genes. As in mouse, the human gene families VCX and
VCY are also testis-specific, and may also be involved in meiotic drive (Lahn and Page, 2000).
These X-Y gene families are lineage-specific, consistent with rapid evolution due to meiotic
drive: VCXis detected only in simian primates, and Sly, Ssty and Srsy only in murid rodents. The
121
scale of amplification on the mouse MSY is much greater than that of the sequenced primate
MSYs. However, the mouse may not be exceptional: the cat and horse MSYs also appear to have
highly amplified gene families (Murphy et al., 2006; Paria et al., 2011). To determine the true
phylogenetic range of lineage-specific acquisition and amplification of X-Y genes, SHIMS
assemblies must first be constructed for the X and Y chromosomes across more mammals.
Genetic conflict between the X-Y acquired ampliconic genes may contribute to hybrid
sterility and consequent reproductive isolation. It was previously proposed (Frank, 1991; Hurst
and Pomiankowski, 1991), and recently demonstrated in Drosophila(Phadnis and Orr, 2009;
Tao et al., 2001), that segregation distortion can cause male hybrid sterility. In Mus, hybrid
sterility loci map at or near ampliconic regions on the X chromosome, including Slx and SixlI
(Elliott et al., 2001, 2004; Good et al., 2008; Mueller et al., 2013), and they may result from
incompatibilities between X and Y chromosomes (Campbell et al., 2012). Further study of the
rapidly co-evolving, lineage-specific X-Y ampliconic gene families will be needed to test the
proposed link between X-Y genetic conflict and hybrid sterility in mammals.
THE MOUSE Y CHROMOSOME AS AN EXPERIMENTALLY TRACTABLE SYSTEM
The mouse MSY sequence provides unprecedented opportunities for experimentation. Its two
major sequence classes - acquired and amplified, versus ancestral, single-copy - require distinct
experimental approaches.
The three gene families that are intermingled and massively amplified on the mouse Y
long arm cannot be deciphered by the previous major experimental approach, which utilizes
massive, naturally occurring deletions of the long arm: all three intermingled gene families
would be deleted, which would preclude parsing the roles of individual gene families. It would
122
also be challenging to use a traditional genetic targeting approach to ablate all members of any
one of the gene families. At present, the most feasible approach is to target members of
individual gene families by knockdown. Knowledge of the mouse MSY sequence and structure
that emerged early during this project led investigators to adopt knock-down strategies to target
Sly and its X homologs, Slx and Six1i (Cocquet et al., 2009, 2010, 2012). These approaches have
recapitulated sperm defects and sex ratio distortion observed with MSY long-arm deletions, and
enabled further investigations of gene function. The other massively amplified X-Y gene
families remain to be similarly investigated, and the sequence presented here can guide
knockdown strategies.
The prominent amplification on the mouse MSY long arm presents a novel opportunity
for biochemical studies of chromatin. Unbiased profiling of chromosomal proteins, by purifying
proteins associated with a specific DNA locus, has been hindered by the inability to purify
sufficient amounts of a target region. One successful strategy took advantage of telomeric
sequences, which are abundant relative to most DNA loci (Dejardin and Kingston, 2009). The
mouse long-arm amplicons, comprising 3% of the mouse haploid genome, constitute a
significantly enriched substrate for such unbiased chromatin protein-profiling strategies.
The mouse MSY sequence also provides a foundation for genetic manipulation of the
ancestral, single-copy genes. Until recently, efforts at targeted mutagenesis of Y genes met with
poor success. As such, studies have relied on Y chromosome translocations, deletions, and
transgenesis, which are not optimal for dissecting individual gene functions (Burgoyne, 1998;
Mazeyrat et al., 2001; Vernet et al., 2011, 2012; Yamauchi et al., 2014). The sequence we
present, in combination with new genetic targeting methods such as TALENs and CRISPR, has
already enabled genetic targeting of Y single-copy genes in mice (Wang et al., 2013a, 2013b).
123
The research community can now explore the diverse biology of the male-specific chromosome
in the premier mammalian genetic model, armed with a comprehensive, high-quality reference
sequence.
124
EXPERIMENTAL PROCEDURES
Fingerprint analysis of C5 7BL/6J BA C libraries
BAC fingerprint analysis (Soderlund et al., 2000) confirmed that the mouse MSY contained
highly repetitive sequences: male-specific fingerprint contigs displayed 162-fold depth, whereas
single-copy Y sequence was expected to display 5-fold depth. Fingerprints for individual BACs
demonstrated, however, that individual BACs were internally non-repetitive: each BAC yielded
a complex library of fingerprint fragments, and the total of fragment sizes for each BAC added
up to the approximate size of the BAC insert. If each BAC were internally repetitive, we would
expect to observe a simple library of fragments whose sizes sum to much less than the size of the
BAC's insert.
BAC selection and sequencing ISingle-haplotype iterative mapping and sequencing
We sequenced 746 BACs from the RPCI-24 C57BL6/J library and 232 BACS from the CHORI36 C57BL6/J library (Table SI). BAC selection and sequencing occurred in two phases. In the
first phase, we aimed to identify non-overlapping BACs for seed contigs. 8 STSs (364, 365, 372,
373, 379, 383, 384, 386, Data S1) were designed using the sequence of one typical BAC (RP24507D23) which was available before the project started. All BACs with end sequences matching
RP24-507D23 were tested with the appropriate STSs. We identified 121 BACs containing
unique variants, which were selected for sequencing, and were used to seed the initial contigs. In
the second phase, we aimed to expand contigs. Unique SFVs were identified at the end of each
contig, and all BACs with end sequences best matching this contig and which would extend the
contig were tested for the SFV. Out of clones matching these criteria, we selected for sequencing
one clone with at least 30 kb overlap and which would add most new sequence. In some cases,
125
the overlap proved not to be real, in which case the BAC was treated as a new contig, and SFVs
were again identified. This process was iterated until no more new BACs could be found. The
number of contigs, and therefore points of extension, initially increased to 175 in the first round
of iteration, but then gradually coalesced to 19 final contigs. Both phases of BAC selection
depended heavily on availability of high quality BAC end sequences.
We also identified and sequenced three clones each, representative of the long-arm amplicons,
from CHORI-33 (AKR/J), CHORI-26 (CAST/EiJ), and CHORI-35 (SPRET/EiJ) (Table S5).
Radiation hybrid mapping
215 STS markers were tested on a 25000-rad panel consisting of 93 hybrid clones (Table S2,
Data Si). An RH map was constructed using RHMAPPER (Slonim et al., 1997).
Calculation of sequence accuracy
The Washington University sequencing center error rate was estimated to be 1 in 50 kb (Church
et al., 2009). However, as 65% of our sequence is covered redundantly by two BACs, we were
able to identify and resolve all discrepancies in redundantly covered regions, so that the error rate
*
for these regions is zero. Therefore, the final error rate is estimated to be 0.35 * 1/50000 + 0.65
0 = 1/143000, or 1 in 143 kb.
Sequence assembly and gap-filling
To create a model assembly for analyses, the 19 sequence contigs were ordered and oriented by
RH mapping, and joined with estimated gap sizes. Gaps that fell within the two regions of
outstanding identity (7 Mb of 99.999% identity and 4.5 Mb of 99.995% identity) were filled with
sequence from the corresponding segment of the other almost-identical repeat unit.
126
Estimation of total Rbmy gene copy number and array size
We obtained about 370 kb of sequence containing 10 intact ORFs for Rbmy. As sequence
obtained across the Rbmy array was incomplete, we estimated the total Rbmy gene copy number
and array size as follows. We performed hybridization with probes for Rbmy on the RP24
library, and identified 38 Rbmy-positive BACs. The RP24 library has an average clone size of
155 kb, and average genome coverage of 10.8X or Y chromosome coverage of 5.4X. Thus, we
calculated the total size of the Rbmy array to be approximately 1.1 Mb. Assuming an average
repeat unit size of 37 kb, and that each repeat unit contains an intact ORF for Rbmy, we estimate
that there are 30 copies of Rbmy.
Interspersed repeats
Interspersed repeats were electronically identified with RepeatMasker (Smit et al., 1996).
Dotplots
Triangular dotplots (representing intrachromosomal sequence similarity) and square dotplots
(representing interchromosomal sequence similarity) were generated by a custom Perl script
available at http://pagelab.wi.mit.edu/material-request.html.
Electronicfractionation plot
Intrachromosomal similarity, or percent identity to other MSY sequences, was determined by
using custom Perl code that used BLAST to compare all 5 kb sequence segments, in 2 kb steps,
to the entire remainder of the MSY sequence.
Identification of genes and transcription units
127
We identified genes and transcripts as previously described (Skaletsky et al., 2003). Our general
criteria for a protein-coding gene are (1) experimental evidence of transcription together with (2)
a predicted open reading frame (ORF) of greater than 170 amino acids. Transcriptional units
without significant ORFs are considered to be non-coding transcripts.
Sly: Sly is a spliced gene with many possible splice variants. We considered an individual gene
member to be intact if all exons are present as in the consensus, splice sites are intact, and the
open reading frame (ORF) is at least two-thirds the size of the consensus ORF. We did not
estimate the number of pseudogenes due to the difficulty of classifying various splice variants or
scattered exons as pseudogenes.
SstyJ and Ssty2: As Sstyl and Ssty2 are not spliced, we considered an individual gene member to
be intact if the ORF is at least 90% the size of the consensus ORF. We considered as
pseudogenes those which have ORFs less than 90% but greater than one-third of the consensus
ORF. We find 281 Sstyl pseudogenes and 85 Ssty2 pseudogenes.
Srsy: We identified two forms of Srsy. The majority form, with 135 copies, has an ORF of 363
amino acids, whereas 62 copies have a longer ORF of up to 742 amino acids. As Srsy is not
spliced, we considered an individual gene member to be intact if the ORF is at least 90% the size
of the major consensus ORF (363 amino acids). We considered as pseudogenes those which have
ORFs less than 90% but greater than one-third of the consensus ORF. We find 242 Srsy
pseudogenes. We note that while we consider Srsy to have protein-coding potential, as it has a
sizable open reading frame translating into 363 amino acids, it has relatively low levels of
transcription in the tissue panel we examined, compared with typical protein-coding genes.
128
Prsslyand Teyorfl: Prssly (Accession no. KJ780361) and Teyorfl (KJ780362) were initially
identified from a Celera alternate assembly unplaced contig, assembled from whole genome
shotgun sequence from mixed Mus musculus strains (NW_001034423.1). Markers for both genes
were strictly male specific when tested on C57BL6 male and female genomic DNA, leading us
to conclude that both genes were located on the Y chromosome. We were unable to identify both
genes within the CHORI-36 and RPCI-24 C56BL6/J BAC libraries, although we were able to
identify them in BAC 45G04 from CHORI-29. Nevertheless, we confirmed the integrity of both
genes in C56BL/6 by sequencing from C57BL/6Tac adult testis cDNA. We additionally
determined the location of both genes by DNA FISH and RH mapping. DNA FISH using BAC
probe CH29-45G04 showed that both genes are located on the distal tip of the Y chromosome
short arm. RH mapping reveals that both genes are linked to the most distal marker in our RH
map of the mouse Y short arm. Because the Celera contig containing the two genes also contains
telomeric sequence, we infer that both genes are very near the distal tip of the short arm, adjacent
to the telomere.
Transcriptionunits AK006152 and KC] 70991: KCJ 70991 and AK006152 are two related testis
transcripts that are 94% identical. We identified KC1 70991 in our genomic sequence; we could
not find AK006152, which was described previously (Ferguson et al., 2009). However, we were
able to assemble and identify both transcripts from 454 sequence reads of cDNA generated from
C57BL/6JTac adult testis. RH mapping confirms the presence of AK006152, and places it near
the distal tip of the short arm, in proximity to KC170991.
129
Transcription of ampliconic gene families
To determine which individual members within the Sly, SstyJ, and Ssty2 gene families are
transcribed, we identified SFVs that distinguish individual members of each gene family, and
looked for RNAseq reads that contained these SFVs. We found evidence of transcription of at
least 34 of 126 intact Sly genes, 27 of 85 intact SstyJ genes, and 73 of 221 intact Ssty2 genes. In
addition, we found evidence of transcription of 32 of 281 SstyJ pseudogenes, and 42 of 85 Ssty2
pseudogenes. We could not confidently determine transcription of individual copies of Srsy, as
Srsy is expressed at very low levels.
Sequence differences between C57BL/6J and C57BL/6Tac
Discrepancies between the sequence, which was obtained from C57BL/6J, and the RH map,
which was obtained from C57BL/6Tac, suggested that approximately 6 Mb of sequence that was
present in one copy in C57BL/6J was present in two copies in C57BL/6Tac. The most
parsimonious explanation for the difference was that a deletion had occurred in C57BL/6J,
following a prior duplication in the common ancestor of the two strains. We confirmed this using
quantitative PCR to measure the copy number of the putative deleted region compared to regions
identical between the two strains.
Chromosomal fluorescence in situ hybridization / FISH analyses
FISH assays were performed on mouse embryonic fibroblasts using probes for the C57BL6/J
Mus musculus musculus Y chromosome. Mouse embryonic fibroblasts were derived from
embryos of C57BL6/NTac, Mus musculus domesticus (AKR/J), Mus musculus castaneus
(C57BL6/NTac x CAST/EiJ), and Mus spretus (SPRET/EiJ). Metaphase FISH analyses were
performed as previously described (Saxena et al., 1996).
130
Gene expression analyses
We measured expression across mouse tissues using previously published mRNA-seq data:
Kitw/Kitwv and Kit/Kitw testis, SRA060831; ovary, SRX135150; all other tissue, SRP016501
(ENCODE Project Consortium, 2012; Merkin et al., 2012; Mueller et al., 2008). Normalized
expression for each gene was calculated as in Bellott et al., 2014. For multi-copy or ampliconic
gene families, we counted the number of reads that aligned to any member of the gene family.
See Extended Experimental Procedures for details.
Multiple alignments andphylogenetic analyses
To align mouse MSY repeat units, we used Fast Statistical Alignment (Bradley et al., 2009). For
phylogenetic analyses of X-Y ampliconic gene families, nucleotide sequences for members of
each gene family were aligned using Clustalw (Larkin et al., 2007),and phylogenetic trees were
generated by DNAML in the Phylip package (Felsenstein, 1989).
Ethics statement
All experiments involving mice conformed to ethical principles and guidelines approved by the
Committee on Animal Care at the Massachusetts Institute of Technology (Institutional Animal
Care and Use Committee no. 0711-075-14).
131
132
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138
SUPPLEMENTARY TABLES
Table S1
BAC clones, GenBank accession numbers and nucleotide positions in mouse
MSY sequence assembly, Related to Experimental Procedures
Table S2
Radiation hybrid mapping data vectors, Related to Experimental Procedures
Table S3
Transcripts of the mouse Y chromosome, Related to Table 1
Table S4
Positions of long-arm amplicon Red Yellow Blue core blocks, Related to
Figure 2.4
Table S5
BAC clones sequenced from related Mus species, Related to Experimental
Procedures
Tables Si-S5 available at Soh and AlfMldi et al. (2014).
139
SUPPLEMENTARY DATA
Data S2.1
Zipped file containing the following sequences:
1
Sequence Family Variants used to identify BAC clones for sequencing, Related to
Experimental Procedures
2
STS markers used to generate RH map, Related to Experimental procedures
3
FASTA file of mouse MSY sequence assembly, Related to Figure 2.1
4
Rbmy sequences, Related to Table 2.1
5
Sly sequences, Related to Table 2.1
6
Srsy sequences, Related to Table 2.1
7
Sstyl sequences, Related to Table 2.1
8
Ssty2 sequences, Related to Table 2.1
9
Sequence for Chr 3 transposition, Related to Figure 2.4
10
Sequence for 200kb Rbm3Jy insertion, Related to Figure 2.4
11
Sequences for Sly, Ssty, and Srsy identified in AKR/J, M. castaneus, M. spretus,
Related to Figure 2.7
12
Six sequences, Related to Table 2.1
13
SixIl sequences, Related to Table 2.1
14
Srsx sequences, Related to Table 2.1
15
Sstx sequences, Related to Table 2.1
Data S2.1 available at Soh and Alfdldi et al. (2014).
141
Data S2.2
Annotated sequence of mouse MSY
Background colors represent sequence classes: pseudoautosomal (green); ancestral single-copy
(yellow); ampliconic (blue); other (gray); and heterochromatic (pink stripes). Gaps shown in
white. Bottom bar of bright red, blue, and yellow stripes represents the substructure of each long
arm repeat unit. All sequence features and BACs drawn to scale. (A) Major features. Black
arrows represent various repeat units on the short arm; gray arrows represent two regions of
extended and high identity on the long arm; green arrows represent locations of chromosome 3
transpositions. (B) Positions of all intact, actively transcribed genes. Plus (+) strand above, minus
(-) strand below. (C) Positions of pseudogenes. (D) G+C content (%) calculated in a 100-kb
sliding window with 1-kb steps. (E) SINE, LINE, ERV densities, calculated in a 200-kb sliding
window with 1-kb steps. (F) Sequenced MSY BACs. Each bar represents the size and position of
one BAC clone, labeled with the library identifier. BAC clones with no prefix are from the
RPCI-24 library. BAC clones with prefix "E" are from the CHORI-36 library. Black bars
represent BAC clones used for the tiling path, dark gray bars represent other finished BAC
clones, and light gray bars represent unfinished BAC clones.
142
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A
CHAPTER 3.
GENE REGULATORY PROGRAM FOR MEIOTIC INITIATION IN
THE FETAL OVARY
Y. Q. Shirleen Soh, Jan Philipp Junker, Mark E. Gill, Jacob L. Mueller, Alexander van
Oudenaarden, David C. Page
AUTHOR CONTRIBUTIONS
Y.Q.S.S., J.P.J., M.E.G., J.L.M, A.v.O., and D.C.P planned the project. Y.Q.S.S., M.E.G., and
J.L.M. collected and analyzed RNAseq data, Y.Q.S.S. and J.P.J collected and analyzed smFISH
data, J.P.J. provided software for smFISH analysis, Y.Q.S.S., and D.C.P. wrote the paper.
ACKNOWLEDGEMENTS
I would like to thank Gregoriy Dokshin and Katherine Romer for comments.
157
ABSTRACT
The chromosomal program of meiotic prophase must be preceded and enabled by the regulated
induction of meiotic prophase genes. This gene regulatory program is poorly understood,
particularly in organisms with a segregated germline. We characterized the gene regulatory
program of meiotic prophase as it occurs in the mouse fetal ovary. We identified 106 genes
expressed specifically in pre-meiotic to pachytene germ cells, and characterized the regulation of
these genes by 1) retinoic acid (RA), which induces meiosis, 2) Dazl, which is required for germ
cells to gain competence to respond to RA, and 3) Stra8, which is a downstream target of RA
and is required for the chromosomal program of meiotic prophase. Initial induction of practically
all meiotic prophase genes requires Dazl. In the presence of Dazl, RA induces at least two
pathways: one Stra8-independent, and one Stra8-dependent. Genes vary in their induction by
Stra8, spanning three classes: fully Stra8-independent (Class 1), partially Stra8-independent
(Class 2), and fully Stra8-dependent (Class 3). Thus, Stra8 regulates the entirety of the meiotic
chromosomal program but plays a more nuanced role in governing the gene expression program.
We propose that Stra8-independent expression of the Class 1 and Class 2 genes enables the
stockpiling of selected meiotic structural proteins prior to the commencement of the
chromosomal program. Unexpectedly, we discovered that Stra8 is required for prompt downregulation of itself and Rec8. Germ cells that have expressed and down-regulated Stra8 are
refractory to further Stra8 expression. Negative feedback of Stra8, and subsequent refractoriness
to further Stra8 expression, may ensure a single, restricted pulse of Stra8 expression. Taken all
together, these experiments reveal a gene regulatory logic by which germ cells prepare for the
chromosomal program of meiotic prophase, and ensure that it is induced only once.
159
INTRODUCTION
In sexually reproducing organisms, germ cells undergo meiosis, a specialized cell division
program that produces haploid gametes. The reductive segregation of chromosomes depends
upon a complex series of chromosomal events that occur during meiotic prophase. This
chromosomal program must be supported by expression of a large suite of genes. A genomewide description of this gene expression program, and how it is regulated, has not been available
for mammals or other animals with specialized sex cells, or germ cells. Indeed, the best existing
model for such a gene regulatory program is that of budding yeast (Chu et al., 1998; Kassir et al.,
2003; Primig et al., 2000; Smith and Mitchell, 1989).
The chromosomal program of meiotic prophase, including events such as laying down of
meiotic cohesins, synapsis between homologs, and homologous recombination, has been the
subject of intense study (Cohen et al., 2006; Handel and Schimenti, 2010; Marston and Amon,
2004). Investigations of these processes in mammals have relied principally upon identifying
mouse orthologs of proteins that have demonstrated meiotic functions in lower eukaryotes, and
that are well conserved amongst sexually reproducing species (Schurko and Logsdon, 2008;
Villeneuve and Hillers, 2001). However, not all proteins involved in the meiotic chromosomal
processes are well conserved among eukaryotes, and identifying these exceptions has proven
challenging (Kumar et al., 2010). Identification of a gene set associated with mammalian meiotic
prophase would provide an orthogonal means of discovering poorly conserved or even novel
proteins involved in the mammalian meiotic chromosomal program.
Studies in a mammalian system are also required if we are to understand how the gene
expression program of mammalian meiotic prophase is regulated; the regulation of meiotic
initiation is poorly conserved. For instance, between mouse and budding yeast, the regulatory
160
logic of meiotic initiation appears similar, but the molecular identities of the regulators are not
conserved (van Werven and Amon, 2011). In both mouse ovarian and testicular germ cells,
meiosis is initiated by retinoic acid (RA) (Anderson et al., 2008; Bowles et al., 2006; Koubova et
al., 2006), a signaling molecule restricted to chordates (Fujiwara and Kawamura, 2003). RA
induces Stra8, a vertebrate-specific gene that encodes a putative helix-loop-helix-containing
transcription factor (Baltus et al., 2006; Bowles et al., 2006; Ghyselinck et al., 2006; Koubova et
al., 2006; Oulad-Abdelghani et al., 1996; Zhou et al., 2008a, 2008b). Stra8 is required for all
chromosomal events of meiotic prophase assayed, including cohesion, synapsis, and
recombination (Anderson et al., 2008; Baltus et al., 2006). In mouse fetal ovarian germ cells,
induction of Stra8 by RA requires the germ cell-expressed competence factor Dazl (Lin et al.,
2008). Dazl, which encodes an RNA binding protein expressed in postmigratory XX and XY
germ cells, is required for germ cells to gain competence to respond to developmental cues,
including RA (Gill et al., 2011). Thus far, the roles of RA, Stra8, and Dazl have largely been
assayed with respect to the chromosomal program of meiotic prophase; their potential roles in
regulating the gene expression program have not been examined systematically.
We sought to elucidate the gene regulatory program of meiotic prophase. We used the
mouse fetal ovary as a model for two reasons. First, germ cells in the fetal ovary initiate and
progress through meiotic prophase with greater synchrony than in the postnatal or adult testis.
All germ cells in the fetal ovary initiate meiosis around embryonic day 13.5, progress through
meiotic prophase during subsequent fetal development, and arrest at diplotene of meiotic
prophase before birth. Initiation and progression of meiotic prophase occurs in an anterior-toposterior wave: Stra8 and meiotic prophase genes are expressed first in the anterior before
extending towards the posterior (Bullejos and Koopman, 2004; Menke et al., 2003). We
161
therefore took advantage of the relative synchrony of cell state over time and space to finely
dissect initiation and progression of meiotic prophase. Second, the roles of Dazl, RA, and Stra8
in meiotic initiation are well defined in the fetal ovary. To determine how the gene expression
program is regulated by Dazl, RA, and Stra8 we examined expression in wild-type and mutant
animals. We used whole-gonad, genome-wide transcriptome profiling, to obtain a global
description of gene expression, and followed up with targeted single-cell, single-transcript
measurements to precisely quantify elements of regulatory control at the level of individual germ
cells.
We identified a set of 106 genes associated with meiotic prophase, as assayed in fetal
ovarian germ cells. We characterized how Dazl, RA, and Stra8 regulate this gene expression
program, thus complementing our previous understanding of how they regulate the chromosomal
program. From these data, we discerned two elements of gene regulatory logic centered on
Stra8, a key inducer of the chromosomal program. Initial induction of genes requires Stra8independent and Stra8-dependent pathways. After gene induction, Stra8 is required for
subsequent down-regulation of its own expression. We propose that these regulatory elements
respectively account for how germ cells prepare for and ensure a single induction of the
chromosomal program of meiotic prophase.
162
RESULTS
IDENTIFICATION OF THE GENE EXPRESSION PROGRAM OF MEIOTIC PROPHASE
To identify and catalog the gene expression program of meiotic prophase as it occurs in the fetal
ovary, we performed genome-wide transcriptome profiling by RNA-seq on whole fetal ovaries at
embryonic days 12.5, 14.5, and 16.5 (E12.5, E14.5, and E16.5). At these timepoints, ovarian
germ cells are in pre-meiotic, leptotene (early meiotic prophase), and pachytene (mid-late
meiotic prophase) stages, respectively (Borum, 1961; Speed, 1982). At each timepoint, we
determined expression in gonads of wild-type and germ cell-depleted (Kitw/Kitv) mice (Handel
and Eppig, 1979), so as to identify germ-cell-dependent genes. During this embryonic period,
testicular germ cells do not initiate meiosis, but instead enter and remain in mitotic G0/G 1 arrest
(McLaren, 2003). We therefore also profiled expression from wild-type fetal testes, at the same
timepoints, to identify ovary-enriched genes. We defined "meiotic prophase genes" as those
meeting criteria as follows.
Since genes involved in meiotic prophase should be expressed leading up to or during
prophase, we required that genes be expressed in wild-type ovaries at one or more of the three
timepoints (E 12.5, E 14.5, or E 16.5) at greater than 5 Fragments Per Kilobase of transcript per
Million mapped reads (FPKM). Additionally, since only germ cells might be expected to express
genes required for meiotic prophase, we also required that genes be at least 2-fold enriched (FDR
adjusted p value, q < 0.01) in wild-type over germ-cell-depleted ovaries. A total of 530 genes
met these two criteria (Fig. 3.1, Table S3.1). There was one conspicuous absence: Rec8, a
meiotic cohesin, was not germ-cell-enriched. We verified by single-molecule fluorescent in situ
hybridization that Rec8 was indeed expressed in ovarian somatic cells as well as germ cells (Fig.
3.2), and added it to the 530 genes. Of the 531 genes, about half, including Stra8, were up-
163
regulated between E12.5 and E14.5, then down-regulated by E16.5, suggestive of functions
restricted to early meiotic prophase. About a quarter of the 531 genes are up-regulated between
E12.5 and E14.5 and remain elevated at E16.5; these genes include Sycp3, which encodes a
synaptonemal complex protein. A small number of genes are not up-regulated until E 16.5,
suggestive of functions later in meiotic prophase. A final quarter of the 531 genes are highly
expressed at E 12.5 and progressively down-regulated by E 16.5. These include many
pluripotency markers, including Pou5fi (Oct4), Nanog, and Sox2, and reflect the downregulation of a pluripotency program as germ cells enter meiosis (Pesce et al., 1998; Western et
al., 2005; Yamaguchi et al., 2005).
To winnow this list of 531 ovarian germ cell genes down to those functioning in meiotic
prophase, we required that genes fulfill additional criteria. Since testicular germ cells do not
embark on meiosis until well after birth, we required that gene expression be at least 2-fold
higher (q < 0.01) in fetal ovary than in fetal testis. Since genes with meiotic functions should be
up-regulated as germ cells enter and progress through meiosis, we also required that genes be at
least 2-fold up-regulated (q < 0.01) between E12.5 and E14.5 or E16.5. By imposing these two
additional filters, we obtained a final set of 106 genes (Fig. 3.1), which we shall refer to as the
gene expression program of meiotic prophase.
Of these 106 genes, 54 genes have previously been implicated in meiotic prophase by
independent, lower-throughput methods. For 33 of these 106 genes, loss-of-function mutants
have been examined for fertility defects; defects in meiotic prophase were reported for 32 of the
33 genes tested in this manner (Text S3.1). For 22 of the remaining 73 genes, detailed
descriptions of RNA or protein expression patterns are publicly available, and all are consistent
with functions in meiotic prophase. Thus, among 106 genes implicated in meiotic prophase
164
through our systematic, whole-genome RNA-seq analysis, 54 genes (of 55 tested) are
substantiated by prior studies.
These findings suggest that many of the remaining 51 (of 106) genes represent novel and
uncharacterized genes involved in meiotic prophase. Review of the published literature indicates
that our RNA-seq analysis captured most meiotic prophase genes that are expressed specifically
in meiotic germ cells. Of 21 genes for which mutant germ cells have been reported to arrest at
leptotene, zygotene, or pachytene stages of meiotic prophase (as cataloged by Handel and
Schimenti, 2010), 14 are represented in our list of 106 genes. The seven genes with meiotic
prophase arrest phenotypes that we failed to identify by RNA-seq analysis are either ubiquitously
expressed (such as Cyclin-dependent kinase 2, Cdk2) or are expressed in both ovarian and
testicular germ cells (such as Piwi-like RNA-mediated gene silencing 2, Piwil2).
165
Figure 3.1
RNA-seq of wild-type and germ cell-depleted fetal gonads identifies a meiotic
prophase-associated gene set
Relative expression of 531 ovarian germ cell-enriched genes in E12.5, E14.5, and E16.5 wildtype (Kit/Kit') ovary. The 531 genes comprise 530 genes expressed in wild-type ovary (>5
Fragments Per Kilobase of transcript per Million mapped reads, FPKM), and enriched in wildtype over germ-cell-depleted ovary (Kit/Kitw') (fold-change > 2, q <0.01 at either E 12.5, E14.5,
or E16.5). Rec8 is additionally included for a final set of 531 genes. Gene expression was
measured by RNA-seq and represented as log transformed and mean centered FPKM. Genes
(rows) are organized by hierarchical clustering using the Pearson correlation metric and complete
linkage. Black bars to right of gene expression heat map represent 106 genes that fulfilled
additional criteria of being up-regulated between E 12.5 and E 14.5 or E 16.5 in the ovary (foldchange > 2, q < 0.01), and being enriched in ovary over testis (fold-change > 2, q < 0.01).
166
W-
W-
'-
ww w
531 genes
expressed in
fetal ovary
and enriched in
wild-type over
germ-cell-depleted
ovary
106 genes
up-regulated
in fetal ovary
from E12.5 to E16.5,
and enriched in
ovary over testis
Relative
expression
1.0
0.5
0.0
-0.5
-1.0
0 FPKM
167
Figure 3.2
Rec8 is expressed in both somatic and germ cells
E14.5 fetal ovaries stained DAPI, SSEA 1 (immunofluorescence), Rec8 (single molecule
fluorescent in situ hybridization, smFISH), and Dazi (smFISH). Single transcripts of Rec8 and
Dazi are detected as punctate signals by smFISH. Germ cells are outlined in red.
168
MEIOTIC PROPHASE GENE EXPRESSION IS WHOLLY DEPENDENT ON DAZL, BUT RANGES
FROM STRA
8-INDEPENDENT TO STRA 8-DEPENDENT
We next sought to determine how the meiotic prophase genes are activated. Stra8 was previously
shown to be required for meiotic initiation, as primarily assayed by the meiotic chromosomal
program. Dazl and RA are germ-cell-intrinsic and -extrinsic factors required for induction of
Stra8, and thus for initiation of the chromosomal program. The roles of these factors in
regulating the program of gene expression are largely unknown. We first determined whether
meiotic prophase genes are regulated by Dazl and Stra8 by examining gene expression by RNAseq in whole E14.5 Dazl-deficient (Dazl -/-) and Stra8-deficient (Stra8 -/-) ovaries compared to
corresponding homozygous wild-type controls (Fig. 3.3, Table S3.2).
Dazl is required for germ cells to acquire competence to respond to RA. Dazl-expressing
germ cells respond to RA by expressing Stra8 and initiating meiosis. Dazl is also more broadly
required for the processes of gametogenesis, which encompass meiosis, the sex-specific cellular
differentiation events of oogenesis and spermatogenesis, and the down-regulation of
pluripotency markers (Gill et al., 2011; Lin et al., 2008). Given Dazl's broad role in competence
for gametogenesis, we predicted that Dazl would be required for induction of the meiotic
prophase gene expression program. Indeed, we find that expression of practically all meiotic
prophase genes (101 of 106) is significantly diminished if not eliminated in Dazl-deficient
ovaries (expression in Dazl-deficient ovaries < wild-type ovaries, FDR-adjusted p value, q <
0.05) (Fig. 3.3, Table S3.2).
Stra8 is required for the meiotic chromosomal program, including loading of meiotic
cohesins, such as REC8, and assembly of the synaptonemal complex proteins, including SYCP3.
However, although the REC8 and SYCP3 proteins do not localize to chromosomal axes in Stra8-
169
deficient germ cells, the proteins are nevertheless expressed (Baltus et al., 2006). In fact, Rec8
expression can be induced in testicular germ cells by RA in the absence of Stra8 function
(Koubova et al., 2014). These results suggested that while Stra8 might regulate the entirety of the
meiotic chromosomal program, it might have a more restricted role in governing the gene
expression program. We aimed to clarify the extent to which Stra8 regulates the meiotic gene
expression program.
We found that expression of the 101 Dazl-dependent genes ranged across a wide
spectrum of Stra8-dependency. For slightly over half of the 101 genes, including DmcJ, which is
required to repair meiotic double-strand breaks, expression appeared to be fully dependent on
Stra8. Expression of these genes was reduced in Stra8-deficient ovaries to levels as low as in the
Dazl-deficient ovary (Fig. 3.3, Table S3.2). Expression of the remaining genes appeared to be
partially dependent on, or in a few cases, largely independent of Stra8. Some genes, such as
Sycp3, were expressed at lower levels in Stra8-deficient ovaries compared to wild-type ovaries,
but still at higher levels compared to Dazl-deficient ovaries. At the Stra8-independent extreme of
the spectrum is Rec8, whose levels are not only undiminished in Stra8-deficient ovaries, but in
fact are modestly increased.
Thus, RNA-seq analyses of whole Dazl-deficient and Stra8-deficient ovaries suggests a
model of gene induction whereby Dazl is required for induction of the meiotic prophase gene
expression program via at least two pathways: a Stra8-independent pathway, and a Stra8dependent pathway.
170
Figure 3.3
RNA-seq of Daz and Stra8-deficient fetal gonads reveals Stra8-independent
regulation of meiotic prophase genes
Relative expression of 106 meiotic prophase-associated genes in E14.5 wild-type, Stra8deficient, and Dazl-deficient ovary. Gene expression was measured by RNA-seq and represented
as log transformed and mean centered FPKM. Genes (rows) are arranged from least to most
down-regulated in the Stra8-deficient ovary relative to the Dazl-deficient ovary, with the
exception of the bottom five rows which are not significantly down-regulated in the Dazldeficient ovary. 13 genes, listed to the right of the gene expression heat map, were selected for
follow-up by smFISH.
172
to
W)
W)
qW
qW
q
Rec8Sycpl
SYvp2
GmI5O4
106
13 genes
followed up
by smFISH
meiotic
prophase
genes
Smclb
D6Mm5e
S61g3
Rekive
Dmc
_
expression
S1.0
0.5
0.0
-0.5
-1.0
173
STRA 8-INDEPENDENT AND STRA 8-DEPENDENT PATHWAYS ACT ADDITI VELY IN INDIVIDUAL
CELLS
Whole-gonad RNA-seq analysis provides genome-wide breadth in characterizing the gene
expression program. However, because this method averages across a population that includes a
diversity of both germ cells and somatic cells, our observations may not accurately reflect events
in individual germ cells. Specifically, we wondered whether our observation that some genes
appeared partially Stra8-independent by RNA-seq actually reflected a partial reduction in gene
expression in all Stra8-deficient cells. If so, this would indicate that Stra8-independent and
Stra8-independent pathways act additively in individual germ cells. Alternatively, our RNA-seq
observation could be explained by a subset of Stra8-deficient germ cells retaining wild-type
levels of gene expression, with other Stra8-deficient germ cells having greatly reduced levels of
gene expression.
Distinguishing between these two scenarios required measurement of gene expression
with single-cell resolution. We used single molecule FISH (smFISH) to quantify gene expression
in single cells in situ. smFISH (fluorescence in situ hybridization) involves multiple short
fluorescently-labeled oligonucleotide probes that collectively bind along the same target
transcript to detect and localize each target mRNA molecule as a punctate signal (Raj et al.,
2008) (Fig. 3.4A). These punctate signals can be quantified to determine the number of
transcripts per cell volume (transcript density) (Fig. 3.4B). We selected 13 genes, spanning a
spectrum of Stra8-dependencies as measured by RNAseq in E14.5 ovaries (Fig. 3.3), for
examination by smFISH in germ cells of E14.5 wild-type, Stra8-deficient, and Dazl-deficient
ovaries. Selected genes include the meiotic-specific cohesins Rec8, Smcib, and Stag3; the
synaptonemal complex proteins Sycpl, Sycp2, and Sycp3; DmcJ and Msh5, which are involved
174
in double-strand break repair; and Hormadi, which promotes homolog alignment and
synaptonemal complex formation. We also included Meil and Miap, which exhibit defects in
meiotic prophase when mutated, and Gm1564 and Ugt8a, which are presently uncharacterized.
Through these smFISH studies of individual germ cells, we confirmed that all 13 genes
were Dazl-dependent, and that they defined a wide spectrum of Stra8-dependence. Although the
reality is most likely that genes fall on a continuum of Stra8-dependence, for conceptual
simplification and ease of discussion, we will refer to genes as Class 1 - fully Stra8-independent,
Class 2 - partially Stra8-independent, and Class 3 - fully Stra8-dependent (Fig. 3.4C - E). Rec8
fell into Class 1, fully independent of Stra8 expression (Fig. 3.4C, 3F). Expression of Rec8 in
Stra8-deficient germ cells, as a population, was in fact slightly higher than in wild type, an
observation we later explored. DmcJ, Msh5, Hormadi, meil, and Miap fell into Class 3, fully
dependent on Stra8 (Fig. 3.4E, 3.4H, 3.5A). Their expression in Stra8-deficient germ cells was
reduced (compared to wild-type germ cells) to the same degree as in Dazl-deficient germ cells.
Sycp3, Sycp2, Sycpl, Stag3, Smcib, Gm1564, and Ugt8a fell into Class 2, partially independent
of Stra8 expression (Fig. 3.4D, 3.4G, 3.5A). Their expression in Stra8-deficient germ cells was,
as a population, significantly lower than in wild type, but significantly higher than in Dazldeficient germ cells. We always observed a unimodal distribution of gene expression, which is
consistent with gene expression being reduced in each germ cell. The direction and relative
magnitudes of gene expression changes as measured by smFISH and RNA-seq are consistent for
all 13 genes (Fig. 3.5B).
175
Figure 3.4
Single molecule FISH analysis corroborates 3 classes of gene regulation at
the level of individual germ cells
(A) Detection of single transcripts of Stra8 and Dazl by smFISH in E 14.5 fetal ovary. Single
transcripts are visible as punctate signals. Germ cells are also co-stained for SSEAI, and DAPI.
(B) Top: Individual images are stitched together for an entire E 14.5 ovary. Red signal in the
stitched image represents Stra8 transcript. Bottom: Scatterplot of Stra8 transcript density in
single germ cells, along the anterior-posterior axis of the ovary. Transcript density is the number
of transcripts per cell normalized by cell size (#/um-3).
(C), (D), (E) Models for 3 classes of regulation, and classification of 13 representative meiosis
genes into the three classes by smFISH data. (C) Genes that are fully Stra8-independent, (D)
genes that require both Stra8-independent and a Stra8-dependent pathway to be fully expressed,
and (E) genes that are fully Stra8-dependent.
(F), (G), (H) Distributions of transcript densities in E14.5 wild-type (red), Stra8-deficient (blue)
and Dazl-deficient (green) germ cells, for (F) Rec8, a representative of Class 1, (G) Sycp3, a
representative of Class 2, and (H) Dmcl, a representative of Class 3. Asterisks represent
significant differences between the means of the distributions (p< 0.05, t-test on average
transcript densities of biological replicates).
(I) Representative scatterplot of transcript densities of Stra8 againstRec8 in E 14.5 wild-type
ovarian germ cells.
(J), (K) Representative scatterplots of transcript densities of Sycp3 againstRec8 (J), and Dmc]
against Rec8 (K) in E 14.5 Stra8-deficient ovarian germ cells. (I), (J), (K) Correlation coefficients
for biological replicates provided in S3 Table.
176
A
-
B
E
U: 8
O
0.2
00 0
0
00
0
0.1
00
A
D
Class 2
Class 3
E
DazI
Dazi
Dazl
RAc-A
RA
RA
Stra8
Stra8
I
Dmc1, Hormad1, Msh5,
Meil, Miap
Sycp3, Sycp2, Sycpl,Stag3,
Smc1b, Gm1564, Ugt8a
F
H
G
0.3
Sns.
1 0.2
0.2
'c3
0.1
-
R=0.75, p=10-5
e
0.2
o
.'
**,
0.15
0
0.1
S.
e
0.1
.S
0.2
0.3
Rec8 transcript
density (#lpm3
)
3
0
177
--
F% U.IJ I,
0.05
0.1 0.2 0.3
Rec8 transcript
density (#/pmi
V i
0
I0
0.1
)
0
0.2
4-
Stra8 -/-
0.2
R=0.81, p=10
f0
0.1 to.*
type
K
Stra8 -/-
Wild type
0.3
Wild Stra8 Dazi
Wild Stra8 Dazi
type 4-
Wild Stra8 Dezi
type 4-/-
I
0.1
0
0
0.
0
0.2
0.3
*
Rec8 Stra8
j
0
6
0
0.1 0.2 0.3
Rec8 transcript
density (#/pm 3
)
Class 1
C
Figure 3.5
Transcript densities of meiotic prophase genes as measured by single
molecule FISH
(A) Violin plots representing distribution of transcript densities in wild-type (red), Stra8 mutant
(blue) and Dazl mutant (green), for 13 selected meiotic prophase genes. Asterisks represent
significant differences between the means of distributions (t-test on average transcript density of
a population of germ cells from independent biological replicates). n.s. represents not significant
differences (p > 0.05). For Meil and Msh5, expression in Stra8 and Dazl mutants were at an
average of less than the equivalent of 1 transcript per cell, hence, difference between Stra8 and
Dazl mutants was not tested. Genes were grouped into class 1, 2, and 3 based on significant
differences in expression between wild type, Stra8-deficient, and Dazl-deficient germ cells as
described in main text. Sycp2 transcript densities do not differ significantly between wildtype
and Stra8-deficient germ cells. Nevertheless, we placed it in class 2 based on subsequent
analyses that show that by E15.5, it is expressed at significantly lower levels in Stra8-deficient
compared to wildtype germ cells (Fig 3.9, p = 0.0088 at E15.5).
(B) Scatterplot of log2 fold-change of gene expression in E14.5 Stra8 mutant over wild-type
ovaries against Dazl mutant over wildype ovaries for 106 meiotic prophase genes, as measured
by RNA-seq. 13 selected meiotic prophase genes that were also examined by smFISH are
highlighted in color. Red, blue, and green represent class 1, 2, and 3 genes respectively, as
defined by smFISH.
178
A
class i
Rec8
0.3
0.25
02
0.15
U Wildtype
U Stra8 mutant
0.1
Dazi mutant
0.05
0
Class 2
Sycp2
Sycp3
025
0.2
0.15
*
025
0.2
*
0.
*
0,15
0
0
0.2
0.25
0.2
0.15
0.15
0
0
Ugt8a
0.15
0.2
0.3
0.25
02
0.15
0.15
0.05
0.05
005
0
04
.51
0
0.3
*
Gm1564
Smclb
0.3
0.25
Stag3
SycpI
0.3
0.25
0.3
0.3
.056
0.1
0
Class 3
n.s.
0.15
0.1
0.3
025
0.3
*
0.25
n.S.
02
0.15
0.1
ns
02
0.15
.067
0.05
0.1
1
0.
Meil
Miap
Hormad1
Dmci
02
Msh5
0.1
.076
0
0.05
0
.6i
20
.6
N
. Class
*
-5
0
5
log2 ( DazKO / WT)
179
I
- Class 2
Class 3
SINGLE-CELL CORRELA TION OF STRA 8-INDEPENDENT GENE EXPRESSION WITH
EXPRESSION OF RA-INDUCED GENE
What is the role of RA in regulating the meiotic prophase genes? It was previously shown that
RA induces Stra8 (Bowles et al., 2006; Koubova et al., 2006). Therefore, Stra8-dependent
induction of Class 2 and 3 genes would depend, indirectly, on RA. Does RA also regulate the
Stra8-independent induction of Class 1 and 2 genes? We previously showed that RA induces
Rec8 in the absence of Stra8 (Koubova et al., 2014), and we presently quantitatively demonstrate
the full independence of Rec8 expression from Stra8. By extension, we hypothesized that RA is
responsible for Stra8-independent induction of not just Rec8, a Class 1 gene, but also of the class
2 genes.
An ideal test of this hypothesis would be to eliminate RA in vivo in the fetal ovary. This
was not technically feasible, so we instead sought evidence of RA regulation by analyzing gene
expression in hundreds of individual germ cells, and using endogenous variation in expression of
a RA-induced gene in these hundreds of germ cells as a read out of cell response to RA. If
variation in expression of an RA-induced gene reflects the individual cell's response to RA, then
expression of two RA-induced genes across hundreds of individual germ cells should be
positively correlated. To test this, we examined variation in expression of the two known
independently RA-induced genes, Stra8 and Rec8. We found that Rec8 transcript density is
indeed positively correlated with Stra8 transcript density in germ cells of E14.5 fetal ovaries
(Fig. 3.41; R = 0.75, p = 10-54).
We proceeded to use variation of Rec8 expression as a read out of RA response, so as to
determine if Stra8-independent expression of genes is due to RA. If so, then expression of the
gene, in the absence of Stra8, should be correlated with that of Rec8. We quantified expression
180
of each Class 2 gene alongside Rec8, in hundreds of individual Stra8-deficient germ cells at
E14.5. Expression of Sycp3, Sycp2, Sycpl, Stag3, Gm1564, and Ugt8a is positively correlated
with Rec8 expression (Fig. 3.4J, 3.6, Table S3.3). As expected, for Class 3 genes, which are fully
Stra8-dependent, residual expression in the absence of Stra8 did not correlate with Rec8
expression (Fig. 3.4K, 3.6, Table S3.3). These results are consistent with the Stra8-independent
pathway being regulated by RA, either directly or indirectly.
181
Figure 3.6
Single cell correlation of expression of meiotic prophase genes compared to
Rec8
Representative scatterplots of transcript densities of 12 meiotic prophase genes from class 2 and
3 (y-axis) against Rec8 (x-axis). Transcript densities were measured in E14.5 Stra8 mutant
(blue), and Dazl mutant (green). Correlation coefficients for both plots shown and of biological
replicates are given in Table S3.
182
Class 2
0
0.2
0 .2
0.2
I 0
Sycp3 0.1
Sycp2
*
Sycp1
*
*
0
00Ott
0.1
0o**
*
.1
0
:.0
0, -Q
%
.1
0.2
0.3
0.2
0.1
Rec8
0o
Rec8
0.2
-
Smclb
*.
0.2
1P
0
Gm1564
0 .1
0
0.1
.
*
-
*
Stag3
0
0
-0 .1
0.2
0.1
00
0.3
0.3
Rec8
*
0.2
0.2
0.1
Rec8
-0
0.3
0.2
0.1
0.2
0.3
Rec8
Rec8
0.1
Ugt8a
0.05
0.1
0.2
0.3
Rec8
Class 3
0.2
0.15
*:
.
-
01.-S
8
%00.1
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Meil
0.1
0.1
0-05
Msh5 0.05
0.2
00
0.4
*..-
00
. 0
.&*O
0.2
0.3
1R1e1
Rec8
183
02
Rec8
M1ap
01
O
0.1
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Rec8
Rec8
.
Hormad1
*
Dmcl
0.2
0.2
0.1
0.3
STRA8-INDEPENDENT PATHWAY ENABLES MAXIMAL AND EARLY GENE EXPRESSION
Our model of gene regulation inferred from E 14.5 fetal ovaries led us to predict two
consequences for gene expression over time. First, we reasoned that, for Class 2 genes, both
Stra8-independent and Stra8-dependent pathways might be required to attain maximal levels of
gene expression. If so, expression of Class 2 genes in Stra8-deficient germ cells would not reach
peak wild-type levels, even after a prolonged time. Second, we considered the possibility that
RA-dependent, Stra8-independent induction of Class 1 and 2 genes might function to induce
genes that are required early in meiotic prophase, in anticipation of Stra8. If so, Class 1 and 2
genes might be induced in parallel with Stra8, and before Class 3 genes.
To determine the temporal dynamics of gene expression with fine resolution, we took
advantage of previous observations that fetal ovarian germ cells initiate and progress through
meiotic prophase in an anterior-to-posterior wave (Bullejos and Koopman, 2004; Menke et al.,
2003). Stra8, Dmc], and Sycp3 expression have been observed to be induced first in germ cells
in the anterior before germ cells in the posterior. Therefore, measuring gene expression as a
function of anterior-posterior position in addition to time should provide finer resolution of
events than would time alone. To formally test the hypothesis that the anterior-posterior axis is a
proxy for time, we compared expression changes of 531 germ cell-enriched genes over time
(between E12.5 and E13.5, anterior third of ovaries only), and over space (between posterior and
anterior thirds of E13.5 ovaries). We find that gene expression changes over both time and space
are indeed highly correlated (Fig. 3.7, Table S4), validating our spatiotemporal approach.
184
*
0
*
0
C.4
W C0*
-
WI
-6
-4
-2
0
2
6
4
)
(
Iog2 E13.5A/ E13.5P
ime
Figure 3.7
Correlation of changes over time versus over anterior-posterior position
Scatterplot of log2 fold-change in expression of 531 ovarian germ cell-enriched genes in time
(E13.5 anterior over E12.5 anterior) against space (E13.5 anterior over E13.5 posterior).
185
We determined gene expression over a spatiotemporal axis, using Stra8 expression in
wild-type germ cells as a reference, as follows. We measured the transcript density of Stra8 in
individual germ cells at El 1.5, E12.5, E13.5, E14.0, E14.5, E15.0, E15.5, and E16.5. For each
timepoint, we calculated the average transcript density along the longitudinal axis, from the
posterior (germ cells at least advanced state) to anterior (germ cells at most advanced state) (Fig.
3.8A). We then joined these average expression traces from consecutive timepoints to create a
continuous trace of average transcript density along a spatiotemporal axis (Fig. 3.8B). Using this
approach, we quantified the Stra8 pulse of expression in the wild-type ovary, which was
previously observed semi-quantitatively, by whole-mount in situ hybridization (Menke et al.,
2003).
We applied this spatiotemporal analysis to characterize expression dynamics of the subset
of 13 meiotic prophase genes in wild-type, Stra8-deficient, and Dazl-deficient germ cells. First,
we asked if Class 2 genes indeed required both Stra8-independent and Stra8-dependent pathways
to attain maximal levels of gene expression. We found that, in Stra8-deficient germ cells, Class 2
genes failed to reach expression levels seen in wild type even when given an additional one to
two days after expression peaks in wild type. For example, expression of Sycp3 in Stra8deficient germ cells began to dip at E16.5, without having reached the peak levels of expression
achieved (at E15.5) in wild-type germ cells (Fig 3.8B, 3.9). Thus, the Stra8-independent pathway
is crucial to ensure full expression of Class 2 genes.
Second, we asked if Stra8-independent induction of Class 1 and 2 genes might enable
early gene expression. We found that induction of four Class 1 and 2 genes - Rec8, Stag3,
Smcib, and Gm1564 - indeed occurred early, and contemporaneous with Stra8. Half-maximal
expression of these genes preceded or coincided with half-maximal expression of Stra8 (Fig.
186
3.8D, 3.9). In contrast, all five of the Class 3 genes tested reached half-maximal expression after
Stra8 had done so. Thus, the Stra8-independent pathway is able to induce expression of some
Class I and 2 genes in parallel with Stra8.
A STRA8-DEPENDENT PROCESS IS REQUIRED FOR SUBSEQUENT DOWN-REGULA TION OF
STRA8 AND REC8
Spatiotemporal analysis of Rec8 expression in Stra8-deficient germ cells unexpectedly revealed
that in the absence of Stra8, germ cells expressed Rec8 at slightly higher levels as a population
(Fig. 3.4F, 3.8E), and Rec8 expression persisted for at least a day longer than in wild type.
Therefore, a Stra8-dependent process is required for the subsequent down-regulation of Rec8.
By our measurements, Rec8 and Stra8 are induced and subsequently down-regulated with
nearly identical dynamics. Therefore, we wondered if Stra8 down-regulation also requires Stra8
function. To measure Stra8 promoter activity in the Stra8-deficient germ cells, we measured
expression of a lacZ reporter knocked into the endogenous Stra8 locus (Baltus et al., 2006). We
compared this to lacZ expression in Stra8 heterozygotes, where one functional copy of Stra8 is
present. As with expression of Rec8, expression of lacZ in the homozygous Stra8 knockout
persisted for at least a day longer than in the heterozygous Stra8 mouse (Fig. 3.8F, 3.10). Thus,
we have identified a novel Stra8-dependent event, which is the down-regulation of Stra8 and
Rec8 (Fig. 3.8G).
187
Figure 3.8
Spatiotemporal analysis demonstrates role of Stra8-independent pathway in
inducing maximal and early gene expression, and identifies Stra8-dependent downregulation of Stra8 and Rec8
(A) Construction of spatiotemporal plot of Stra8 average transcript densities along the anterior-
posterior axis of ovaries at El 1.5, E12.5, E13.5, E14.0, E14.5, E15.0, E15.5, and E16.5. To
construct a smooth average transcript density trace, timepoints are overlapped based on Stra8
transcript density levels. Bold line indicates mean of distribution, light band indicates one
standard deviation about mean.
(B, C) Spatiotemporal plot of Stra8 (B) and Sycp3 (C) expression in wild-type (red), Stra8deficient (blue) and Dazl-deficient (green) germ cells.
(D) Timing of 50% maximal induction of genes for Class 1, 2, and 3 genes (filled brown circles).
Time is represented by a spatiotemporal axis (x-axis), same as in (A). Time of 50% Stra8
induction is represented by open black circle.
(E) Spatiotemporal plot of Rec8 expression in wild-type (red), Stra8-deficient (blue) and Dazldeficient (green) germ cells. Rec8 average transcript densities are significantly higher in Stra8deficient compared to wild-type germ cells at E14.5, E 15.5, and E 16.5 (p< 0.05, t-test on average
transcript densities of biological replicates).
(E) Spatiotemporal plot of lacZ expression in endogenous Stra8 locus in Stra8-deficient (blue)
and Stra8 heterozygote (wild type/lacZ) (black) germ cells, normalized for per allele.
Normalized lacZ transcript densities differ significantly at E15.5, and E16.5 (p< 0.05, t-test on
average transcript densities of biological replicates).
(F) Model representing Stra8-dependent down-regulation of Stra8 and Rec8.
188
A
Stra8 expression over time
and anterior-posterior position
B
straB
0.15 Wild
type
StraBDazl-
0.2[
10.15
0.1
0.1
0.05
E16.5
E11.5 E12.5
0
E11 5 E12.5 E13.5 E14.5 E15.5 E16.5
E11.5 E12.5 E13.5 E14.5 E15.5 E16.5
spatlotemporal axis
SycP3
C
spaotemporal axis
D
Timing of 50% maximum
gene expression
Dazl
-
0.15 Wild type
0.11
4
3.
L
2
U 1~
0.05
.
I)
E15.5
E13.5
20.05
8. osrS
E13.5
00
E14.5
E15.5
Spatiotemporal axis
E11.5 E12.5 E13.5 E14.5 E15.5 E16.5
- m-- Iax-s
ReO8
E
StraS-Iacz
F
0.01
Wild typ
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Stra
)
t
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cci 0.05 t
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v
E12.5 E13.5 E14.5 E15.5 E16.5
E11.5 E12.5 E13.5 E14.5 E15.5 E16.5
spatiotemporal axis
spatiotemporal axis
G
Rec8 l-Stra8
189
Figure 3.9
Spatiotemporal analyses of gene expression by single molecule FISH
Spatiotemporal plot of average transcript densities of class 2 and 3 genes along the anterior-
posterior axis of ovaries at El 1.5, E12.5, E13.5, E14.0, E14.5, E15.0, E15.5, and E16.5 (Rec8 is
shown in Figure 3.8).
190
Class 2
Sycp3
Sycp1
Sycp2
0.1
0.1
0.1
0.12
0.15
0.1
0.08
0.1
0
0.06
0.05
0.
0.04
0.
0.02'
0.
0
0*
0
Eli.5 E12.5 E13.5 E14.5 E15.5 E16.5
E11 .5 E12.5 E13.5 E14.5 E15.5 E16.5
Gm1564
Stag3
0.1
0
.11
0.1
0.1
0.0E
0.:
0.
0
El1.5 E12.5 E13.5 E14.5 E15.5 E16.5
0.09
0.0
0 E11.5 E12.5 E13.5 E14.6 E15-5 E16.5
0.
EII.5 E12-5 EM3. E14.5 ElS.5 E16.5
Ugtma
0.1
0.08
0.08
0.04,
0.02
E1.5 E12.5 E13.5 E14.5 E15.5 E16.5
Class 3
Hormadi
Dmc1
M1ap
0.15
0.
0.1
0.
0.06
0.5
0.
0.04
0.02,
0
0
E11.5 E12.5 E13.5 E14.6 E15.5 E16.5
E1.5 E12.5 E13.5 E14.5 E15.5 EI.5
Msh5
Meil
0.03
0.04
0.03
0.02
0.02'
0.01.
0.01,
0
--
A
E11.5 E12.5 E13.5 E14.5 E15.5 E16.5
0
E11.5 E12.5 E13.5 E14.5 E15.5 E16.5
191
0
E11.5 E12.5 E13.5 E14.5 E15.5 E16.5
Stra8*'
0.03 f
0
.5
R=0.53, p=10-"
0
0
0 00
0
000
0.02
01
0
Replicate:
R=0.59, p=10-
o0
0%00
0
0
Ca)
C
0.01
0
N
ik
0
toso**
0.1
0.2
Stra8 transcript density
Figure 3.10
Single molecule FISH analyses of Stra8 and Stra8-lacZreporter
Representative scatterplot of transcript densities of Stra8-lacZreporter (y-axis) against Stra8 (xaxis) from E14.5 Stra8 wild type/lacZ heterozygote.
192
AFTER DOWN-REGULA TING STRA8, GERM CELLS ARE REFRACTORY TO FURTHER STRA8
EXPRESSION
The observation that Stra8 expression is rapidly down-regulated after its initial induction led us
to wonder if, in addition to down-regulating Stra8, germ cells become refractory to subsequent
induction of Stra8.by RA. If so, wild-type germ cells that have expressed Stra8 once should not
be able to express Stra8 again, even if they were provided with a second (exogenous) dose of
RA. To test this prediction, we administered exogenous RA to pregnant mice at E 15.5, when
most germ cells have down-regulated Stra8. We then measured expression of Stra8 a day later,
at E16.5 (Fig. 3.11). Stra8 expression was not increased in ovarian germ cells of fetuses that
received RA, compared to fetuses that did not receive RA. As a control, we tested if RA was able
to induce Stra8 in E15.5 testicular germ cells. We expected that since testicular germ cells do not
ordinarily express Stra8 until after birth, they would be able to induce Stra8 expression if they
receive RA at an earlier time. We found that at E16.5, Stra8 expression was induced about 20fold in testicular germ cells of fetuses that received RA, compared to fetuses that did not receive
RA. Thus, fetal ovarian germ cells that have down-regulated Stra8 expression are refractory to
re-expressing Stra8 even when exposed to RA.
193
Ovary
0.3
Testis
_
-*-
+
-
0.2
0.1
RA
Figure 3.11
-
+
cc
RA is unable to induce Stra8 in germ cells that have induced and down-
regulated Stra8
Distributions of Stra8 transcript densities in in E16.5 ovary (pink), and testis (blue), with and
without exogenous RA administered at E15.5. Asterisks represent significant differences
between the means of the distributions (p< 0.05, t-test on average transcript densities of
biological replicates).
194
SEQUENTIAL A) INDUCTION OF SYCP3 AND B) STRA8-DEPENDENT NEGATIVE REGULATION
OF REC8 ALONG THE ANTERIOR-POSTERIOR AXIS OF THE E14.5 OVARY
Earlier, we measured expression of Rec8 and Sycp3 expression in hundreds of individual E14.5
germ cells that lacked Stra8, and found that Rec8 and Sycp3 expression was correlated, implying
their co-regulation by a Stra8-independent pathway (Fig. 3.4J). We were initially surprised to
find, upon performing the same analysis in E14.5 wild-type germ cells (Fig. 3.12A), that Rec8
and Sycp3 expression was not correlated in the presence of Stra8. We reasoned that the
differences we observed between these wild-type and Stra8-deficient germ cells should be due to
Stra8-dependent processes. Indeed, we found that two Stra8-dependent processes identified thus
far - partial induction of Sycp3, and negative regulation of Rec8 - explain the differences we
observed. We dissected the germ cells' progression over meiotic prophase by examining germ
cells in the posterior third, middle third, and anterior third of the ovary separately (Fig. 3.12B),
and observed that these two processes occurred sequentially.
Initially, as observed in germ cells in the posterior of the ovary, Sycp3 expression is
induced by both Stra8-independent and Stra8-dependent pathways. We expected that, due to
Stra8-dependent induction of Sycp3, wild-type germ cells would express Sycp3 at higher levels
than Stra8-deficient germ cells. Indeed, we observed that Sycp3 expression was higher in wildtype germ cells than in Stra8-deficient germ cells (Fig. 3.4J, 3.12A). Rec8 and Sycp3 expression
are positively correlated, reflecting that the dominant process at this time is the induction of
meiotic gene expression. As meiosis progresses, germ cells continue to up-regulate Sycp3, so that
Sycp3 expression is highest in the most anterior germ cells of the ovary (Fig. 3.12B). Because
Stra8 is required for subsequent down-regulation of Rec8, we expected that Rec8 expression
would be down-regulated in the most advanced germ cells, located toward the anterior of the
195
ovary. Indeed, we observed that germ cells in the anterior of the ovary expressed Sycp3,at peak
levels, while Rec8 was expressed at lower levels than in more posterior germ cells (Fig. 3.12B).
As a consequence of down-regulation of Rec8 but not Sycp3 in the most advanced germ cells,
Rec8 and Sycp3 expression is no longer correlated. Thus, the conclusions arising from our earlier
analyses are corroborated by positionally-informed single-cell correlation analysis in the E14.5
wild-type ovary.
196
A
B
E14.5 Wild type
00
0
0
~0.2
caa
Trajectory of
0
0
0
000
00
06 b
posterior
0P
00d
middle
anterior
gene expression
0
%
%
*1~*
.~
0
.1~..~
.3
*2
p...
I...
.- I.
4'.'.,'
*
.
4"
0
0
0;0
0
0.1
~0I.
02
0.3
)
Rec8 transcript
density (#/pm3
Figure 3.12
Sycp3 and Rec8 expression in single germ cells from E14.5 ovaries reflects
Stra8-independent induction of Sycp3, and Stra8-dependent down-regulation of Rec8
(A) Representative scatterplot of transcript densities of Sycp3 againstRec8 in E14.5 wild-type
ovarian germ cells.
(B) Breakdown of scatterplot by cell position. The trajectory of gene expression for an individual
germ cell is inferred from gene expression at posterior, middle, and anterior positions.
197
DISCUSSION
The regulated induction of meiotic prophase genes is a prerequisite for the chromosomal
program of meiotic prophase. We report here a mammalian gene regulatory program for meiotic
prophase as it occurs in fetal ovarian germ cells. We identified 106 genes that fulfill stringent
criteria for association with meiotic prophase. A quarter of these genes have been shown
previously to be required for successful meiotic prophase. The remaining three quarters remain
uncharacterized and represent promising candidates that may play similarly critical roles during
meiotic prophase. Meiotic prophase genes are induced initially by RA, in the presence of Dazl,
via Stra8-independent and Stra8-dependent pathways (Fig. 3.13A). Subsequently, downregulation of Stra8 and Rec8 occurs via a Stra8-dependent process (Fig. 3.13B). We propose that
these two elements of gene regulation enable germ cells to prepare for the chromosomal program
of meiotic prophase, and to ensure that the chromosomal program is induced just once.
INDUCTION OF MEIOTIC PROPHASE GENES VIA STRA8-INDEPENDENTAND
STRA8-
DEPENDENT PATHWAYS REPRESENTS A MULTI-OUTPUT FEEDFORWARD LOOP THAT
ENABLES A TEMPORAL ORDER OF GENE ACTIVATION
At the onset of meiotic prophase, meiotic prophase genes are induced by Dazl, RA, and Stra8,
organized in two branching pathways (Fig. 3.13A). RA, in the presence of Dazl, induces the
meiotic prophase genes. Gene induction can occur via a Stra8-independent pathway or a Stra8dependent pathway; these pathways function both separately and additively. Expression of some
genes requires only the Stra8-independent pathway (Class 1), some require both Stra8independent and Stra8-dependent pathways (Class 2), and some are fully Stra8-dependent (Class
3).
199
A
Dazr
RA
Class I
Class 2
Rc8
_Sycp3
_4pnc1
sdyp1
3
_ Hond1
j+Slg
Class 3
_r+Afh5_
B
r* Rec8
Figure 3.13
Model for induction of meiotic prophase gene expression
(A) Model for induction of meiotic prophase genes by Dazl, RA, and Stra8. In the presence of
Dazl, RA induces meiotic prophase genes via Stra8-independent and Stra8-dependent pathways.
Genes range a spectrum of Stra8-dependency. For simplification, we refer to this spectrum as
three classes: Class I - fully Stra8-independent, Class 2 - partially Stra8-independent and
partially Stra8-dependent, and Class 3 - fully Stra8-dependent. Representatives of each gene
class are shown.
(B) Model for subsequent down-regulation of Stra8 and Rec8 expression. A Stra8-dependent
process is required for down-regulation of Stra8 and Rec8 expression.
200
These genetic insights lead us to two speculative hypotheses regarding molecular
mechanisms of meiotic prophase gene regulation: (1) The Stra8-dependent pathway is mediated
directly by STRA8, a putative basic helix-loop-helix transcription factor, and (2) the Stra8independent pathway is mediated directly by RA receptors (RARs). Transcriptome data from
both whole gonads (this study) and sorted germ cells (Lesch et al., 2013) support this possibility:
fetal ovarian germ cells initiating meiosis express all three RARs (RAR alpha, beta, and gamma)
and their heterodimeric partners, the retinoid X receptors (RXR alpha, beta, and gamma).
Potential redundancies among the RARs and RXRs complicate genetic interrogation of the roles
of the RARs. The possibility that STRA8 and RARs directly regulate gene expression can
instead be tested by chromatin-immunoprecipitation-sequencing (ChIP-seq) of RA receptors and
STRA8 in germ cells that are initiating meiosis. We predict that Class 1 genes will be bound by
RARs but not STRA8, that Class 3 genes will be bound by STRA8 but not RARs, and that Class
2 genes will be bound by both RARs and STRA8. A ChIP-Seq study in embryonic stem cells
identified RAR binding of both Stra8 and Rec8 promoter regions (Mahony et al., 2011). Of
course, it is also possible that the Stra8-independent and Stra8-dependent pathways are mediated
indirectly by as-yet unidentified germ cell-expressed factors.
The branching regulatory model described above is reminiscent of a motif termed a feed
forward loop (FFL), which has been shown to generate a temporal order of gene activation (Kalir
and Alon, 2004). An FFL comprises an upstream regulator, in this case RA, which regulates a
downstream regulator, in this case Stra8. Both the upstream regulator, RA, and the downstream
regulator, Stra8, regulate multiple downstream targets - in this case, the meiotic prophase genes.
Genes respond to input from either the upstream or downstream regulator, or both. Modulating
the activation strengths of upstream versus downstream regulators can generate a temporal order
201
of gene activation: genes with greater input from the upstream regulator are activated earlier, and
genes with greater input from the downstream regulator are activated later. Consistent with such
an outcome, we observe that a subset of Class 1 and 2 genes, which are fully or partially induced
by the Stra8-independent pathway, are expressed earlier than Class 3 genes and with timing of
induction close to that of Stra8 induction.
We propose that Class 1 and 2 genes may be induced earlier so as to prepare cells for the
meiotic chromosomal events triggered by Stra8. Indeed, we observe that Class 1 and 2 genes
include almost all known cohesins and synaptonemal complex proteins, which structurally
associate with meiotic chromosomes and may therefore be required early, and in large quantities.
Early expression of cohesin and synaptonemal complex proteins, prior to initiation of the meiotic
chromosomal program, may be a common feature of both sexes and across species. In mouse
testicular germ cells, Sycpl, Sycp2, Sycp3 are expressed as early as in mitotic spermatogonia
(Wang et al., 2001). In C. elegans and D. melanogaster, Rec8 and synaptonemal complex
proteins respectively are also expressed during the amplifying mitotic divisions preceding
meiosis (Christophorou et al., 2013; Joyce et al., 2013; Pasierbek et al., 2001).
Induction of Class 2 genes by Stra8-independent and Stra8-dependent pathways has
important implications for achieving the necessary levels of gene expression. Precise regulation
of gene dosage has been shown to be important for meiotic chromosomal processes. In the
mouse, heterozygous loss of function for either one of the cohesins Smclb and Rec8 causes
perturbations in the formation of the synaptonemal complex and affects synapsis and
recombination between homologs (Murdoch et al., 2013). Therefore, although either the Stra8independent pathway or the Stra8-dependent pathway is sufficient for partial gene expression,
202
the combination of the two pathways is likely required to achieve optimal levels of gene
expression and chromosomal function.
DoWN-REGULATION OF STRA8 VIA A STRA8-DEPENDENT PROCESS PRESENTS NEGATIVE
FEEDBACK LOOP THAT MAY ENSURE ONE-TIME INITIATION OF MEIOSIS
Subsequent to their initial induction, Stra8 and Rec8 expression rapidly declines. We discovered
that this down-regulation depends on Stra8. It remains to be determined whether this occurs
directly via Stra8 activity as putative transcription factor, or indirectly as a consequence of
progression of cell state. We propose that Stra8-dependent down-regulation of itself and Rec8
may serve to limit gene expression to their appropriate window of function. In particular, Stra8dependent down-regulation of itself represents a negative feedback loop that prevents a
prolonged induction of the chromosomal program of meiotic prophase. In addition, we found
that ovarian germ cells that have down-regulated Stra8 are refractory to re-expressing Stra8 even
in the presence of exogenous RA, which may prevent re-initiation of the chromosomal program.
In yeast, an analogous negative feedback loop is postulated to restrict supernumerary rounds of
DNA replication and nuclear division. IME 1, a transcription factor that initiates the yeast meiotic
transcriptional program, induces IME2, which restricts expression of IMEl and destabilize IMEl
protein (Guttmann-Raviv et al., 2002; Smith and Mitchell, 1989). Absence of IME2 results in
prolonged IME expression and additional rounds of DNA synthesis and nuclear division (Foiani
et al., 1996).
IMPLICATIONS FOR THE GENE REG ULA TORY PROGRAM OF MEIOTIC PROPHASE IN THE
MALE
203
Similarities between ovarian and testicular germ cells suggest that the gene regulatory program
as inferred from fetal ovarian germ cells may be common to both sexes. In both sexes, RA
induction of Stra8 has been shown to be required for initiation of the meiotic prophase
chromosomal program (Anderson et al., 2008; Baltus et al., 2006; Koubova et al., 2006). RA and
Stra8 could therefore also regulate gene expression in the male. In testicular germ cells entering
meiosis, Stra8 is also rapidly induced at pre-leptotene then rapidly down-regulated by leptotene
(Zhou et al., 2008a), suggesting the possibility that there is also Stra8 negative feedback.
However, several aspects of regulation in the male remain unclear. For instance, in the male,
RA-STRA8 signaling regulates not only meiotic initiation but also spermatogonial differentiation
(Endo, Romer, and Anderson et al., 2015). In fetal ovarian germ cells, competence to respond to
RA and initiate meiosis requries Dazl (Lin et al., 2008); in testicular germ cells, Dazl's role in
meiotic competence remains unknown. The gene regulatory program of meiotic prophase in the
male must be determined in the context of the distinct role of Stra8 and, and the unexplored
functions of Dazl.
IMPLICA TIONS FOR IN VITRO GERM CELL DERIVA TION
Our findings have practical implications for in vitro derivation of germ cells. First, our results
provide a blueprint to guide efforts in recapitulating the gene regulatory program of
gametogenesis in vitro. Second, our results substantiate previous criticisms against taking
expression of meiotic genes as sufficient evidence of meiosis (Handel et al., 2014; Oatley and
Hunt, 2012). By explicitly interrogating the regulation of the gene expression program and
chromosomal program by the same factors, we showed that the two programs are regulated
distinctly. Specifically, the meiotic prophase chromosomal program requires Stra8 function, but
a subset of the gene expression program is induced independently of Stra8. Our findings thus
204
underscore the insufficiency of simple gene expression for meiotic progression. Rather, both the
gene regulatory and the chromosomal programs are mandatory for successful meiosis.
205
I
EXPERIMENTAL PROCEDURES
Mice
Germ cell-depleted (Kitw/Kitwv) and homozygous wild-type control (Kit/Kit') were generated
by crossing C57BL/6J-Kitw' (The Jackson Laboratory) males to WB/ReJ-Kitw (The Jackson
Laboratory) females (Mintz and Russell, 1957). KitWand Kitw' alleles were genotyped as
previously described (Tanosaki and Migliaccio, 1997; Waskow et al., 2002). Stra8-deficient
(Stra8-), Dazl-deficient (Dazl-) and homozygous wild-type control embryos were generated by
heterozygote matings of DaztmiHic (Ruggiu et al., 1997) and Stra8tm]Dp (Baltus et al., 2006) mice
respectively. DazltmIc, Stra8tmDcp, and wild-type mice used are of C57BL/6 background.
DaztHmic and Stra8tmDPalleles were genotyped as previously described (Baltus et al., 2006;
Ruggiu et al., 1997). All experiments involving mice were approved by the Committee on
Animal Care at the Massachusetts Institute of Technology.
RA treatment
500mg/kg of body weight all-trans RA (Sigma-Aldrich, St Louis, MO), dissolved at 30mg/ml of
corn oil, was administered to pregnant mice via gavage.
Embryonic gonad collection and sexing
Timed matings were set up by housing female mice with male mice overnight. Noon of the day
when a vaginal plug was evident was considered EO.5. For mRNA-seq, embryonic gonads were
dissected away from mesonephroi. For mRNA-seq from E12.5 and E13.5 anterior and posterior
ovaries, dissected gonads were dissected into thirds, and only the anterior and posterior thirds
were used. For smFISH, embryonic gonads were dissected with mesonephroi intact to provide
207
anterior-posterior orientation. For embryos E13.5 and older, sex of tissues was determined by
scoring presence or absence of testicular cords. For El 1.5 and E 12.5 embryos, sex was
determined by PCR as previously described (Menke et al., 2003).
mRNA-seq sample preparation
For all mRNA-seq experiments, total RNA (-lug) was extracted from embryonic gonads using
Trizol (Invitrogen) according to manufacturer's protocol. For Kitw/Kitw' and Kit/Kit' embryonic
gonads and E 12.5 and E 13.5 embryonic ovary thirds, hemoglobin transcripts were selectively
removed from total RNA using GLOBINclear (Ambion) according to manufacturer's protocol.
Libraries were prepared using the Illumina mRNA-Seq Sample Preparation Kit according to
manufacturer's protocol. Libraries were sequenced on the Illumina Genome Analyzer II platform
to obtain 36-base pair single reads. For E14.5 Dazl-deficient, Stra8-deficient, and wild-type
control ovaries, libraries were prepared using the Illumina TruSeq RNA Sample Preparation Kit.
Libraries were multiplexed and sequenced on the Illumina HiSeq 2000 platform to obtain 40base pair single reads. RNA-seq data for KitW/Kit'v and KitC/KitC, E12.5 and E13.5 embryonic
ovary thirds, and E 14.5 Dazl-deficient, Stra8-deficient, and wild-type control ovaries have been
deposited under accession numbers ###.
mRNA-seq on gonads from Kitv/Kitwv and Kit /Kit' embryos was performed on two biological
replicates for each condition. mRNA-seq on anterior and posterior thirds of ovaries from E12.5
and E13.5 wild-type embryos was performed on two replicate pools for each condition, where
each pool consisted of ovary thirds from eight embryos. mRNA-seq on E14.5 Dazl-deficient and
Stra8-deficient ovaries was performed on three biological replicates each, with paired
homozygous wild-type controls.
208
mRNA-seq data analysis
Reads were aligned to the mouse genome (mm9) using TopHat (Trapnell et al., 2009), allowing
only unique alignments (option -gI). We counted reads mapping to the Refseq annotated gene
set using htseq-count (Anders et al., 2014). Fold-changes and FDR-corrected p values, q, for
differentially expressed genes were calculated using edgeR, using tagwise-dispersions and
normalizing for library complexity (Robinson et al., 2010). FPKMs were calculated using
Cufflinks (Trapnell et al., 2010). Hierarchical clustering was performed using Cluster 3.0 on log
transformed and mean centered FPKMs, using the Pearson correlation metric and complete
linkage (de Hoon et al., 2004), and visualized using Treeview (Saldanha, 2004).
Single-molecule Fluorescent In Situ Hybridization (smFISH)
Probe design, synthesis, and coupling were as previously described (Peterson et al., 2012; Raj et
al., 2008). Probes sequences are provided in S2 Text. Gonads were fixed in 4% PFA/PBS for 2
hours at 4 'C, incubated overnight in 30% sucrose/4% PFA/PBS at 4 'C, then embedded in
O.C.T. compound. Frozen blocks were sectioned at 8 pm thickness, fixed in 4% formaldehyde at
room temperature for 15 minutes, rinsed in PBS, and dehydrated overnight in 70% ethanol at 4
*C. The hybridization procedure was performed as previously described (Peterson et al., 2012;
Raj et al., 2008). FITC-coupled anti-SSEA-1 antibody (BD Biosciences 560127) was added to
the hybridization step at 1:30 to identify germ cells. In all experiments, germ cells were
identified by either smFISH for Dazl or Oct4, in combination with SSEA1 immunostaining,
and/or DAPI nuclear staining. Counting of individual mRNA particles, image stitching, and data
analysis was performed using custom Matlab software as previously described (Peterson et al.,
2012; Raj et al., 2008).
209
To depict distributions of transcript densities for each group, we pooled biological replicates in
one violin plot. Comparison of groups was performed by comparing means of at least two
biological replicates from at least two litters using the two-sample t-test. To depict correlations
between pairs of genes in individual cells, we show one representative biological replicate, but
calculate Spearman correlation coefficient for each biological replicate. To depict average
transcript densities over space and time, we pooled biological replicates. At each timepoint, we
determined average transcript densities from the posterior to anterior of the ovary for 100
windows of size 0.2 of the total length of the ovary. The average transcript density traces of
consecutive timepoints were joined together from posterior to anterior. Using average transcript
density traces of Stra8 as a guide, we overlapped some timepoints by shifting along the x-axis in
order to maximize overlap between the average expression traces for Stra8. We determined shifts
using Stra8 expression, and applied the same shifts to spatiotemporal plots for all other genes.
210
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SUPPLEMENTARY INFORMATION
SUPPLEMENTARY TABLES
Table S3.1
Expression of 531 ovarian germ cell enriched genes in E12.5, E14.5, E16.5,
wildtype and KitWwwv ovaries and testes
Table S3.2
Expression of 106 meiotic prophase genes in wild type, DazI-deficient, and
Stra8-deficient ovaries
Table S3.3
Pairwise gene correlations in E14.5 ovaries
Table S3.4
Expression of 531 ovarian germ cell enriched genes in E12.5 and E13.5
anterior and posterior ovaries
Table S3.5
smFISH probe sequences
Tables S3.l-S3.5 available upon request
215
SUPPLEMENTARY TEXT
Text S3.1
Available data on 106 meiotic prophase genes
Number of genes for which a mutant has been analyzed, and a meiotic defect found: 33
Number of genes for which expression pattern has been characterized (in detail, not by
microarray or next-generation sequencing screens), and expression corresponding to meiotic
prophase found: 53
Gene
1700013H 16Rik
Mutant analyzed
Defect in
Expression
Expression corresponds to
meiotic
pattern
meiotic prophase
prophase
characterized
No
References
Yes
Yes, from leptotene to
(1)
Yes
pachytene. Contains Corl
domain.
Yes
(2-4)
Yes
(5)
(Xlr6)
4930528F23Rik
(Meiob)
AsfI b
Yes
Ccdc155
No
Yes
Yes
(6)
Ccnb3
No
Yes
Yes
(7)
Cyld
Yes
Yes
Not examined in germ
cells
Spermatogenes
is defects,
No
(8)
meiotic
prophase not
characterized
DiPasl
No
Yes
Yes
(9,10)
Mlap (D6Mm5e)
Yes
Yes
Yes
Yes
(11,12)
Ddb2
Yes
Fertile, but
No
No
(13)
with defects in
apoptosis in
spermatogenic
Dmct
Yes
cells
Yes
Yes
Yes
(14,15)
Dmrtc2
Yes
Yes, male only
Yes
Yes
(16)
Ecsit
Not examined in germ
cells
Yes
Yes
Yes
(3)
Yes
Yes, but persists beyond
(17)
Figla
Defect in
ovarian follicle
meiotic prophase
formation
Fmrlnb
No
Yes
Gpat2
No
Gprl9
No
Hfml
Yes
Yes
No
Hormadl
Yes
Yes
Yes
Yes
(21-23)
Hormad2
Yes
Yes
Yes
Yes
(24,25)
Hsf2bp
No
Yes
Yes
(3)
1118
Not examined in germ
Yes
Claimed but data unclear
(26)
Incal
cells
Yes
Yes
Yes
(3)
No. KO mice
Yes
(3)
Yes
Most likely
(18)
Yes
Most likely
(19)
(20)
were viable and
fertile.
Mei I
Yes
1__
Yes
Yes
1
217
Not examined with cellular
1resolution
(27,28)
No
Yes
Yes
(29)
Msh4
Yes
Yes
Yes
Yes
(30)
Msh5
Yes
Yes
No
Mirl8
(31,32)
Mtl5
No
Yes
Yes
(33-35)
Poln
No
Yes
Yes
(36)
Prdm9
Yes
Yes
Yes
Yes
(37-41)
Rad211
Yes
Yes
Yes
Yes
(42-45)
Rec8
Yes
Yes
Yes
Yes
(46-48)
Rhox13
No
Yes
Yes
(3,49)
Setdb2
No
Yes
Yes
(50)
Slc25a31
Yes
Yes
Yes
Yes
(51,52)
Smclb
Yes
Yes
Yes
Yes
(53-56)
Spata22
Yes
Yes
Yes
Yes
(57)
Spata5
No
Yes
Yes
(58)
Spdya
No
Yes
Yes
(3)
Spol
Yes
Yes
Yes
(59-62)
Stag3
No
Yes
Yes
(63,64)
Stra8
Yes
Yes, both
Yes
Yes
(65-67)
Sycel
Yes
Yes, both
Yes
Yes
(68,69)
Syce3
Yes
Yes, both
Yes
Yes
(70)
Sycpl
Yes
Yes, both
Yes
Yes
(71,72)
Sycp2
Yes
Yes, in males,
females are
Yes
Yes
(73,74)
Sycp3
Yes
Yes, in males,
females are
Yes
Yes
(75-78)
Syn2
Not examined in germ
Yes
Yes in fetal ovary and testis
(3)
Yes
Yes, in adult testis
(79,80)
Yes
Yes
(81,82)
Yes
Yes, both
(83,84)
Yes
subfertile
subfertile
cells
Males are
subfertile with
post-meiotic
defects,
females are
fertile
Males have
post-meiotic
defects,
females are
Taf7l
Yes
TexiO1
Yes
Texl 1
Yes
Yes, in males,
females are
Tex12
Yes
Yes
Yes
Yes
(85,86)
Tex15
Yes
Yes, male only,
female are
Yes
Yes
(87,88)
Tsc22d3
Yes
Yes
Yes, male
(89-91)
TsgalO
No
Yes
Yes, male
(92,93)
fertile
subfertile
fertile
Yes, male only
(may be
spermatogonial
differentiation
defect?),
female fertile.
218
Wbp2nl
No
Yes
Yes, male.
(94)
Zfp541
No
Yes
Yes, male
(95)
170001F14Rik
No
No
1700123101Rik
No
No
3110057012Rik
No
No
4930432K21Rik
No
No
4930447CO4Rik
No
No
4930455F23Rik
(Ccdc181)
4930524B15Rik
No
No
No
No
4933416CO3Rik
No
No
4933427DO6Rik
No
No
AdarbI
No
BC049762
Not examined in germ
cells
Not examined in germ
cells
No
BC051142
No
No
Caprin2
No
Ccdc36
No
Not examined in
germ cells
No
Ccdc73
No
No
Ccdc79
No
No
Cdkl2
No
CntdI
No
Not examined in
germ cells
No
Crebl2
No
No
Dennd4a
No
No
Dopey 1
No
No
Eaf2
No
Fbxo47
Not examined in germ
cells
No
Fhl4
No
No
(CcdcI72)
Aspa
No
No
No
Gm13718
No
No
Gm1564
No
No
H2-D1
No
Haus8
Not examined in germ
cells
Not examined in germ
cells
No
Larplb
No
No
Lypd4
No
No
Not examined in germ
No
Pet2
No
No
Phka2
No
No
Pparg
No
No
H2-K1
Madd
cells
No
No
219
Pramell
No
No
Rad5lap2
No
No
Rbpms2
No
No
Reg2
No
No
RibcI
No
No
Spryd3
No
No
Sycpl-psl
No
No
Syngr4
No
No
Taf9b
No
No
Tex16
No
No
TktlI
Not examined in germ
No
Usp32
cells
Not examined in germ
cells
No
No
Zcwpwl
No
No
Ugt8a
No
As previously noted, many knock-out phenotypes are sexually dimorphic - males are often
infertile and display arrests in meiosis, whereas females are fertile or subfertile (96).
220
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228
CHAPTER 4.
CONCLUSIONS
229
SEQUENCE OF THE MOUSE Y CHROMOSOME
The sequence of the mouse MSY identifies sex chromosome conflict as a powerful driving force
in the evolution of genomic architecture and structure. Further comparative studies of sex
chromosomes will allow us to determine the extent to which X-Y genetic conflict is prevalent
and influences the evolution of genomes and drives speciation. Our work enables the mouse as a
model for studying the mechanisms of X-Y genetic conflict, and also to genetically interrogate
the functions of Y-chromosome genes, sequences, and genomic structures.
X-Y GENETIC CONFLICTAS A DRIVING FORCE IN SEX CHROMOSOME EVOLUTION
The mouse MSY demonstrates the diversity and rapid evolution of mammalian Y chromosomes.
The three sequenced primate MSYs, diverged up to 25 million years, revealed two major forces
underlying mammalian Y chromosome evolution: degeneration of the ancestral sequences, and
acquisition and amplification of testis-expressed genes. In the mouse MSY, diverged from the
primate MSYs about 90 million years, these forces appear to have played out to an extreme
degree. The mouse MSY retains fewer ancestral genes than the primate MSYs. Instead, the
mouse MSY is predominantly composed of an acquired and amplified 500 kb amplicon, which
makes up a total of 86.4 Mb or 96.5% of its length.
Several explanations have been offered for the amplifications observed on sex
chromosomes. Recombination between Y chromosome palindrome arms in human is thought to
help preserve functional copies of Y genes (Rozen et al., 2003). Amplification of X genes is
thought to have evolved to compensate for the repressive chromatin environment affecting the X
chromosome in postmeiotic cells following meiotic sex chromosome inactivation (Mueller et al.,
2008). The mouse Y amplicons appear to have arisen in response to another phenomenon:
231
meiotic drive. Each of the three most highly amplified gene families has similarly acquired and
amplified X homologs. This co-amplification of X-Y genes is highly reminiscent of the
DrosophilaX and Y-linked multicopy genes Stellate and Suppressor ofStellate, which are
hypothesized to be a meiotic driver and suppressor pair (Hurst, 1992, 1996; Palumbo et al.,
1994). A meiotic driver is a locus within a genome that causes the preferential transmission of
itself over another allele. The evolution of a meiotic driver is often accompanied by evolution of
a suppressor of the meiotic driver. Consistent with the meiotic drive hypothesis, mice with partial
deletions of the Y long arm exhibit sex-ratio distortion: they produce more female than male
offspring (Conway et al., 1994; Moriwaki et al., 1988).
An often-asked question is, why has X-Y co-amplification occurred only in mouse? The
underlying assumption is that sex chromosome meiotic drive, and co-amplification of X-Y
genes, is an oddity of a few lineages. A more pertinent question at this point is, rather, how
prevalent is sex chromosome meiotic drive and co-amplification of X-Y genes? Indeed, within
the sequenced Y chromosomes, the most extensive X-Y co-amplification occurs in mouse. Some
have also noted that sex chromosome meiotic drive has been observed mostly only in rodents
and in Diptera (Helleu et al., 2014; Jaenike, 2001). However, occurrences of amplifications on
sex chromosomes, and of meiotic drive are probably underestimated. It is hard to detect meiotic
drive, because any driver that causes deviation from a 1:1 sex ratio will rapidly be suppressed.
Thus, meiotic driver and suppressor pairs will remain cryptic, unless revealed by genetic crosses
between lineages with incompatibilities in driver and suppressor pairs. Similarly, coamplification of X-Y genes is also underestimated, because Y chromosomes are typically not
sequenced, and X-chromosome ampliconic sequences tend to be poorly assembled by wholegenome shotgun approaches. In human, a small case of co-amplification between the acquired
232
genes VCX and VCY (Lahn and Page, 2000). High-quality sequencing of sex chromosomes may
reveal additional potential cryptic cases of sex chromosome meiotic drive. Only after we have
determined the prevalence of co-amplification of sequences on the sex chromosome, can we then
understand the forces that drive it.
MECHANISMS OFMEIOTIC DRIVE IN THE MOUSE
The details and mechanism by which drive occurs between the mouse X and Y
chromosomes are largely unknown. Mice with partial deletions of the Y long arm are subfertile,
and their sperm have abnormal heads. The resulting sex ratio distortion may result at various
points between gamete formation and development of the next generation, via perturbation in the
ratio of X- to Y-bearing sperm, the ratio of zygotes formed from X- versus. Y-bearing sperm, or
the survival of these zygotes. The defect for the mouse appears to be in differential fertilization
ability of X- versus Y-bearing sperm, as fertilization of eggs by intracytoplasmic sperm injection
(thus circumventing the natural process of fertilization) restores a balanced sex ratio (Ward and
Burgoyne, 2006). However, the number of X- and Y-bearing sperm has not yet been directly
counted.
Our mouse MSY sequence identified three gene families that might account for meiotic
drive. At least two of the three gene families have homology to chromosome- and spindlebinding proteins. It could be possible, therefore, that direct interaction of these gene products
with the chromosomes or spindle is involved in the meiotic-drive phenotype. Sly and its X
homologs Slx/Slxl-i are homologous to a synaptonemal complex protein Sycp3, and contain a
Corl domain that mediates chromatin interactions. Ssty/Sstx are homologous to a spindlin, which
associates with the meiotic spindle in the mouse oocyte.
233
The function of one of these genes, Sly and its X homologs, Slx/Slxl-], has begun to be
investigated via knock-down (Cocquet et al., 2009, 2010, 2012). Knocking down Sly
recapitulates the Y long-arm deletion phenotype of sex-ratio skewing to females; knocking down
Slx/Slxl-J results in sex-ratio skewing to males. Knocking down both families at once restores
sex-ratio balance. Thus, Sly/Six/Six-1 at least partially accounts for the meiotic drive phenotype..
Sly appears to be required for normal postmeiotic silencing of the sex chromosomes: knockdown of Sly leads to up-regulation of sex chromosome genes in postmeiotic germ cells is
associated with a loss of repressive epigenetic marks, such as trimethylated histone H3
(H3K9me3). Knock-down of Slx/Slxl-1 leads instead to the deregulation of mostly autosomal
genes. Knock-down of both X and Y gene families partially restores gene expression to wildtype levels. How misregulation of gene expression might result in sex-ratio distortion is still
unclear. Two other highly amplified gene pairs, Ssty/Sstx, and Srsy/Srsx, have yet to be
investigated.
IMPLICA TIONS FOR HYBRID STERILITY AND SPECIA TION
X-Y genetic conflict not only has consequences for genome evolution, but may drive
evolution of hybrid sterility and consequently speciation. When driver-suppressor pairs evolve in
isolation in diverging lineages, and individuals from recently diverged lineages subsequently
hybridize, incompatibility between independently evolved driver-suppressor pairs may cause
sterility in hybrid offspring. Consistent with this hypothesis, F 1 hybrid males produced by
asymmetric crosses between M musculus musculus mothers and M musculus domesticus fathers
display phenotypes similar to Y long-arm deletion or Sly-deficient mice (misregulation of X
genes and sperm head defects), and are sterile (Good et al., 2010). Again, the two other highly
234
amplified gene pairs, Ssty/Sstx, and Srsy/Srsx, have yet to be investigated for their contribution to
hybrid sterility.
EXPERIMENTA L IMPLICA TIONS FOR THE MOUSE MSY AND AMPLICONIC SEQUENCES
The mouse MSY sequence opens up a mammalian Y chromosome to experimentation in a
genetic model organism. Sequences are now available to guide genetic targeting of single-copy
sequences by TALENs and CRISPR (Wang et al., 2013a, 2013b), and knock-down of
ampliconic gene families by RNAi (Cocquet et al., 2009, 2010, 2012). The amplification of the
mouse Y long arm, representing 3% of the mouse haploid genome, also provides an opportunity
for biochemical studies of chromatin, such as purification of proteins associated with the longarm sequences (Ddjardin and Kingston, 2009).
We demonstrate, through sequencing of the long-arm ampliconic sequences, that the
SHIMS strategy, complemented by radiation hybrid mapping and DNA FISH, can resolve
sequences similar to 1 in 100,000 base pairs. Applications of SHIMS to additional sex
chromosomes and other ampliconic regions of the genome may yield further unanticipated
discoveries.
235
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238
THE GENE REGULATORY PROGRAM OF MEIOTIC PROPHASE IN THE FETAL OVARY
Our work elucidates the gene regulatory program of meiotic prophase, distinct from but required
for the chromosomal program for meiotic prophase. We identify elements of regulatory logic that
might account for how germ cells prepare for meiosis, and attempt meiosis once only. Arising
from these elements of regulatory logic are new pathways and hypotheses for further
investigation.
SUBPROGRAMS OF MEIOTIC PROPHASE
We suggest that meiotic prophase is comprised of distinct programs: A chromosomal program by
which cohesins are loaded, chromosomes synapse, and crossing over is achieved, and a gene
regulatory program by which the expression of genes required for the chromosomal program is
regulated. The vast majority of studies of meiotic prophase do not explicitly distinguish the two.
Most often, the term "meiotic prophase" implicitly refers to the chromosomal program that
directly produces haploid gametes. Less attention has been paid to how meiotic genes are
regulated. Sometimes, the two programs are conflated.
Here, we characterize explicitly the gene regulatory program of meiotic prophase as it
occurs in fetal ovarian germ cells. We identify genes expressed during meiotic prophase, and
determined how they are regulated by the same factors previously examined for their regulation
of the chromosomal program. Thus, we provide a direct means of comparison between the gene
regulatory versus the chromosomal program for meiotic prophase.
239
ROLES OF DAZL, RA, AND STRA8 IN REGULATING THE GENE EXPRESSION PROGRAM
VERSUS THE CHROMOSOMAL PROGRAM
Our study reinforces and elaborates upon the roles of three factors previously described to be
necessary for initiation of the meiotic chromosomal program. RA is the extrinsic signal shown to
induce the chromosomal program. Response to RA requires Dazl, the gametogenesis competence
factor. RA, in the presence of Dazl, induces Stra8, which is required for initiation of the
chromosomal program.
We demonstrate that Dazl is required not only for competence to initiate the meiotic
chromosomal program, but also for competence to express the entire gene expression program.
Stra8, without which the entire chromosomal program fails to initiate, does not similarly regulate
the entire gene expression program. A subset of genes are expressed fully or partially
independently of Stra8. Regulation of the chromosomal program is therefore distinct from
regulation of the gene expression program. While the chromosomal program is wholly dependent
on Stra8, only part of the gene expression program requires Stra8.
Two ELEMENTS OF GENE REGULATORY LOGIC ORGANIZE INDUCTION OF THE MEIOTIC
PROPHASE GENE EXPRESSION PROGRAM
We find that regulation of the meiotic prophase is organized by two elements of
regulatory logic: branching pathways, and negative feedback.
Induction of the meiotic prophase genes occurs via at least two branching pathways. RA,
in the presence of Dazl, induces expression of Stra8, together with a subset of the meiotic
prophase genes. Stra8 in turn induces expression of more genes. We classified the genes into
three classes: Class 1 genes are fully expressed in the absence of Stra8, Class 2 genes require
240
both the Stra8-independent pathway and the Stra8-dependent pathway to be fully expressed, and
Class 3 genes are fully dependent on Stra8 for expression.
Such a branching regulatory architecture is reminiscent of and might represent a
multioutput, coherent feedforward loop (FFL) with a sum input function, which is capable of
activating a battery of genes in a temporally defined order. Such a FFL has been examined in the
context of the flagella biosynthesis transcription network in Escherichiacoli (Kalir and Alon,
2004). This FFL comprises two regulators, FlhDC and FliA, which activate a battery of flagella
genes via cis-regulatory interactions with their promoters. FlhDC sits at the top of this network
and activates FliA and flagella genes, and FliA in turn also activates flagella genes. FlhDC and
FliA differ in their relative strength of interactions at each flagella gene promoter, and their cisregulatory inputs are summed to generate transcription at each gene. This FFL is capable of
activating genes in a temporal sequence as follows: Initially, FlhDC activates genes in a
hierarchy of strengths, based on the strength of cis-regulatory inputs from FlhDC. When FliA is
also activated by FlhDC, FliA induces further activation of flagella genes. For genes with strong
activation by FlhDC, the relative effect of this later dose of FliA is small. However, for genes
with weaker FlhDC activation and strong FliA activation, the late dose of FliA makes a large
contribution to the activation dynamics, resulting in a late rise in expression. The resultant
activation order of the flagella genes corresponds to the order in which the gene products are
assembled to form the flagellar motor.
The branching pathways of the meiotic gene regulatory program correspond to such a
FFL in the following way: The upstream regulators Dazl and RA, regulate a downstream
regulator Stra8, and also the meiotic prophase genes. Stra8 in turn also regulates the meiotic
prophase genes. The output expression of each meiotic prophase gene is a summation of RA and
241
STRA8 inputs. Modulating the relative activation strengths of RA versus STRA8 can account for
the observed spectrum of genes across the three classes. If the Stra8-independent pathway acts
more directly to induce genes than the Stra8-dependent pathway, this can result in Stra8independent genes being induced earlier and Stra8-dependent genes being induced later. We
propose that the Stra8-independent pathway does enable class 1 and 2 genes to be expressed
early, in preparation for the meiotic chromosomal events triggered by Stra8. Indeed, we find that
a subset of class 1 and 2 genes are induced almost in parallel with Stra8. Class 1 and 2 genes
include almost all known cohesins and synaptonemal complex proteins, all of which structurally
associate with meiotic chromosomes and may therefore be required early, and in large quantities.
A mechanism for such a FFL would be if RA receptors (RARs) and STRA8 directly
activated transcription of the meiotic prophase genes. We might hypothesize that direct binding
of the promoter by the transcription factor RA receptor (RAR), and/or the putative transcription
factor Stra8, drives gene expression. For Class 1 genes, fully-independent of Stra8, we might
expect promoters to be bound only by RARs, but not Stra8. For Class 3 genes, fully-dependent
on Stra8, we might expect promoters to be bound only by Stra8. For Class 2 genes, which
require both Stra8-dependent and Stra8-independent pathways to be fully expressed, both RAR
and Stra8 may bind. We can determine RAR and Stra8 binding sites by chromatin
immunoprecipitation of RAR and Stra8 and sequencing (ChIP-seq). These experiments are likely
challenging given limited germ cell numbers. Prior observations point to the possibility that
RARs may directly bind promoters to promote expression in germ cells. RAR binding sites,
known as RA response elements, have been identified within the promoter of the RA-responsive
gene Stra8 (Giuili et al., 2002). RARs have also been shown to bind to the Stra8 promoter during
242
RA-induced differentiation of ES cells into motor neurons (Mahony et al., 2011), but not yet in
germ cells.
Unexpectedly, we discovered that Stra8 induces down-regulation of its own expression
after initial induction. Stra8 is also required for down-regulation of Rec8 expression. Therefore,
down-regulation of gene expression may not be a passive process, but requires active downregulation induced by Stra8.
How does Stra8 induce down-regulation of itself and Rec8 subsequent to meiotic
initiation? Stra8 may directly down-regulate itself and Rec8 at the transcriptional level. To test
this, we can begin by determining where Stra8 binds by ChIP-seq. Alternatively down-regulation
may be an indirect result of a gene expression cascade accompanying dramatic changes in cell
state or competency. We would predict then that a Stra8-dependent gene might down-regulate
Stra8. A recent paper reports that Sohihi and Sohlh2 directly bind to and down-regulate Stra8
when Sohihi and Sohlh2 are transfected into cells in culture (Desimio et al., 2015). Additionally,
it was previously noted that, in both Sohihi and Sohlh2 mutants, Stra8 appears to persist in
ovaries of newborns three to four days after when Stra8 should have been down-regulated (Choi
et al., 2008; Pangas et al., 2006). However, expression of Sohihi and Sohlh2 are not Stra8dependent. Hence, while they may suppress Stra8 expression in a newborn ovary, they are
unlikely to be responsible for the Stra8-dependent down-regulation we observe at fetal
timepoints.
Why should Stra8 be strictly regulated in a pulse? One explanation might be that
prolonged or supernumerary Stra8 pulses might in turn result in prolonged or supernumerary
attempts to initiate the meiotic chromosomal program, with disastrous consequences. To test this
243
hypothesis, we would need to determine how Stra8 is down-regulated, and examine the
consequences of preventing its down-regulation. An alternate way to test this would be to
ectopically force prolonged Stra8 expression. A conditionally activatable Stra8 transgene could
be created by inserting a lox-stop-lox cassette between a ubiquitous promoter and the Stra8
coding sequence. Stra8 could then be conditionally activated in germ cells by a tamoxifeninducible Cre driven by the Mvh promoter (John et al., 2008).
PARALLELS TO OTHER SYSTEMS
Aspects of the gene regulatory network we have identified in fetal ovarian germ cells
may be conserved to testicular germ cells. The key regulators are shared between the sexes: Dazl
and Stra8 are expressed in both ovarian and testicular germ cells entering meiosis, and RA
induction of Stra8 is required for meiotic initiation in both sexes (Anderson et al., 2008; Baltus et
al., 2006; Koubova et al., 2006; Menke et al., 2003; Ruggiu et al., 1997; Seligman and Page,
1998). In testicular germ cells a subset of meiotic genes, including Sycpl, Sycp2, Sycp3, and
Spol , have also been observed to be expressed prior to meiotic prophase. In the case of
testicular germ cells, expression is detectable as early as in mitotic spermatogonia (Keeney et al.,
1999; Wang et al., 2001). An additional parallel is that Stra8 expression is induced in preleptotene germ cells and rapidly down-regulated by leptotene (Zhou et al., 2008). However, key
differences also exist between the sexes. In testicular germ cells, Stra8 is expressed during and
plays a role at two key transitions: meiotic initiation, and spermatogonial differentiation (Endo et
al., 2015). Induction of Stra8 by RA in a testicular stage-specific manner also requires
competence on the part of the germ cell, but the competence factor is presently unknown. Dazl,
which is required for ovarian germ cells to gain competence to respond to RA, has not yet been
investigated as a competence factor in postnatal testicular germ cells. Further investigation is
244
required to determine how the ovarian germ cell meiotic gene regulatory program relates to that
in testicular germ cells, given these distinctions.
Early expression of meiotic proteins such as cohesins and synaptonemal complex
proteins, prior to initiation of the meiotic chromosomal program, may be a common feature of
germ cells in organisms in addition to mouse. In both C. elegans and D. melanogaster,both
cohesins and synaptonemal complex proteins have been observed to be expressed during
amplifying mitotic divisions prior to meiosis (Christophorou et al., 2013; Joyce et al., 2013;
Pasierbek et al., 2001). Early expression of synaptonemal complex is thought to aid chromosome
pairing, which begins during the mitotic divisions preceding meiosis.
A negative feedback loop in meiotic initiation, analogous to Stra8-induced downregulation of itself, may exist in yeast. In yeast, IME 1, a transcription factor, initiates the yeast
meiotic transcriptional program. Among its targets is IME2, which restricts expression of IMEl
(Smith and Mitchell, 1989). Absence of IME2 results in an additional round of DNA synthesis
during meiotic prophase, which is suggested to be a result of extended IME expression (Foiani
et al., 1996). These studies in yeast thus point to important functional consequences of not
properly down-regulating the signal for initiating meiosis.
PRACTICAL IMPLICA TIONS FOR ASSAYS OF MEIOTIC PROPHASE
The chromosomal program and the gene expression program have been conflated most often in
the field of in vitro derivation of germ cells. Those most steeped in studies of the meiotic
chromosomal program demand (Handel et al., 2014; Oatley and Hunt, 2012) or acknowledge
(Nicholas et al., 2009; Novak et al., 2006) the need for functional assays of the chromosomal
events. However, many in vitro studies continue to show expression of meiotic genes as evidence
245
of successful meiosis (Hubner et al., 2003; Nayernia et al., 2006; White et al., 2012). This has
resulted in many unsubstantiated claims that "meiosis" has been achieved in culture.
Our findings thus have practical implications for the field of in vitro derivation of germ
cells. By formally demonstrating that for a substantial subset of the meiotic prophase genes,
meiotic expression and function during the chromosomal program is regulated distinctly, we
highlight the insufficiency of simple gene expression for meiotic progression. Rather, regulation
of both gene expression and the chromosomal program must be coordinated for successful
meiosis.
CATA LOG OF NOVEL MEIOTIC PROPHASE GENES
Finally, our catalog of 106 genes meiotic prophase (as assayed in ovarian germ cells)
should be useful to identifying novel genes with meiotic functions. A quarter of the genes have
demonstrated roles in meiotic prophase, while the remaining three quarters are thus far
uncharacterized. The timing of gene induction, as well as how the gene is regulated, may provide
clues to potential function. For example, genes induced early and in parallel with Stra8 may
encode regulatory or chromosomal proteins required early during meiotic prophase. In-depth
studies of these genes may yield key insights into the processes of mammalian meiotic prophase.
246
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Ruggiu, M., Speed, R., Taggart, M., McKay, S.J., Kilanowski, F., Saunders, P., Dorin, J., and
Cooke, H.J. (1997). The mouse Dazla gene encodes a cytoplasmic protein essential for
gametogenesis. Nature 389, 73-77.
Seligman, J., and Page, D.C. (1998). The Dazh gene is expressed in male and female embryonic
gonads before germ cell sex differentiation. Biochem. Biophys. Res. Commun. 245, 878-882.
Smith, H.H.E., and Mitchell, A.P.A. (1989). A transcriptional cascade governs entry into meiosis
in Saccharomyces cerevisiae. Mol. Cell. Biol. 9.
Wang, P.J., McCarrey, J.R., Yang, F., and Page, D.C. (200 1). An abundance of X-linked genes
expressed in spermatogonia. Nat. Genet. 27, 422-426.
White, Y. a R., Woods, D.C., Takai, Y., Ishihara, 0., Seki, H., and Tilly, J.L. (2012). Oocyte
formation by mitotically active germ cells purified from ovaries of reproductive-age women.
Nat. Med. 18, 413-421.
Zhou,
Q., Nie,
R., Li, Y., Friel, P., Mitchell, D., Hess, R. a, Small, C., Griswold, M.D., and
Biosciences, M. (2008). Expression of stimulated by retinoic acid gene 8 (Stra8) in
spermatogenic cells induced by retinoic acid: an in vivo study in vitamin A-sufficient postnatal
murine testes. Biol. Reprod. 79, 35-42.
249
APPENDIX
251
A. RETINOIC ACID ACTIVATES TWO PATHWAYS
REQUIRED FOR MEIOSIS IN MICE
Jana Koubova, Yueh-Chiang Hu, Tanmoy Bhattacharyya, Y. Q. Shirleen Soh, Mark E. Gill,
Mary L. Goodheart, Cathryn A. Hogarth, Michael D. Griswold, David C. Page
Author contributions
Conceived and designed the experiments: JK MDG DCP. Performed the experiments: JK YCH
TB YQSS MEG MLG CAH. Analyzed the data: JK YCH TB YQSS MEG CAH MDG. Wrote
the paper: JK DCP.
Published as Koubova, J., Hu, Y.-C., Bhattacharyya, T., Soh, Y.Q.S., Gill, M.E., Goodheart,
M.L., Hogarth, C.A., Griswold, M.D., and Page, D.C. (2014). Retinoic acid activates two
pathways required for meiosis in mice. PLoS Genet. 10, e1004541.
253
I
OPEN a
ACCESS Freely available online
".'PLOS
IGENETICS
Retinoic Acid Activates Two Pathways Required for
Meiosis in Mice
Jana Koubova 1,2, Yueh-Chiang Hu1 , Tanmoy Bhattacharyyal, Y. Q. Shirleen Soh1,2 , Mark E. Gill',
2 3
,
*
Mary L. Goodheart'', Cathryn A. Hogarth , Michael D. Griswold', David C. Page'
1 Whitehead Institute, Cambridge, Massachusetts, United States of America, 2 Department
of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts,
United States of America, 3 Howard Hughes Medical Institute, Whitehead Institute, Cambridge, Massachusetts, United States of America, 4Center for Reproductive
Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington, United States of America
Citation: Koubova J, Hu Y-C, Bhattacharyya T, Soh YQS, Gill ME, et al. (2014) Retinoic Acid Activates Two Pathways Required for Meiosis in Mice. PLoS Genet 10(8):
e1004541. doi:10.1371/journal.pgen.1004541
Editor: R. Scott Hawley, Stowers Institute for Medical Research, United States of America
Received February 13, 2014; Accepted June 13, 2014; Published August 7, 2014
Copyright: 2014 Koubova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Howard Hughes Medical Institute, the National Institutes of Health, and the Whitehead Institute Abraham Siegel
Fellowship (to JK). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: dcpage@wi.mit.edu
Introduction
-
Most eukaryotes reproduce sexually, with life cycles that display
an alternation of diploid and haploid phases. The generation of
haploid cells from diploid cells is achieved through meiosis,
featuring a single round of DNA replication (meiotic S) followed
by two rounds of division (meiosis I and meiosis II).
In all sexually reproducing organisms, including fungi, plants,
and animals, cells of the germ line activate the meiotic program
when conditions are opportune and appropriate to the species'
reproductive strategies. In yeast, for example, the meiotic program
is initiated only when diploid cells are starved for nutrients and
cannot proliferate. In mammals, the meiotic program is initiated
only after the specialized cells of the germ line have migrated to
the gonad. The timing of mammalian meiotic initiation differs
dramatically between the sexes. In males, meiotic initiation does
not commence until a spermatogonial stem cell population has
been established, well after birth. In females, meiosis is initiated
shortly after the germ cells have entered the gonad, during fetal
development.
In mice, the published data are consistent with a model whereby
an extrinsic meiosis-initiating signal - retinoic acid (RA) -- induces
transcription and expression of a single meiotic factor - Stra8
which in turn governs the meiotic program [1 4]. In the ovary,
induction of Stra8 in fetal germ cells expressing Dazl, an intrinsic
factor, is required for meiotic DNA replication and the subsequent
events of meiotic prophase [2,5,6]. In fetal testes, this process is
temporarily blocked: CYP26BI, a cytochrome p45 0 enzyme,
PLOS Genetics I www.plosgenetics.org
degrades RA, preventing expression of Stra8 and thus precluding
meiotic initiation [1,3,7]. After birth, RA induces Stra8 in
testicular germ cells, leading to meiotic initiation [3,4].
Although the currently accepted model in mice postulates that
RA induction of Stra8 may be necessary and sufficient for meiotic
initiation [8], evidence suggests that other, independent factors are
also at play: germ cells in Stra8-deficient fetal ovaries express Rec8
[2], encoding a meiosis-specific component of the cohesin
complex. Rec8 is required for completion of sister chromatid
cohesion, proper synapsis, and chiasmata formation [9,10]. We
decided to examine how Rec8 expression is regulated during the
meiotic transition and whether RA plays a role in its expression.
Our investigation proceeded by first comparing the patterns and
regulation of Rec8 and Stra8 expression and then exploring
important differences with respect to their roles in driving meiotic
initiation. We discovered that RA activates meiosis in two
independent ways, both of which require Dazl expression in the
germ cells.
Results
Rec8, like Stra8, is expressed in an anterior-to-posterior
wave in fetal ovaries
We first sought to investigate how Rec8 expression is initiated in
the germ cells of fetal ovaries. If Rec8 is regulated like Stra8 and
other early meiotic markers, it should initiate expression in an
anterior-to-posterior pattern between E12.5 and E16.5 [5,11,12].
Using whole mount in situ hybridization, we discovered that Rec8
August 2014 1 Volume 10 | Issue 8 1 e1004541
(a
CMIW.@dM
rk
Retinoic Acid Activates Two Pathways Required for Meiosis in Mice
Stra8-deficient fetal ovaries express Rec8 [2], we conclude that RA
induces Rec8 in fetal ovaries independently of Stra8.
In fetal testes, RA-mediated upregulation of Rec8 requires
DazI
We next considered whether RA regulation of Rec8 expression
resembles that of Stra8 in other respects. Germ cells in wild-type
fetal testes express Stra8 when exposed to high levels of exogenous
RA [3], but germ cells in Dazl-deficient testes do not [6]. Thus,
during meiotic initiation, the germ cells must express Daz in order
to respond to RA signaling. We tested whether RA-mediated
upregulation of Rec8 expression similarly requires Dazl. We used
quantitative RT-PCR to compare Rec8 expression levels in E12.5
Dazl-deficient testes cultured for two days with or without RA
added to the medium (Figure 2B). We found that, unlike Stra8,
Rec8 is expressed, albeit at very low levels, in wild-type and Dazldeficient testes. However, similarly to Stra8, Rec8 expression was
significantly upregulated by RA treatment in wild-type but not
Dazl-deficient testes (Figure 2B). Thus RA-induced upregulation
of Rec8 in embryonic testes depends on Daz.
RA induces Rec8 expression in adult testes independently
of Stra8
RA also regulates Stra8 expression and meiotic initiation in
germ cells of postnatal testes [3,4]. We examined whether Rec8
followed a similar pattern to Stra8 here as well. Since retinoic acid
is a metabolite of vitamin A, vitamin A-deficient (VAD) mice can
be used to evaluate the effects of dramatically reduced RA
signaling on postnatal testes. We removed testes from several
vitamin A-deficient adult males and VAD males with restored RA
signaling (24 hours post RA injection) and evaluated Rec8 and
Stra8 transcripts by quantitative RT-PCR. Like Stra8, Rec8
transcription was dramatically increased 24 hours after injection of
RA (Figure 2C). These results demonstrate that RA regulates Rec8
transcription in adult testes in vivo, as in fetal ovaries; this
signaling event is shared between the sexes.
To test whether this Rec8 upregulation in postnatal testes was
Stra8-dependent, we examined Rec8 expression in Stra8-deficient, VAD testes before and after injection of RA. While the
Stra8-deficient, RA-deficient VAD testes expressed very little
Rec8, restoration of RA resulted in dramatically increased
expression of Rec8 (Figure 2D). Thus, as in fetal ovaries, RA
induces Rec8 expression in postnatal testes independently of Stra8.
expression does unfold this way from E13.0 to E16.0 (Figure IA).
These findings suggested that Rec8, like Stra8, could be a target of
RA signaling. Furthermore, since Daz expression is required for
ovarian germ cells to respond to RA signaling, perhaps, as with
Stra8 expression, expression of Rec8 requires both DAZL and
RA. We tested this new model (Figure 1B) in fetal ovaries, fetal
testes and adult testes.
RA induces Rec8 in fetal ovaries
We examined if RA signaling was required for Rec8 expression
in the germ cells of fetal ovaries. We harvested ovaries at E12.5
and cultured them for two days in the presence of the RA receptor
pan-antagonist BMS-204493 and then evaluated expression of
both Stra8 and Rec8 using quantitative RT-PCR. BMS-204493
antagonizes all three RAR isotypes [13] and prevents RA signaling
in fetal ovaries without killing the germ cells. We discovered that
BMS-204493 dramatically lowered Rec8 expression, similar to
Stra8 (Figure 2A), indicating that, in wild-type fetal ovaries, RA
signaling is required for the germ cells to express Rec8. Taking
these results together with our laboratory's previous finding that
A
9A
B
sfreB
RA+
+
DaZI\
R
901
Melo s
Rec8
Figure 1. In fetal ovaries, Rec8is expressed in an anterior-to-posterior wave. A) Rec8 expression pattern from E12.5-E16.0 in fetal gonads. B)
Proposed model: RA signaling regulates meiotic initiation in mouse germ cells in parallel pathways through Stra8 and Rec8. In all panels, testes are at
left and ovaries at right.
doi:10.1371/journal.pgen.1004541.gOOl
PLOS Genetics I www.plosgenetics.org
2
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Retinoic Acid Activates Two Pathways Required for Meiosis in Mice
EXY Control
B
A
I XY Control + RA
13.0
* XX Control
* XX Control + BMS-204493
* XY Dazi -/" XY Dazi -/- +RA
2.5
T
2.0
1.5
$ 1.5
1.0
1.0
* 0.5
0.5
0
StS8
0
Rec8
D
C
N
Rec6
N XY VAD Stra8
-
EXY VAD
EXY VAD + RA
Ann
Stra8
I XY VAD Stra8 -- +RA
3
8
,
20
6
S15
4
10
2
5
0
0
Stra8
Rec8
Rec8
Figure 2. In fetal ovaries and postnatal testes, RecS is a target of RA signaling. Quantitative RT-PCR analyses of A) Stra8 and Rec8
transcription in E12.5 ovaries cultured in control medium or with pan-RAR inhibitor added, B) Stra8 and Rec8 transcription in E14.5 control and Dazldeficient testes cultured in control medium or with RA added (Stra8 is undetectable in Daz-/-; indicated by asterisks), C) Stra8 and Rec8 expression
in RA-restored or control adult VAD testes compared to pre-injection, contralateral testes, and D) Rec8 expression in Stra8-deficient VAD adult testes,
without and with RA restoration.
doi:10.1371/journal.pgen.1004541.g002
induction of Rec8 in Cyp26bJ-deficient fetal testes is independent
of Stra8.
RA induces Rec8 expression in Cyp26b 1-deficient fetal
testes independently of Stra8
Germ cells in Cyp26bl-deficient fetal testes express Stra8 and
several other early meiotic factors at the same time as they do in
fetal ovaries because of uninhibited RA signaling [1,7,14](Figure
S1). However, whether STRA8 protein is expressed and, if so,
whether it influences other early meiotic factors has not been
determined. We developed a system of single- and double-mutant
mice with which to analyze in vivo the effects of RA signaling on
germ cells in the presence and absence of STRA8. We found that
STRA8 protein is expressed in Cyp26bl-deficient fetal testes but
not in double-mutant Cyp26bl -deficient/Stra8-deficient testes
(Figure 3A). We then assayed Rec8 expression in single- and
double-mutant fetal testes using quantitative RT-PCR. In both
cases, Rec8 expression is higher than in wild type, achieving
similar levels in single- and double-mutant samples (Figure 3B).
High expression levels in the double mutant indicate that RA
PLOS Genetics I www.plosgenetics.org
DNA replication, DNA double-strand break formation,
and upregulation of Dmcl are all dependent on STRA8
induction in Cyp26b 1-deficient fetal testes
In our studies above, we have established that RA regulates
Rec8, and that it does so in parallel to its other known target,
Stra8, in fetal ovaries, adult testes and in Cyp26bJ -deficient fetal
testes (Figure IB). Drawing on the comparative model we used to
examine Rec8 expression in fetal testes, we explored whether RA
regulates other early meiotic factors in parallel to Stra8.
We first tested whether ectopic RA signaling is sufficient to drive
DNA replication in germ cells of fetal testes, and, if so, whether
this effect is also mediated through STRA8. The thymidine analog
5-bromo-2-deoxyuridine (BrdU) can be incorporated into newly
synthesized DNA during S phase. We injected BrdU into pregnant
3
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Retinoic Acid Activates Two Pathways Required for Meiosis in Mice
A
Cvn2hl -/-
Contm
Cyp28bI -/
Stra8 -/-
STRA8
GCNA
B
3.0
2.5
:C
0
I
IT
2.0
" XX Control
" XY Control
* XY Cyp26b1 -1E XY Cyp26bI -/- Stra8 -I-
1.5
E
1.0
0
0.5
0
Rec8
Figure 3. In Cyp26b1-deficient/Stra8-deficient fetal testes, Rec8 is induced by RA signaling. A) Fluorescent immunohistochemical staining
for STRA8 protein (green) and GCNA (red) in E15.5 testes of the indicated genotypes (400 x). Scale bar: 50 gm. B) Quantitative RT-PCR analysis of Rec8
transcription in E14.5 gonads of the indicated genotypes.
doi:10.1371/journal.pgen.1004541.g003
females, dissected E16.5 fetal gonads and immunostained them
with anti-GCNA (a germ cell marker) and anti-BrdU antibodies.
In wild-type animals, testicular germ cells have arrested in GO/Gi
by E16.5. We can therefore detect ectopic germ cell proliferation
in response to STRA8 upregulation by assaying for ongoing DNA
replication in E16.5 fetal gonads. BrdU incorporation was evident
in germ cells of Cyp26bl -deficient testes (Figure 4A), consistent
with transition towards meiosis. In contrast, GCNA-positive germ
cells of double-mutant Cyp26bl-deficient/Stra8-deficient testes
were uniformly negative for BrdU at E16.5. We conclude that the
DNA replication observed in germ cells of Cyp26bl-deficient fetal
testes at E16.5 depends on and is mediated through STRA8
(Figure 4A).
We then determined if RA is sufficient in fetal testes to induce
DNA double strand breaks (DSBs), which are required for meiotic
recombination [15-19], and if the generation of these DSBs is
mediated through STRA8 induction. We assayed for the presence
of yH2AX, a phosphorylated histone variant that localizes to
DSBs, by immunostaining at E15.5, when DSBs are first observed
[20]. Cyp26bl-deficient testes displayed many germ cells positive
for yH2AX, suggesting that DSBs are induced by RA (Figure 4B).
In contrast, we rarely observed yH2AX-positive germ cells in
PLOS Genetics I www.plosgenetics.org
double-mutant Cyp26bl-deficient/Stra8-deficient testes (Figure 4B). This result suggests that the induction of DSBs in
Cyp26bl-deficient testes is driven by ectopic RA and STRA8.
Since DSBs arise not only during meiotic recombination but
also during apoptosis [21], and apoptosis has been reported in
Cyp26b1-deficient testes [7], we tested whether YH2AX-positive
germ cells observed in Cyp26bl-deficient testes represent meiotic
and not simply apoptotic events. We first generated double mutant
Cyp26bl-deficient/Bax-deficient embryos. Bax is a proapoptotic
gene, and its deletion has been shown to suppress apoptosis in
germ cells [14,22,23](Figure S2). Staining in double-mutant
Cyp26bl-deficient/Bax-deficient testes revealed many yH2AXpositive germ cells (Figure 4B), confirming that most yH2AXpositive germ cells observed in Cyp26bl -deficient testes represent
meiotic rather than apoptotic DNA DSBs. Formation of meiotic
DNA DSBs thus represents another portion of the meiotic
pathway that is STRA8-mediated.
Meiotic DSBs are processed by DMC1, an ortholog of the
bacterial strand exchange protein RecA, which commences
expression early during meiotic initiation. We compared the
effects of RA on Dmcl expression in Cyp26bl-deficient testes and
in double-mutant (Cyp26bl-deficient/Stra8-deficient) testes. The
4
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Retinoic Acid Activates Two Pathways Required for Meiosis in Mice
A
Control
Cyp26b 1IStra8-1-
Cyp26bl-l-
BrdU
GCNA
DAPI
B
Cyp26b1-Stra8-/-
Cyp26b1--
Control
Cyp26b1-IBax-f-
yH2AX
GCNA
C
1.5
10
* XX Control
EXY Control
" XY Cyp26b1 -I" XY Cyp26bI -/- Stra8 -I-
1.0
0.5
0
Dffc1
Figure 4. In Cyp26bl-4eficient testes, STRAB induces Dmcl expression, DNA replication and DNA double-strand break formation. A)
Fluorescent immunohistochemical staining for BrdU (green) and GCNA (red) in E16.5 testes of the indicated genotypes (400 x). Scale bar: 50 gm. B)
Fluorescent immunohistochemical staining for yH2AX protein (green) in E15.5 testes of the indicated genotypes (400 x). Scale bar: 50 Rm. C)
Quantitative RT-PCR analysis of Dmcl transcription in E14.5 gonads of the indicated genotypes.
doi:10.1371/journal.pgen.1004541.g004
Cyp26bl -deficient testes displayed increased levels of Dmcl, while
levels of Dmcl in double-mutant testes were similar to controls
(Figure 4C). Thus, RA is sufficient to drive Dmcl expression in
fetal testes in vivo, but this induction requires mediation by
STRA8.
In summary, it appears that RA induction of STRA8 in fetal
testes is required for all of the above-tested markers/processes
during early meiosis, with the notable exception of RA-regulated
Rec8 expression.
PLOS Genetics I www.plosgenetics.org
Stra8 and Dmcl are expressed independently of Rec8
)
To exclude the possibility that induction of Stra8 and its
downstream target Dmcl depends on Rec8 function, we examined
Stra8 and DmcJ expression in Rec8-deficient (Rec8-'81" 8
ovaries and testes [9]. As expected, we found no significant
difference in Stra8 and Dmcl expression levels between control
and Rec8-deficient E13.5 ovaries (Figure 5A). Similarly, we
detected STRA8 and DMC I proteins in both control and Rec8deficient adult testes (Figure 5B). We conclude that RA induction
5
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Retinoic Acid Activates Two Pathways Required for Meiosis in Mice
A
2.0
ISi'S
C
1.5
1.0
I
TT
T
* XX Control
* XX Rec8*
0.5
0
B
Stra8
Dmc1
Control
Stra8 -/-
Recdm
Stra8 4-
Rec8"
I
co
(0
Control
Figure 5. Stra8 and Dmcl expression in male and female germ cells is independent of Rec& A) Quantitative RT-PCR analysis of Stra8 and
Dmcl transcription in E13.5 Rec8-deficient and control ovaries. B) Colorimetric immunohistochemical staining for STRA8 and DMC1 proteins in
8
/meis adult testes. Scale bar: 10 pm.
control, Stra8-/-, and Rec
doi:10.1371/joumal.pgen.1004541.g005
How Rec8 expression is induced by RA remains elusive. Stra8's
promoter region contains two putative RA Response Elements
(RAREs), suggesting that RA could be turning on this gene
directly [25]. A chromatin immunoprecipitation-sequencing
(ChIP-Seq) study in embryonic stem cells identified RAR
binding sites in both Stra8 and Rec8 promoter regions,
suggesting that Rec8 may also be regulated by RA directly
[261. Intriguingly, in the same study, Dmcl, which is dependent
on STRA8, does not show such RAR binding sites, consistent
with Stra8 and Rec8 being regulated directly, unlike Stra8's
downstream targets.
What purpose does RA upregulation of REC8 serve? It may
ensure that Rec8 is expressed during pre-meiotic S phase so that its
product can be incorporated into the meiotic cohesin complex that
joins sister chromatids. Indeed, germ cells in Rec8-deficient mice
later show defects that can be traced to its cohesion function
incorrect synapsis topology and failure at chromosome segregation
and chiasmata formation [9,10]. Recent studies also suggest a role
for cohesins in direct regulation of gene expression by novel
mechanisms involving DNA looping [27,28]. It is presently
unknown if Rec8 is a direct transcriptional regulator. However,
of Stra8, and its downstream targets, is independent of and occurs
in parallel with RA induction of Rec8.
Discussion
PLOS Genetics I www.plosgenetics.org
-
Our findings lead us to conclude that RA plays a broad and
encompassing role in regulating and coordinating the transition from mitosis to meiosis in mouse germ cells, in both fetal
ovaries and postnatal testes. Surprisingly, RA accomplishes
this by independently inducing both Stra8 and Rec8, which
both play critical roles in the earliest stages of meiosis. The
discovery that RA induction of Stra8 in Cyp26bJ-deficient
fetal testes mediates DNA replication, DSB formation, and the
expression of recombinase Dmcl provides critical details about
the Stra8 pathway. Moreover, Stra8 induction was recently
shown to be required for SYCP3 expression in Cyp26bldeficient testes [24]. Rec8 induction is the first component of
the molecular program of meiotic initiation shown to be Stra8independent in mice. Now that Rec8's independent induction
has been established, its expression pattern and function invite
deeper investigation.
6
August 2014 1 Volume 10 1 Issue 8 1 e1004541
Retinoic Acid Activates Two Pathways Required for Meiosis in Mice
Rec8 null animals exhibit partial embryonic lethality and fail to
thrive [10], phenotypes hard to reconcile with an exclusive role in
germ cell meiotic cohesion.
The mechanisms that govern meiotic initiation have been
explored most thoroughly in yeast, and these studies offer
interesting parallels to our findings in mice. In both yeast and
mice, the decision to initiate the meiotic program is taken prior to
pre-meiotic DNA replication [2,29]. Our finding that RA
regulates Rec8 is consistent with an early role of RA in this
transition, since at least in budding yeast, REC8 associates with
chromosomes from late G 1 phase [30]. In addition, in both yeast
and mice, the decision to initiate meiosis requires an extrinsic
signal and an intrinsic competence factor [1,3,6,31,32]. In yeast,
the extrinsic signal - nutrient depletion -- activates multiple
molecular pathways in parallel, and these converge on IME1,
which is required for upregulating the expression of meiosisspecific transcripts. However, IME1 is not sufficient to induce
meiosis in yeast [33,34]. Our studies show that, analogously, RA
activates at least two pathways by regulating Stra8 and Rec8
independently. While many early meiotic processes described so
far hinge on STRA8, STRA8 may not be sufficient for meiosis in
mice. The search for additional RA targets will likely yield further
insights into the networks governing transition from mitosis to
meiosis in mammals.
Materials and Methods
Ethics statement
All experiments involving mice were approved by the Committee on Animal Care at the Massachusetts Institute of Technology.
Targeted disruption of the Cyp26bl gene
Cyp26bJ-deficient mice were generated by deleting a 2.9-kb
portion of the gene (including exons 4, 5, 6, and the coding region
of exon 7) by homologous recombination in embryonic stem (ES)
cells (Figure Sl). A Cyp26bJ/PGK-Neo targeting construct was
assembled using PCR products amplified with Advantage HF2
polymerase (Clontech) using mouse (C57BL/6J) genomic BAC
RP24-470013 (GenBank Accession AC159337) as template. The
targeting construct was linearized and electroporated into v6.5 ES
cells [35]. Cells harboring the construct were selected using
neomycin (Invitrogen). ES cell colonies were screened by PCR for
homologous integration at both the 5' and 3' arms of the
construct. Clones that tested positive by both PCR assays were
confirmed by Southern blot analysis using EcoRV and Ndel
restriction endonucleases.
Correctly targeted ES cell clones were injected into Balb/c or
C57Bl/6N blastocysts and transferred to pseudopregnant Swiss
Webster females. Germline transmission was obtained with one
clone, and the resulting homozygous embryos displayed anomalies
of limb, eye, and facial development and died at birth, as
previously described [7,36]. Embryos were genotyped by PCR,
(primer sequences available in Note Sl).
Additional mutant mouse strains
Mice carrying the DazlTM'Hg" allele [37] were generously
provided by Howard Cooke, MRC Human Genetics Unit,
Western General Hospital, Edinburgh, UK, and Dazl-deficient
mice were generated as described previously [6,38]. Stra8deficient mice were generated as described previously [2,4].
Bax-deficient mice were generated by mating Bax
1sJ'1
mice
obtained from The Jackson Laboratory (Bar Harbor, ME). Rec8deficient mice were generated by mating Rec8'ei8 + mice [9],
PLOS Genetics I www.plosgenetics.org
which were generously provided by John Schimenti, Cornell
University, Ithaca, New York.
Mouse embryo collection and in situ hybridization
Mouse embryos used in whole mount in situ hybridizations and
gonad cultures were obtained from matings between CDI random
bred mice (Charles River Labs). Noon of the day when vaginal
plug was recorded was considered EO.5. Whole mount in situ
hybridizations with the Stra8 probe were performed as previously
described [3,39]. Digoxigenin riboprobe for Rec8 was generated
by amplifying cDNA fragments by RT-PCR from Rec8
(NM_020002.2: bases 274 865), and inserting them into TA
cloning vector pCR4-TOPO (Invitrogen). Plasmid was linearized
with Spel or Notl and transcribed with T7 or T3 respectively to
make the antisense and sense probes.
RT-PCR
For experiments involving Rec8-deficient mice, total RNAs
were prepared from gonads using the RNeasy plus Micro RNA
isolation kit (QIAGEN), and reverse transcription was carried out
using the high-capacity cDNA reverse transcription kit (Applied
Biosystems). For all other experiments, total RNAs were prepared
using TRIzol (Invitrogen) extraction followed by DNase (Ambion)
treatment, and reverse transcription was carried out using the
RETROscript reverse transcription kit (Life Technologies). The
resulting total cDNAs were analyzed quantitatively using SYBR
Green PCR reagents (Applied Biosystems) with primers for Dmcl,
Rec8, Stra8, or Daz. Expression profiles were tested in triplicate
on at least two litters of embryos on an ABI 7500 instrument
(Applied Biosystems). Data were analyzed using the comparative
Ct (AACt) method and one-tail, unpaired student T test
(significance cutoff p<0.01). Results were normalized to Rps2
(VAD experiments on adult testis), Daz (Rec8-mutant experiments on embryonic ovary), and Hprt (all other experiments).
Primers were selected from PrimerBank [40] (Note Sl).
Immunofluorescent studies of tissue sections
Fetal gonads were dissected in phosphate buffered saline (PBS),
fixed in 4% paraformaldehyde overnight at 4'C, embedded in
paraffin and sectioned. Slides were incubated with anti-GCNA
IgM (courtesy of G. Enders, undiluted supernatant), anti-STRA8
(Abcam. 1:100), and anti-phosphoH2A.X (Upstate Cell Signaling
Solutions, 1:250 dilution). Colorimetric staining was performed
using ABC reagents (Vector Laboratories) and developed with
DAB peroxidase substrate (Vector Laboratories).
Sections were mounted in Vectashield Medium with DAPI
(Vector Laboratories), and fluorescent staining was obtained using
Texas-Red or FITC-conjugated secondary antibodies (Jackson
Immunoresearch Laboratories, 1:500 dilution).
Immunohistochemical studies of tissue sections
Adult testes were fixed in Bouin's solution overnight at 4'C,
washed with PBS and 70% ethanol, embedded in paraffin, and
sectioned at 5 gm thickness. Slides were matured overnight, dewaxed, rehydrated, and heated in 10 mM sodium citrate buffer
(pH 6.0) for antigen retrieval. Sections were incubated in 3%
hydrogen peroxide for 5 min and blocked in 2.5% normal horse
serum (Vector Laboratories) for 80 minutes at room temperature.
Later, slides were incubated overnight with anti-STRA8 (Abcam,
1:500) or anti-DMCI (Santa Cruz Biotechnology, 1:50 dilution).
The following day, slides were washed three times in PBS and
incubated with anti-rabbit ImmPRESS peroxidase reagent (Vector
Laboratories) for 30 minutes. The slides were later developed
August 2014 1 Volume 10 1 Issue 8 1 e1004541
Retinoic Acid Activates Two Pathways Required for Meiosis in Mice
using a DAB substrate kit (Vector Laboratories) for 1 minute. The
slides were counterstained with Mayer's hematoxylin for 5 minutes and washed in running water, dehydrated, and mounted with
Permount (Fisher Scientific).
spermatogenesis (as judged by pre-injection testicular histology)
were injected with 100 gl of 7.5 mg/ml all-trans retinoic acid
(Sigma) in 10% ethanol/90%sesame oil solution. The animals'
remaining testes were harvested 24 h after injection. In contrast,
both testes were harvested from two Stra8-deficient VAD animals
at the same time (one was analyzed histologically to confirm
depletion) and compared to testes harvested from two RA-restored
Stra8-deficient animals. Quantitative RT-PCR analysis was
performed, in triplicate, using Stra8 and Rec8 primers, and
Rps2 was used as a normalization control (primer sequences in
Note Sl).
TUNEL analysis
Apoptotic cells were detected in paraffin sections of fetal testes
using the Fluorescein in situ Cell Death Detection Kit (Roche
Applied Science) and mounted in Vectashield Medium with DAPI
(Vector Laboratories).
BrdU incorporation
Supporting Information
Pregnant females were injected with 5-bromo-2-deoxyuridine
(BrdU) solution (50 mg/kg) at 18.5 days post coitum. Six hours
later, fetal gonads were dissected. Gonads were then fixed in 4%
paraformaldehyde overnight at 4'C, embedded in paraffin, and
sectioned. Prior to antibody application, sections were treated with
denaturing reagent (3.5N HCl) for 2 min. Incorporated BrdU was
detected using anti-BrdU (Accurate Chemical & Scientific Corp.,
1:500 dilution) in anti-GCNA IgM supernatant.
Figure S1 Targeted disruption of the Cyp26bl locus on mouse
chromosome 6. A) Homologous recombination removes exons 4,
5, 6 and the coding portion of exon 7, and replaces them with a
loxP-PGK-Neo-loxP selection cassette. B) Correctly targeted ES
cell clones were confirmed by Southern blot analysis (E, EcoRV;
N, NdeI). Positions of 5' (red) and 3' (green) probes are shown in
embryos exhibit defects in limb
part A. C) E14.5 Cyp26bI -/and facial development as previously reported (Yashiro et al.,
2004). D) Whole-mount in situ hybridization with Stra8 probe
reveals staining in Cyp26bI - / - testes. E) Quantitative real-time
PCR shows increase in Stra8 expression levels in Cyp26bl - /testes.
(PDF)
Mouse fetal gonad culture
Pregnant female mice were sacrificed by cervical dislocation and
embryos were removed into PBS solution. After determining tail
somite number, fetal ovaries and mesonephroi were dissected. One
gonad from each embryo was then placed in a 35 W droplet of
culture media (DME +10% FBS) supplemented with either 5 gM
pan-RAR inhibitor BMS-204493 (Bristol-Myers Squibb) or all
trans RA (Sigma) dissolved in ethanol in a Petri plate. Control
media contained vehicle (ethanol) alone. Petri plates were then
inverted and placed within larger plates containing water and
incubated at 37'C with 5% CO 2. Media was replaced after
24 hours. After 48 hours, tissue was removed from media,
mesonephroi were dissected off and ovaries were placed individually into TRIzol reagent (Invitrogen). Samples were then
processed for quantitative RT-PCR as described above.
Figure S2 Immunohistochemical staining for MVH protein
(red) and TUNEL staining (green) in E15.5 control ovary and
testis, Cyp26bl-deficient testis, and double-mutant (Cyp26bldeficient, Bax-deficient) testis.
(PDF)
Note S1 Primer sequences for genotyping Cyp26bI-deficient
mice and RT-PCR analyses.
(DOCX)
Analysis of Rec8 expression in vitamin-A-deficient testes
Acknowledgments
Adult female mice (129/SvJ) were fed a Vitamin-A-Deficient
(VAD) diet (Harlan Teklad, Indianapolis) for at least 2 weeks
before mating and throughout pregnancy. Their male offspring
were fed a VAD diet for 13-14 weeks. In the first experiment with
wild-type animals, one testis was removed from each animal and
cut into two pieces; one fixed in Bouin's solution for histological
assessment of spermatogenesis and the other placed in TRIzol
(Invitrogen) for RNA extraction to serve as a pre-injection control
in RT-PCR analysis. Incisions were sutured and the animals
recovered for 24 h. Three animals with similarly deficient
We thank Howard Cooke for DazITMHg8/+ mice, John Schimenti for
Rec8meiS/ mice, Ericka Anderson for cDNA from VAD Stra8-deficient
testes, Bristol-Myers Squibb for RAR antagonist BMS-204493, George
Enders for anti-GCNA antisera, and Greg Dokshin, Jennifer Hughes, and
Katherine Romer for critical reading of the manuscript.
Author Contributions
Conceived and designed the experiments: JK MDG DCP. Performed the
experiments: JK YCH TB YQSS MEG MLG CAH. Analyzed the data:
JK YCH TB YQSS MEG CAH MDG. Wrote the paper: JK DCP.
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9
August 2014 | Volume 10 1 Issue 8 1 e1004541
B. LICENSING OF PRIMORDIAL GERM CELLS TO GAMETOGENESIS IS
DEPENDENT UPON THE GENITAL RIDGE
Yueh-Chiang Hu, Peter K. Nicholls, Y.
Q.
Shirleen Soh, Joseph R. Daniele, Jan Philipp Junker,
Alexander van Oudenaarden, David C. Page
Author contributions
Conceived and designed the experiments: YCH PKN YQSS JPJ AvO DCP. Performed the ex-
periments: YCH PKN YQSS JRD JPJ. Analyzed the data: YCH PKN YQSS JRD JPJ. Wrote the
paper: YCH PKN DCP.
Published as Hu, Y.-C., Nicholls, P.K., Soh, Y.Q.S., Daniele, J.R., Junker, J.P., van
Oudenaarden, A., and Page, D.C. (2015). Licensing of Primordial Germ Cells for Gametogenesis
Depends on Genital Ridge Signaling. PLoS Genet. 11, e1005019.
265
.PLOS
GENETICS
RESEARCH ARTICLE
Licensing of Primordial Germ Cells for
Gametogenesis Depends on Genital Ridge
Signaling
Soh1,2 , Joseph R. Daniele, 2 , Jan
Yueh-Chiang Hul1n, Peter K. Nicholls', Y. Q. Shirleen
3
23
C. Page,2,s*
David
,4,
Oudenaarden2,
Philipp Junker ,4, Alexander van
I
1 Whitehead Institute, Cambridge, Massachusetts, United States of America, 2 Department of Biology,
Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 3 Department
of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America,
4 Hubrecht Institute-KNAW (Royal Netherlands Academy of Arts and Sciences) and University Medical
Center Utrecht, Utrecht, Netherlands, 5 Howard Hughes Medical Institute, Whitehead Institute, Cambridge,
Massachusetts, United States of America
na
Current Address: Division of Developmental Biology, Cincinnati Children's Hospital Medical Center,
Cincinnati, Ohio, United States of America
* dcpage@wi.mit.edu
& OPEN ACCESS
CItation: Hu Y-C, Nicholls PK, Soh YQS, Daniele JR,
Junker JP, van Oudenaarden A, et al. (2015)
Licensing of Primordial Germ Cells for
Gametogenesis Depends on Genital Ridge Signaling.
PLoS Genet 11(3): e1005019. doi:10.1371/joumal.
pgen.1005019
Editor: R. Scott Hawley, Stowers Institute for Medical
Research, UNITED STATES
Received: August 26, 2014
Accepted: January 22, 2015
Published: March 4, 2015
Copyright: @2015 Hu et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Abstract
In mouse embryos at mid-gestation, primordial germ cells (PGCs) undergo licensing to become gametogenesis-competent cells (GCCs), gaining the capacity for meiotic initiation
and sexual differentiation. GCCs then initiate either oogenesis or spermatogenesis in response to gonadal cues. Germ cell licensing has been considered to be a cell-autonomous
and gonad-independent event, based on observations that some PGCs, having migrated
not to the gonad but to the adrenal gland, nonetheless enter meiosis in a time frame parallel
to ovarian germ cells - and do so regardless of the sex of the embryo. Here we test the hypothesis that germ cell licensing is cell-autonomous by examining the fate of PGCs in Gata4
conditional mutant (Gata4 cKO) mouse embryos. Gata4, which is expressed only in somatic
cells, is known to be required for genital ridge initiation. PGCs in Gata4 cKO mutants migrated to the area where the genital ridge, the precursor of the gonad, would ordinarily be
formed. However, these germ cells did not undergo licensing and instead retained characteristics of PGCs. Our results indicate that licensing is not purely cell-autonomous but is induced by the somatic genital ridge.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was supported by the Howard
Hughes Medical Institute, where DCP is an
Investigator. PKN holds an Australian National Health
and Medical Research Council CJ Martin Fellowship
(#1053776). The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
Author Summary
During embryonic development, stem cell-like primordial germ cells travel across the developing embryo to the genital ridge, which gives rise to the gonad. Around the time of
their arrival, the primordial germ cells gain the capacity to undertake sexual specialization
and meiosis-a process called germ cell licensing. Based on the observation that meiosis
and sexual differentiation can occur when primordial germ cells stray into the area of the
adrenal gland, the primordial germ cell has been thought to be responsible for its own
371/joumal.pgen.1005019
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Germ Cell Licensing Is Induced by Genital Ridge
licensing. We tested this notion by examining the licensing process in mutant mouse embryos that did not form a genital ridge. We discovered that in the absence of the genital
ridge, primordial germ cells migrate across the developing embryo properly, but instead of
undergoing licensing, these cells retain their primordial germ cell characteristics. We conclude that licensing of embryonic primordial germ cells for gametogenesis is dependent on
signaling from the genital ridge.
Introduction
In mammals, both the testis and ovary derive from a common precursor structure, the bipotential gonad [1]. The development of the bipotential gonad involves two simultaneously occurring processes. The coelomic epithelium on the ventromedial surface of the mesonephros
transforms from a monolayer into a thickened, multilayer epithelial structure, the genital ridge.
Meanwhile, primordial germ cells (PGCs) that have migrated from the base of the allantois
start arriving at the genital ridge, as early as the monolayer stage, and multiply as the genital
ridge thickens. The formation of the bipotential gonad in mouse embryos begins at embryonic
(E) day 10.0 and continues until E11.5-E12.0, when sexual differentiation takes place [2-4].
Migratory PGCs maintain a genomic program associated with pluripotency [5,6]. They express core pluripotency genes (Oct4, Nanog, and Sox2) and are able to form teratomas following their injection into postnatal mouse testes [7]. Around the time of their arrival at the
genital ridge, PGCs undergo a global change in gene expression [8-10]. Specifically, the PGCs
turn on a set of genes that enable them to undergo sexual differentiation and gametogenesis,
and to switch off their pluripotency program. Following this transition, germ cells are referred
to as gametogenesis-competent cells (GCCs), and are poised to initiate meiosis as well as male
or female differentiation [11-13]. Upon the development of the genital ridge into either a testis
or an ovary (at -E12.5 in mouse embryos), GCCs respond to cues from the somatic environment and enter either the spermatogenic or oogenic pathway accordingly. The transition from
PGC to GCC is referred to as germ cell licensing [11], and it represents a critical transformation
of germ cells to a sexually competent state.
One of the genes upregulated in germ cells at the time of licensing is Dazi, which encodes an
evolutionarily conserved and germ-cell-specific RNA-binding protein [14]. In mouse embryos
of C57BL/6 genetic background, germ cell licensing is dependent on Dazl [11,15]. In Dazl-null
embryos, germ cells retain characteristics of PGCs and fail to embark upon the pathways to oogenesis or spermatogenesis in the fetal ovary or testis, respectively. However, what triggers
Dazl expression and germ cell licensing remains unknown.
One hypothesis, based on observational studies, states that licensing is triggered in a cell-autonomous and gonad-independent manner. As PGCs migrate to the genital ridge, a fraction of them are
left in places along the migratory path, such as in the allantois, tail, midline, spinal cord, and adrenal
gland [16,17]. While most of these ectopic PGCs die, those migrating to the adrenal gland survive
until -3 weeks after birth [16,18-20]. Upadhyay and Zamboni [19] observed that these adrenal
germ cells, regardless of the sex of the fetus, enter meiosis according to the schedule of normal ovarian germ cell development. Based on these findings, the authors hypothesized that PGCs transition
into meiotic germ cells (oocytes) in a gonad-independent, and therefore cell-autonomous, manner.
This hypothesis was further supported by several in vitro studies [3,21-23, showing, for instance,
that PGCs isolated from E10.5 mouse embryos of both sexes continue to develop in vitro and initiate meiosis at approximately the same time as meiotic entry occurs in vivo [13,22,23].
PLOS Genetics | DOI: 10. 1371/fjoumal.pgen. 1005019 March 4, 2015
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Germ Cell Licensing IsInduced by Genital Ridge
Previous studies from our lab and others led us to question this hypothesis and suggest an
alternative: PGCs undergo germ cell licensing in response to external signals, upon migration
to the genital ridge. The authors who proposed the cell-autonomous hypothesis considered
E10.5 PGCs to be pre-gonadal germ cells [22,23]. However, we recently showed that the marker
of genital ridge formation, GATA4, is expressed as early as E10.0 [2]. It is plausible that the
E10.5 PGCs used in the in vitro studies had already been exposed to gonadal factors. In addition, the claim that the PGCs in the adrenal gland transition to meiotic germ cells without exposure to the genital ridge belies the fact that the adrenal anlagen and genital ridge derive from
a common precursor, called the adrenogonadal primordium. These two organs are not segregated completely until -El 1.5 [24,25]. Adrenal PGCs would therefore be exposed to the genital
ridge, or its equivalent, during a short interval in their development. These findings raise
doubts about whether the transition of PGCs to meiosis-competent cells is gonad-independent,
or induced by factors shared by the developing gonad and adrenal gland.
Germ cell licensing precedes meiotic entry [11,15]. Since the occurrence of licensing coincides
with the arrival of PGCs at the genital ridge, we suspected that the genital ridge provides extrinsic
signals required for inducing germ cell licensing. Initiation of genital ridge formation depends on
the transcription factor GATA4, which is expressed in the somatic compartment, but not in germ
cells [2]. We therefore utilized Gata4 conditional knockout (cKO) embryos, which lack the genital
ridge, to test the hypothesis of genital ridge-dependent licensing. If true, we would expect that in
the absence of the genital ridge, PGCs would fail to undergo licensing and subsequent meiotic
entry. The result of this study would provide fundamental insight into how germ cells switch off
their pluripotency program and acquire competence for meiosis and sexual differentiation.
Results
Anterior-to-posterior expression of the germ cell licensing marker Daz
The genital ridge develops in an anterior-to-posterior (A-P) direction starting at E10.0 [1,2], as
PGCs are entering the region. Dazl is expressed in germ cells during licensing for gametogenesis
[8,9,11,14,15]. If the genital ridge regulates germ cell licensing, we would expect to find a similar
A-P induction of licensing, along with Dazlexpression. To test this prediction, we quantified Dazl
transcript levels in individual germ cells along the A-P axis of the genital ridge using singlemolecule fluorescence in situ hybridization (smFISH) [26]. We first confirmed that Dazl expression was below the detectable level in migratory PGCs at E9.5, as expected (SlA Fig). When examining post-migratory germ cells at El 1.5, we detected a gradient of Dazl transcript levels along the
genital ridge; expression is highest in the anterior portion and decreases in an A-P direction
(Figs. 1 and S1B). As a comparison, we also quantified the transcript level of Oct4 (Pou5fi), a pluripotency gene that is expressed in both migratory PGCs and post-migratory germ cells. We did
not observe an A-P gradient of Oct4 transcript levels in germ cells. Instead, germ cell Oct4 transcript levels were relatively consistent along the length of the genital ridge. Therefore, the A-P expression pattern of Dazl supports the possibility that the genital ridge induces germ cell licensing.
Genital ridge formation is required for germ cell licensing
We next asked whether germ cell licensing requires the presence of the genital ridge. We examined DAZL expression in transverse sections of E11.5 embryos in which we had prevented genital ridge formation by ubiquitous deletion of Gata4 (Gata4 cKO) through tamoxifen injection
at E8.75 (Fig. 2A, Gata4XI"A; CAG-CreER [2]). Sections were immunostained for SSEA1,
DAZL, and GATA4 expression. SSEA1 was used to identify all germ cells at this time point, as
it identifies both migratory PGCs and post-migratory germ cells. In sections from littermate
controls (Gata4"fl"), we found that the majority of germ cells that had colonized the genital
PLOS Genetics IDOI: 10.1371/fjournal.pgen. 1005019 March 4, 2015
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Germ Cell Licensing IsInduced by Genital Ridge
GENETICS
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embryos (n = 3), as measured by smFISH. (B) Daz/ transcript density was normalized against Oct4 transcript
density for each individual cell to obtain relative DazI:Oct4 transcript density per cell. Lines in plots represent
average transcript density of cells (A) or ratio of densities (B) at a particular A-P position.
doi:1O.371jourma.pgen.1005019.g01
ridge expressed DAZL, consistent with our smFISH data (Fig. 1). In contrast, germ cells in
Gata4 cKO embryos migrated to the ventromedial side of the mesonephros (the location of the
genital ridge in wild type animals) but failed to initiate DAZL expression (95% vs. 4%,
PLOS Genetics I DOI: 10. 1371/fjoumal.pgen. 1005019
March 4, 2015
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ventral
dorsal
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B
C
U Control U Gata4 cKO
(CAG-CreER)
100
60
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80
_
40
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20
0
E
D
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Fig 2. Germ cells in Gata4
cKO embryos do not express DAZL or MVH. (A) Schematic illustration of transverse section through embryo trunk that
contains urogenital ridge. Red boxes indicate areas imaged, shown in B and D. a, adrenal gland; ce, coelomic epithelium; d, dorsal aorta; gr, genital ridge; m,
mesentery. (B and D) Immunofluorescent staining for SSEA1, DAZL, MVH, and GATA4 protein in transverse sections of control (Gata4"'*x) and Gata4
ubiquitous cKO (CAG-CreER) embryos on a mixed genetic background. Representative germ cells are indicated by white arrows. Germ cells mis-migrating
to the adrenal gland (yellow arrows) also express DAZL and MVH. Scale bars: 50 pm. (C and E) Percentage of germ cells that are positive for DAZL or MVH
expression in control and Gata4 cKO (CAG-CreER) embryos. SSEA1 marks all germ cells at this age. Plotted here are means standard deviation from
biological replicates (n > 3 for each genotype). *, P < 0.05 (two-tailed Student's t-test).
doi:10.1371/joumal.pgen.1005019.g002
respectively; Fig. 2B and C). These results indicate that genital ridge formation is essential for
DAZL expression in PGCs, but not for their migration.
Like Dazl, Mouse vasa homolog (Mvh, also known as Ddx4) is also expressed in germ cells
around the time of their arrival at the genital ridge [27]. In Dazl-null embryos of the C57BL/6 genetic background, germ cells do not undergo licensing, but MVH is still expressed in these cells,
suggesting that MVH expression is independent of Dazl [11]. We then examined MVH expression in the germ cells of Gata4cKO embryos. In transverse sections from littermate controls, the
majority of germ cells that had colonized the genital ridge expressed MVH, whereas germ cells
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in Gata4 cKO embryos failed to express MVH (89% vs. 13%; Fig. 2D and E). These results indicate that, like DAZL, MVH expression is also dependent upon genital ridge formation.
To establish that the failure of germ cell licensing was due to the loss of Gata4in somatic tissues, we used an additional Cre line, Osri-CreER,that is expressed in genital ridge (somatic)
precursor cells but not in the germ line. As with the ubiquitous Gata4 cKO described above,
Osri-driven loss of Gata4 in the soma resulted in PGCs migrating to the ventromedial side of
the mesonephros, without initiating either DAZL or MVH expression (S2 Fig). These results
confirm that licensing of germ cells depends upon Gata4function in somatic cells.
Consistent with previous observations that mis-migrated adrenal germ cells can initiate meiosis, we noticed that germ cells that had migrated to the adrenal gland expressed DAZL and
MVH at E 11.5 (Fig. 2B and D). These findings suggest that germ cells in the adrenal gland undergo licensing as they do in the genital ridge, which makes germ cells there capable of entering
meiosis [19,20]. Licensing of the adrenal germ cells is likely to be dependent on factors shared
between the genital ridge and the adrenal, as both organs derive from the same primordium.
To confirm that genital ridge formation is sufficient to induce the germ cell licensing factor
DAZL, we examined embryos lacking either Wt1 or Osri, both of which are required for development and maintenance of the embryonic gonad [28-30]. We found that, in the absence of either Wt1 or Osri, GATA4 is expressed in the coelomic epithelium, and the genital ridge is
initially formed, although its growth is severely retarded and degeneration ensues (S3 and S4
Figs). In both Wtl KO and Osri KO embryos, we observed DAZL expression in germ cells that
migrated to the GATA4-expressing cells of the genital ridge, indicating that the association of
PGCs with the nascent genital ridge is sufficient for licensing (S3 and S4 Figs). Taken together,
our findings demonstrate that genital ridge formation is both necessary and sufficient to induce
expression of DAZL in newly arrived PGCs.
Germ cells in cultured urogenital ridges from Gata4 cKO embryos retain
PGC markers and fail to express GCC markers
Having found that germ cells in Gata4cKO embryos do not express DAZL and MVH, we wondered if these cells fail to transition into GCCs and instead retain characteristics of PGCs, such
as continued expression of the pluripotency gene program and inability to sexually differentiate. Because Gata4 cKO embryos die between El1.5-E12.0, we investigated subsequent germ
cell development in urogenital ridge (UGR) cultures. The UGR was dissected from El 1.5 control and Gata4 ubiquitous cKO embryos, cultured for 3 days, and subjected to paraffin sectioning and immunofluorescent staining. In control UGR cultures (n = 5; 3 XX and 2 XY), all germ
cells that we examined expressed markers seen in GCCs and sexually differentiating germ cells,
including DAZL, MVH, GCNA, and MILI (Fig. 3). In addition, these germ cells switched off
pluripotency markers, including NANOG, OCT4, SOX2, and SSEA1. In contrast, all germ cells
in Gata4cKO UGR cultures (n = 4; 2 XX and 2 XY) retained an expression program similar to
that of PGCs, with NANOG, OCT4, SOX2, and SSEA1 being expressed (Fig. 3). Conversely,
markers of GCCs and sexually differentiating germ cells, including DAZL, MVH, GCNA and
MILI, were not expressed in cultured UGRs from Gata4 cKO embryos.
To confirm that the conversion of PGCs to GCCs depends upon Gata4function in somatic tissues and not the germline, we studied similar UGR cultures generated using the
soma-specific Cre line, Wt]-CreER; Osri-CreER. After 3 days of UGR culture, we observed
that germ cells in controls expressed both DAZL and MVH, whereas germ cells in Gata4
soma-specific cKO tissues expressed neither marker, and instead retained expression of the
pluripotency marker SSEA1 (S5 Fig). These results indicate that the genital ridge is indispensable for licensing of PGCs to GCCs.
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0
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Fig 3. Germ cells In Gata4 cKO embryos retain characteristics of PGCs. Immunofluorescent staining for PGC and GCC marker proteins in transverse
sections of control (Gata4*'1**) and Gata4 cKO (CAG-CreER) urogenital ridge cultures on a mixed genetic background. Arrows indicate representative germ
cells. Scale bars: 50 pm.
doi:10.1371fjoumal.pgen.1005019.g003
Germ cells in Gata4 cKO embryos fail to enter meiosis
A key functional characteristic that distinguishes GCCs from PGCs is their ability to enter meiosis. To investigate whether germ cells from Gata4 cKO embryos are able to enter meiosis, we
performed immunostaining on sections of cultured UGRs for SYCP3 and SSEA1 expression.
We found that in control UGR cultures (n = 5 cultures), germ cells that expressed GCC markers (Fig. 3) showed SYCP3 assembly onto chromosomes-a characteristic of prophase of meiosis I-while the pluripotency marker SSEA1 was not detectable (Fig. 4). In contrast, germ cells
in Gata4 cKO UGR cultures (n = 4 cultures) expressed neither GCC markers (Fig. 3) nor
SYCP3 (Fig. 4). Instead, they continued to express SSEA1, a marker of PGC identity. We conclude that germ cells in Gata4 cKO UGR cultures were not competent to enter meiosis, functionally validating the earlier evidence that these cells had not become GCCs.
Discussion
We have identified a previously unrecognized role of the genital ridge in germ cell development, prior to sex determination. We show that germ cell licensing-the transition of PGCs to
Control
Gata4 cKO
A-
Fig 4. Germ cells in Gata4 cKO embryos do not enter melosis. Immunofluorescent staining for SSEA1,
SYCP3, and GATA4 proteins in transverse sections of control (Gata4+/fox) and Gata4 cKO (CAG-CreER)
urogenital ridge cultures on a mixed genetic background. Inset shows higher magnification of cells. Scale
bars: 50 pm.
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GCCs-is induced by cues from the genital ridge (Fig. 5). If the genital ridge is not formed due
to loss of the somatic transcription factor Gata4, germline cells remain at the PGC stage, failing
to become GCCs and lacking the competence to undergo sexual differentiation and initiate
meiosis. Thus, we provide genetic evidence that the transition of PGCs into meiotic germ cells
is not a purely cell-autonomous process and is instead dependent on the somatic gonad.
The genital ridge-dependent induction of germ cell licensing is compatible with the observation that ectopic germ cells in the adrenal gland also enter meiosis [19]. Given that the genital
ridge and adrenal gland share a common precursor, the adrenogonadal primordium [24,25], it
is plausible that these two organs produce the same factors required to induce licensing and
meiosis. Our finding also helps explain previous studies in which PGCs isolated from E10.5
embryos were shown to initiate meiosis in culture [13,22,23]. Because genital ridge formation
is initiated as early as E10.0 [2], it is likely that a portion of E10.5 germ cells had already been
exposed to the genital ridge environment. These licensed germ cells, therefore, had acquired
meiotic competence by E10.5, before being cultured.
We have previously shown that Dazi is required for licensing of PGCs to GCCs in mouse
embryos from an inbred C57BL/6 genetic background [11]. The present study extends our understanding of germ cell licensing for gametogenesis. We now appreciate that germ cell licensing is induced by the soma; indeed, the soma induces expression in germ cells of at least two
key factors, DAZL and MVH, independently. Our findings also indicate that germ cell licensing
depends upon the genital ridge in embryos of either mixed (Figs 2-4) or C57BL/6 genetic background (82 and S5 Figs). The molecular mechanism by which the genital ridge regulates germ
cell licensing requires further exploration.
We propose that the genital ridge-a somatic structure-induces germ cell licensing. However, we cannot formally exclude the possibility that the failure of licensing in germ cells of
Gata4cKO embryos is a secondary effect of other activities at the genital ridge. The genital
ridge has previously been shown to regulate germ cell motility, as PGCs become nonmotile
after arriving at the genital ridge [18,31]. The genital ridge also produces factors that stimulate
PGC proliferation [28,32,33]. Although germ cells colonize the coelomic epithelium in similar
numbers in control and Gata4cKO embryos at E10.3 [2], Gata4cKO embryos display a reduced number of germ cells at E11.5, likely due to the absence of genital ridge-derived factors
that stimulate proliferation. It is unlikely that this reduction in germ cell numbers is responsible
for the licensing defects observed in the Gata4 cKO, given that other mutants with reduced
germ cell number display qualitatively normal germ cell development in males [34,35]. Similarly, we cannot yet exclude the possibility that systemic defects (e.g., in the gut, liver, or heart)
contribute to the failure of germ cell licensing observed in the Gata4cKO. The generation of a
genital ridge-specific Cre mouse will be required to rule this out.
We can now reconstruct the series of events in soma and germline (Fig. 5) that result in postmigratory PGCs initiating meiosis in the female fetal gonad, in an anterior-to-posterior (A-P)
wave. In the soma, Gata4 expression initiates the transformation of the coelomic epithelium (on
the ventromedial surface of the mesonephros) into the genital ridge, in an anterior-to-posterior
(A-P) wave [2]. Concurrently, PGCs migrate to the developing genital ridge as early as the
monolayer stage. Based on our findings-that Dazl is expressed in germ cells in an A-P progression (Fig. 1), and that this expression is dependent upon the genital ridge (Fig. 2)-we propose
that the progressive A-P development of the genital ridge induces Dazi expression and licensing
in a similar A-P wave. Upon expression of DAZL, GCCs acquire the competence to interpret
retinoic acid as a meiosis-inducing signal [36,37], and then express Stra8, the gene required for
meiotic initiation [15,38], along with Rec8 [39] and Dmcl-all in an A-P manner [40,41].
Licensing for gametogenesis constitutes a major transition during early germ cell development,
allowing PGCs to acquire competence for sexual differentiation and gametogenesis. Our results
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Fig 5. A proposed model for somatic Induction of germ cell differentiation, in three steps. 1) Germ cell specification induced by signals, such as BMP4,
from extraembryonic ectoderm [53,54]; 2) germ cell licensing induced by the genital ridge, which arises from the coelomic epithelium following Gata4
expression; and 3) GCCs embark on either spermatogenesis or oogenesis in response to cues from somatic testis or ovary, respectively [12,13].
doi:10.1371/joumal.pgen.1005019.g005
indicate that the genital ridge triggers germ cell licensing. Thus, PGCs undergo licensing upon
their arrival at the genital ridge, ensuring that gametogenesis occurs at the correct time and place.
Materials and Methods
Mice
All experiments involving mice were approved by the Committee on Animal Care at the Massachusetts Institute of Technology. The following mice were obtained from Jackson Laboratory
(Stock Numbers 008194, 004682, 009061, 009387, 010912 and 002332): Gata4flo"' [42]; CAGCreER (ubiquitously expressed) [43]; Osr1eGFP-CreERt2/+(somatically expressed) [44]; Osr1"'Jan/+ [30]; Wt1creERt2/+ (somatically expressed) [45]; and Wttmulae/+[2 9 ], respectively. In some
cases (as described in the text), these mice were backcrossed to the C57BL/6 strain (Taconic
Farms) for at least 10 generations. Gata4cKO embryos were generated by mating Gata4flOf
females with Gata41'6males carrying the indicated CreER. Where applicable, Gata4/flo" littermate embryos were used as controls. Tamoxifen (Sigma) was dissolved in corn oil (Sigma) at a
concentration of 30 mg/ml. Dams were injected intraperitoneally at 8.75 days postcoitum with
a single shot of tamoxifen (4 mg/40 g body weight) to induce excision of the floxed Gata4 allele.
The injection scheme was optimized for maximum embryo survival and Gata4 excision efficiency [2]. Embryos were collected at the indicated time and immediately genotyped by PCR
according to protocols from the Jackson Laboratory website.
Single-molecule fluorescence in situ hybridization
Whole E9.5 embryos or urogenital ridges dissected from El 1.5 embryos on C57BL/6 genetic
background were fixed 2 hours at 4*C in 4% paraformaldehyde, equilibrated in 30% sucrose/
4% paraformaldehyde in PBS overnight, and frozen and stored in OCT (Tissue-Tek) at -80 0C
before cryosectioning (8 tm thick). Probes were synthesized and hybridization performed as
previously described [26]. Probes to Dazl and Oct4 transcripts were conjugated to Cy5, A594,
or TMR. AlexaFluor 488-conjugated anti-SSEA1 (560271, BD Biosciences) was added to the
hybridization to label germ cells. Images were taken with a Nikon Ti-E inverted fluorescence
microscope equipped with a 100x oil-immersion objective and a Photometrics Pixis 1024B
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CCD camera. We recorded stacks of images (z spacing 0.3 pm) at adjacent x-y positions covering the entire A-P length of the genital ridge. Images were stitched based on stage coordinates,
and stitching coordinates were optimized locally by cross-correlation. Data analysis was performed in MATLAB (MathWorks) using custom-written code. Individual transcript molecules
were identified and counted semi-automatically as previously described [26]. Individual germ
cells were identified and outlined manually using a combination of SSEA1 expression and
DAPI morphology. Dazl probe sequences used in this study are listed in S1 Table. Oct4 probe
sequences were published elsewhere [46].
Urogenital ridge (UGR) cultures
Urogenital ridges, comprised of genital ridges, mesonephroi, primitive kidneys, and dorsal aorta,
were dissected from El 1.5 control and Gata4cKO embryos. The dissected UGRs were cultured
on agar blocks, as previously described [47], for 3 days in Dulbecco's modified Eagle's medium
(DMEM) containing 10% fetal bovine serum, non-essential amino acids, glutamine and penicillin/streptomycin. In this culture condition, as reported previously [13,23,48,49], E11.5 XY germ
cells (which are sexually bipotential) develop toward the oogenic pathway, in part due to impaired testis cord formation and the presence of meiosis-inducing factors in the culture medium.
Immunofluorescence
Immunofluorescent staining of embryonic sections was carried out as described previously
[50]. Briefly, whole embryos or cultured urogenital organs were fixed at 4"C overnight in 4%
paraformaldehyde, paraffin embedded, and sectioned. Slides were then dewaxed, rehydrated,
and antigen-retrieved by microwaving in citrate buffer (10mM sodium citrate, 0.05% Tween
20, pH6.0). After blocking, slides were incubated with primary antibodies at 4*C overnight.
Slides were then incubated with donkey secondary antibodies conjugated to FITC, Rhodamine
Red X or DyLight 649 (Jackson ImmunoResearch) and mounted with ProLong Gold Antifade
reagent with DAPI (Life Technologies).
Primary antibodies against GATA4 (sc-25310, Santa Cruz Biotechnology), DAZL (ab34139,
Abcam), SSEA1 (MAB4301, Millpore), MVH (AF2030, R&D Systems), GCNA (a gift from
George Enders, University of Kansas Medical Center, Kansas City, KS) [51], SOX2 (ab97959,
Abcam), NANOG (IHC-00205, Bethyl Laboratories), OCT4 (560186, BD), MILI (a gift from
Gregory J. Hannon, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) [52], and
SYCP3 (sc-33195, Santa Cruz Biotechnology) were used in the study.
Supporting Information
SI Fig. Post-migratory, but not migratory, germ cells express Dazl. (A) Immunofluorescent
staining for SSEA1 and smFISH for Dazl and Oct4 at E9.5. Dazl expression was not detectable in
migratory PGCs, while Oct4 was. Individual germ cells are outlined by dashed lines. (B) Representative E11.5 whole-gonad image of Dazl and Oct4 transcript molecules marked by smFISH. Dazl
transcript counts are higher in anterior region than in posterior region. Dazl (pseudocolored in
blue, upper panel) and Oct4 (pseudocolored in red, lower panel) mRNA molecules were overlaid
with DAPI staining (white) and identified after raw images were subject to image analysis filtering.
Raw images prior to filtering are displayed in magnified images (middle panels), where mRNA
molecules are visible as white dots within individual germ cells (outlined by dashed lines).
(TIF)
S2 Fig. Germ cells in Gata4 cKO (soma-specific Cre) embryos do not express DAZL or
MVH. Immunofluorescent staining for SSEA1, DAZL, MVH, and GATA4 in transverse
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sections of control and Gata4cKO (OsricreER) embryos on a C57BL/6 genetic background.
Nuclei counterstained with DAPI (blue). Scale bars: 50 im.
(TIF)
S3 Fig. Germ cells in Wtl-deficient genital ridges express DAZL. Genital ridge formation is
initiated in Wtl KO embryos (a gift from Kenneth H. Albrecht), but growth is severely retarded
and degeneration ensues. Immunofluorescent staining of longitudinal sections from wildtype
or Wtl KO urogenital regions shows that PGCs at the genital ridge (GATA4-positive, blue) express DAZL (red, arrows). Yellow dashed lines outline the genital ridge. Autofluorescent red
blood cells are indicated (asterisk). gr, genital ridge. Scale bars: 50 Pm.
(TIF)
S4 Fig. Germ cells in Osri -deficient genital ridges express DAZL. Genital ridge formation is
initiated in Osri KO embryos, but complete degeneration occurs by E15.5 [30]. (A) Immunohistochemical staining for GATA4 in cross-sections of wildtype and Osrl KO embryos at
El 1.5. Genital ridge formation is initiated in Osri KO embryos, but growth is restricted. Inset
shows higher magnification of genital ridge. (B) Immunofluorescent staining for SSEA1,
DAZL, and GATA4 in cross-sections of wildtype and Osrl KO urogenital regions. Representative germ cells positive for DAZL are indicated by arrows. Yellow dashed lines outline the genital ridge. a, dorsal aorta; gr, genital ridge; m, mesentery. Scale bars: 50 tm.
(TIF)
S5 Fig. Germ cells in Gata4 cKO (soma-specific Cre) cultured UGRs do not express DAZL
or MVH. Immunofluorescent staining for SSEA1, DAZL, MVH, and 5-methyl-cytosine (meC)
in transverse sections of control and Gata4 cKO (WO CreER;Osr1creER) cultured UGRs (on a
C57BL/6 genetic background). Nuclei counterstained with DAPI (blue). Inset shows higher
magnification of germ cells. Scale bars: 50 im.
(TIF)
S1 Table. Daz probe sequences. Probe sequences used for smFISH analysis of
Dazl expression.
(DOCX)
Acknowledgments
We thank George Enders for GCNA antibody; Gregory Hannon for MILI antibody; Mary
Goodheart for support with animal care; Kenneth Albrecht for Wtl KO embryos; Kyomi Igarashi for genotyping; and Bluma Lesch and Jennifer Hughes for critical reading of
the manuscript.
Author Contributions
Conceived and designed the experiments: YCH PKN YQSS JPJ AvO DCP. Performed the experiments: YCH PKN YQSS JRD JPJ. Analyzed the data: YCH PKN YQSS JRD JPJ. Wrote the
paper: YCH PKN DCP.
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DOI:10.1371[joumal.pgen.1005019
Genetics II DOI:
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