Fall 2009
Click on this link to access the Powerpoint for Lect. #3
Thurs 9/3/09
Reading: Ch7: 175-207; Ch9: 259-263; Ch19: 593-622
(optional reading listed at end of lecture notes)
Paper for Tues (9/8) discussion: Caceres, L. and Nilson, L.A. (2005).
Production of gurken in nurse cells in sufficient for axis determination in
the Drosophila oocyte. Development 132, 2345-2353.
A. Gametogenesis is the formation of the germ cells that will go on to
form progeny. (Slide 2, 3)
1. The formation of germ cells begins during embryogenesis, and involves
several steps that are independent of the sex of the animal –
b. MIGRATION from their origin to the developing gonads (organs for
sexual reproduction)
2. Once they’ve colonized the gonads, gametes continue a specific
developmental program that involves:
a. reduction of the DNA from diploid to haploid in the mature egg or
sperm called MEIOSIS (we won’t go over meiosis, but read Ch19:
606-611 if you need a refresher)
b. SPERM (the male gametes) become specialized for movement
c. EGGS (the female gametes) become loaded with RNA, protein,
and structures (mitochondria, ribosomes) that will allow early
development to proceed QUICKLY and without extensive reliance on
ZYGOTIC TRANSCRIPTION (the ZYGOTE is the newly fertilized egg
that has not yet divided)
-at this time the main body axes are often set up as well
B. Gamete specification
1. Germ cells are specified by two distinct mechanisms:
a. Induction – in many organisms, including mammals, the cells in an
embryonic area are specified as PRIMORDIAL GERM CELLS (PGC) by
inductive signals from nearby tissues. In the mouse BMP-mediated signaling
(BMP is a signal in the TGF-beta superfamily) from the extraembryonic
ectoderm to embryonic cells nearby starts things off (Slide 4).
b. Autonomous specification by cytoplasmic determinants in the egg that
come to be included in the germ cell cytoplasm. In worms, frogs, and flies this
pole plasm (or polar granules) contain specific RNAs and proteins. (Slide 5-7)
This is because in many organisms (frog and fly are good examples)
early development very much depends on deposition of RNAs and
proteins into the egg by the mother. Thus if the mother is missing a
protein or RNA usually deposited into the eggs, she is fine but cannot
deposit it into her eggs, so they develop abnormally. This is called a
MATERNAL EFFECT MUTATION (Slide 8). It is less common in
organisms such as mammals that do not place many determinants into
their eggs, but with mammals females can carry mutations that affect
their ability to nurture embryos. For example, if a mammalian mother is
missing a gene required for formation of the maternal placenta, she will
be OK but not able to produce offspring. They will die because the
placenta does not form properly.
How could a mutation affect grandchildren?
2. Back to germ cell specification. We will go through the process in flies as
an example:
a. during the nuclear divisions in the early fly embryo, a group of nuclei
migrate to the posterior end where the pole plasm resides. If nuclei are
prevented from reaching this spot no germ cells are formed.
b. the future germ cells (called POLE CELLS) are cellularized slightly
before the rest of the cells.
c. if a gene called germ cell-less is missing, the female fly cannot have
grandchildren, because her embryos do not have proper pole plasm.
Thus the embryos themselves develop, but they lack germ cells and are
sterile. (Slide 9)
3. What is in the pole plasm that specifies germ cells?
This has been a big mystery for many years, and we are still not completely
sure how this works, but it seems that perhaps the autonomous germ cell
determinants prevent transcription and translation. The idea is that the germ
cells are “held” in a primitive or totipotent state by prevention of further
differentiation. This may also be the case for germ cells specified by
inductions – the signals may prevent further development.
a. For example, in worms an early promoter is activated in all
blastomeres except P, which will give rise to germ cells (Slide 10).
b. germ cell less encodes a protein that goes to the nucleus of pole
cells and prevents transcription. How it does this is not clear. It can
also induce some cells to become germ cells if ectopically expressed
(Slide 11).
c. The fly pole plasm includes the protein OSKAR, which is required
for germ cell formation. Oskar can also induce ectopic germ cells. It
seems to localize other required components for germ cell formation
such as NANOS mRNA. Nanos also can block transcription. (Note:
Oskar also specifies the posterior axis of the embryo, see below).
d. Exception: VASA is found in pole plasm and PGC of many
organisms, it encodes a RNA helicase that may translationally activate
a subset of RNAs required for germ cell formation.
C. Gamete migration
1. Once germ cells are specified, they can migrate relatively long distances
from origin to gonad. This migration in some situations may be “passive”, that
is the whole tissue moves relative to others (especially during gastrulation)
and the germ cells are along for the ride. However, there is clearly an “active”
component as well, where germ cells migrate independent of surrounding
2. Details differ among different organisms, and we will use germ cell
migration in the mouse as an example, because we do know some molecular
a. Description – mouse PGCs are induced in the posterior epiblast
around day 6.5 of development (just before gastrulation). They stay there
for several days, then exit on day 9 and wander around for awhile. By
day 10 they begin to migrate up the dorsal mesentery and into the genital
ridge, area that will become the gonad (testes or ovary). (Slide 12, 13)
b. Cellular mechanism - unknown, but fibronectin (FN) likely to be
important, because PGCs that lack the integrin receptor for FN do not
reach the gonads.
c. Molecular control:
1) The cells that line the migration path express a ligand called stem cell
factor (SCF) that promotes survival and proliferation of PGC. The PGC
express the receptor c-kit. This pathway provides for survival and
proliferation during migration and may also provide the spatial cues. (This
pathway is also important in long range migration of neural crest cells and
hematopoietic cells)
2) Recently it was found that a chemokine used for lymphocyte trafficking
called SDF1 (stromal cell derived factor 1) and its receptor CXCR4 are
required for the last migration of PGC into the gonad, but not for earlier
D. Spermatogenesis
We won’t spend much time on spermatogenesis, not because it is not
important but the process of oogenesis is more complex and more intimately
linked to subsequent development of the embryo.
-- Contrasts with Oogenesis – see Table 19.1 (p. 612 Gilbert)
a. continuous production of gametes upon puberty
b. meiosis is completed, then differentiation proceeds
c. most material lost upon differentiation: what is retained is nuclear
material, a structure called the ACROSOME that is important for
fertilization, an energy source (mitochondria), and a highly specialized tail
for movement. (Slide 14)
E. Oogenesis –
PGCs that enter female gonads and survive (there is a massive die-off before
and right after birth) undergo oogenesis, which in addition to producing a
haploid gamete via meiosis, provide the oocyte with nutrition and RNAs and
proteins that usually control early developmental events. In many cases the
major axes of the embryo (anterior-posterior and dorsal-ventral) are set
up. How does this happen?
1. There are different modes for different types of oocytes, two examples of
a. the mammalian oocyte is surrounded by granulosa cells that provide
material to the oocyte via gap junctions, vs. the fly oocyte that is
connected to its nurse cells by cytoplasmic bridges, and is also in the
same clonal lineage with the nurse cells. (Slide 15, 16)
b. the frog oocyte nucleus is transcriptionally active (Slide 17),
producing much of the egg material in this way, whereas the fly oocyte
nucleus supports very little transcription, instead the nurse cells become
polytene and make most egg material.
2. Much is known about fly oogenesis so we will follow that process as an
LOCATION. These movements are critical to setting up the major axes of the
future embryo, the A-P and D-V axes.
a. The fly oocyte develops in a germarium, which consists of the oocyte
and nurse cells they are clonally related to, surrounded by follicle cells.
(Slide 18)
b. The oocyte sits asymmetrically in the germarium because the posterior
follicle cells are more adhesive (more DE-cadherin). This occurs because
cells in the previous germarium (there is a whole line of them like beads on
a string) affect the adhesiveness of these cells via a Notch-Delta signal.
c. The oocyte produces a protein called GURKEN (EGF homolog) that
signals to posterior follicle cells. (Slide 19)
d. This results in a reciprocal signal from follicle cell to oocyte
(UNKNOWN) that acts through PAR-1 in an actin-dependent way to
rearrange the oocyte microtubules so that the growing end (plus end) is at
e. an important anterior determinant is BICOID – its message interacts
with EXUPERANTIA and SWALLOW proteins in the 3’UTR of bicoid, and
this tethers bicoid RNA to DYNEIN which goes to the minus end of the
microtubule or anterior.
f. an important posterior determinant is OSKAR (see above), its RNA
becomes complexed with KINESIN I, a motor protein that moves it to the
plus end of microtubules, or the posterior. There it binds and localizes
nanos RNA.
g. Now the nucleus moves along the microtubules and its Gurken
production induces the dorsal follicle cells. (Slide 20).
h. The paper we will discuss on Tues. (Caceres and Nilson, 2005)
describes experiments that determine the source of the gurken message –
oocyte vs. nurse cells.
i. At fertilization, mRNAs are translated into proteins that form a bicoid
gradient from the anterior and a nanos gradient from the posterior, with
downstream consequences on development, including that of germ cells!
(Slide 21)
Note: There is much controversy as to how much axis specification occurs in
mammalian oocytes. Many papers suggest that at least one axis is set up in
the mouse oocyte. However, a beautiful paper recently published (Hiiragi and
Solter, 2004, Nature 430, p.360) refutes this assertion and shows that there is
no obvious axis information prior to fertilization in the mouse.
F. Fertilization
This is the process whereby the egg and sperm come together and fuse to
combine their haploid DNA content and create a diploid zygote that then
commences development. Of course fertilization differs among organisms, so
in the interest of time we will focus on general aspects of the process. Most
studied model is actually the sea urchin, but lately it seems that many other
organisms where fertilization is harder to study, such as mammals, have
similar events.
1. Overview:
There are 3 major outcomes of fertilization—
a. Union of egg and sperm DNA
b. Prevention of polyspermy (multiple sperm fertilizing one egg)
c. Commencement of developmental processes
2. Details:
a. some (maybe all) sperm respond to chemotactic signals made by eggs
to home to them
b. contact of sperm with the egg is mediated by transmembrane proteins
that are often species specific.
c. this contact starts the ACROSOMAL REACTION (Slide 22) (the
acrosome is a structure above the nucleus of the sperm that contains
proteolytic enzymes), which involves binding of egg jelly compounds to
receptors on the sperm membrane. This binding opens calcium ion
channels and lets Ca++ into the sperm head. The acrosomal membrane
fuses with the sperm membrane. In the mouse this release of proteolytic
enzymes allows the sperm to penetrate the ZONA PELLUCIDA that
surrounds the egg and get to the egg membrane.
d. The sperm and egg membranes then fuse. This event polymerizes
actin that lies just beneath the egg membrane, and this widens a channel
that allows the sperm nucleus and tail to enter the egg.
e. These events initiate a series of changes in the egg that are remarkable
for their rapidity and result in a BLOCK TO POLYSPERMY – some occur
within seconds of sperm-egg binding, others take more time (minutes).
SEE TABLE 7.1 (Gilbert p. 205) for listing of events and relative timing.
The release of CORTICAL GRANULES (the egg equivalent of the
acrosome) sets up the later events, or the SLOW BLOCK TO
POLYSPERMY. Cortical granule release depends on Ca++ ion release
from internal stores (the endoplasmic reticulum). (Slide 23)
f. Ca++ ion flux is also critical to “activate” the egg to commence
development (even tho it may take hours – up to 24 – for the egg and
sperm haploid nuclei to fuse). Ca++ flux requires the formation of IP3 and
DAG (diacylglycerol) from PIP2, which is mediated by PLC-gamma. PLCgamma may be activated via a src family kinase in ways not completely
understood. This activation includes resumption of the cell cycle and
recruitment of RNAs for translation. (Slide 24)
g. The zygote is now ready to begin its own development!
Leatherman, JL, et al. (2002). germ cell-less acts to repress transcription
during the establishment of the Drosophila germ cell lineage. Curr. Biol. 12,
Li, J, Xia, F, and Li, WX. (2003). Coactivation of STAT and Ras is required for
germ cell proliferation and invasive migration in Drosophila. Dev Cell 5, 787798.
Newmark, PA, et al. (1997). mago nashi mediates the posterior follicle cell-tooocyte signal to organize axis formation in Drosophila. Development 124,
Molyneaux, KA, et al. (2003). The chemokine SDF1/CXCL12 and its receptor
CXCR4 regulate mouse germ cell migration and survival. Development 130,
Torres, IL, et al. (2003). A notch-delta dependent relay mechanism
establishes anterior-posterior polarity in Drosophila. Dev Cell 5, 547-558.
Hiiragi T and Solter D (2004). First cleavage plane of the mouse egg is not
predetermined but defined by the topology of the two apposing pronuclei.
Nature 430, 360-364.
Cook HA et al. (2004). The Drosophila SDE3 homolog armitage is required for
oskar mRNA silencing and embryonic axis formation. Cell 116, 817-829.
Riechmann V and Ephrussi A. (2004). Par-1 regulates bicoid mRNA
localization by phosphorylating Exuperantia. Development 131, 5897-5907.
Doerflinger H, Benton R, Torres IL, Zwart MF, St Johnston D. (2006).
Drosophila anterior-posterior polarity requires actin-dependent PAR-1
recruitment to the oocyte posterior. Curr Biol 16, 1090-1095.
Robertson SE, Dockendorff TC, Leatherman JL, Faulkner DL, Jongens TA.
(1999). Germ cell-less is required only during the establishment of the germ
cell lineage of Drosophila and has activities which are dependent and
independent of its localization to the nuclear envelope. Dev Biol 215, 288297.
Leatherman, JL and Jongens, TA. (2003). Transcriptional silencing and
translational control: key features of early germline development. Bioessays
25, 326-335.
Tsang, TE, et al. (2001). The allocation and differentiation of mouse primordial
germ cells. Int. J. Dev. Biol. 45, 549-555.
Huynh J-R and St Johnston D (2004). The origin of asymmetry: early
polarization of the Drosophila germline cyst and oocyte. Curr Biol 14, R438R449.
Becalska AN and Gavis ER. (2009). Lighting up mRNA localization in
Drosophila oogenesis. Development 136, 2493-2503.
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Kocer A, Reichmann J, Best D, and Adams IR. (2009). Germ cell sex
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