Developmental Biology 281 (2005) 240 – 255
www.elsevier.com/locate/ydbio
Genomes & Developmental Control
A POU factor binding site upstream of the Chx10 homeobox gene is
required for Chx10 expression in subsets of retinal progenitor cells
and bipolar cells
Sheldon Rowan, Constance L. CepkoT
Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA
Received for publication 29 June 2004, revised 21 February 2005, accepted 23 February 2005
Available online 2 April 2005
Abstract
Retinal progenitor cells (RPCs) undergo a series of changes over time that affect their competency to produce different cell types at
different times in development. The transcriptional machinery that regulates these changes, as well as associated gene expression changes,
have not been characterized. An analysis of the regulatory region of the retinal homeodomain transcription factor, Chx10, was carried out
using in ovo electroporations in chick and transgenic mice. An RPC enhancer was defined that mediates reporter activity in subsets of RPCs
and directs high-level expression in intermediate and late RPCs. Using bioinformatic and biochemical analysis, a key binding site in this
enhancer was found and was shown to be bound by the POU domain factors, Brn-2 and Tst-1/SCIP, in retinal extracts. Analysis of the Brn-2
expression pattern shows that it is expressed in intermediate and late RPCs, but not early RPCs, and thus partially overlaps with expression of
the reporter activated by the defined Chx10 enhancer. Biochemical analysis also revealed binding of both Chx10 and Brn-2 to an enhancer of
the CNS progenitor cell marker, Nestin. Nestin expression in the retina is restricted to intermediate/late RPC subsets, and genetic evidence is
presented that demonstrates that Chx10 represses Nestin expression in early RPCs. A bipolar cell enhancer for Chx10 also was defined, and a
role for Brn-2 in expression of Chx10 in bipolar cells is predicted. These data identify Brn-2 as a new marker of subsets of RPCs and suggest
a mechanism by which a combination of POU factors and Chx10 define RPC gene expression patterns, such as that of Nestin.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Transgenic mouse; Electroporation; Chick; Bioinformatics; Reporter; Enhancer; Alkaline phosphatase; Nestin; EMSA; Bipolar cell
Introduction
The neuronal and glial cell types of the vertebrate retina
are produced by pools of multipotent retinal progenitor cells
(RPCs) (Holt et al., 1988; Turner and Cepko, 1987; Wetts
and Fraser, 1988). It appears that most, if not all, of the CNS
is formed from multipotent progenitor populations (Cepko
et al., 1997; Edlund and Jessell, 1999). A notable feature of
progenitor cells in the CNS, and likely elsewhere, is their
ability to change over time to generate different populations
of neural cell types (Alexiades and Cepko, 1997; Desai and
McConnell, 2000; Luskin et al., 1988; McConnell, 1988;
T Corresponding author. Fax: +1 617 432 7595.
E-mail address: cepko@receptor.med.harvard.edu (C.L. Cepko).
0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2005.02.023
Pearson and Doe, 2004). RPCs appear to undergo such
intrinsic changes over developmental time as well as
encounter changing milieus of extracellular factors to
generate the different retinal cell types in a conserved order
(reviewed in Cepko et al., 1996; Livesey and Cepko, 2001).
Ganglion cells, horizontal cells, amacrine cells, and cone
photoreceptors are the first generated cell types, while
bipolar cells and Mqller glia are the last-born cell types. Rod
photoreceptors, the most abundant cell type in the mature
retina, are born in a broad developmental window. These
observations have raised the question of the identity of the
molecules that underlie the changes in RPCs over time that
control their competence to make the different cell types.
Transcription factors are expected to play key functions
in determining the intrinsic gene expression program of
progenitor cells. Some transcription factors are likely to
S. Rowan, C.L. Cepko / Developmental Biology 281 (2005) 240–255
determine the ability of a progenitor cell to make particular
cell types, while others may influence the decision to
produce daughter cells that will continue dividing or exit the
cell cycle. Basic helix–loop–helix transcription factors
(bHLH factors) have been implicated as having a role in
controlling exit from the cell cycle, as well as in endowing
competence to produce particular cell types (Cepko, 1999;
Marquardt et al., 2001; Moore et al., 2002; Ohnuma et al.,
2002; Yang et al., 2003). Pax6, a homeobox-containing
transcription factor, has been demonstrated to control the
multipotent state of RPCs. In its absence, the only cell type
that could be generated was the amacrine cell type
(Marquardt et al., 2001). Several other homeobox transcription factors have been identified that may have
important functions in RPCs but are necessary for optic
vesicle development and have not been analyzed later in
development. Chx10 is a homeobox-containing transcription factor expressed in RPCs and in bipolar cells, a type of
retinal interneuron (Burmeister et al., 1996; Liu et al.,
1994). Retinas lacking Chx10 protein show defects in RPC
proliferation, but the RPCs do not lose their multipotency,
as all cell types appear to be generated except for bipolar
cells (Burmeister et al., 1996). Furthermore, the ratios of
different retinal cell types in Chx10 mutant retinas are
abnormal, and a large proportion of cells exhibit markers of
RPCs or Mqller glial cells (Rowan and Cepko, 2004).
These data suggest the possibility of further roles for
Chx10 in cell fate determination, particularly of late-born
cell types.
Overexpression studies have indicated a potential cooperation between homeobox transcription factors and neurogenic bHLH factors in the production of several retinal cell
types (Hatakeyama et al., 2001; Inoue et al., 2002).
Relatively little is known about the targets of progenitor
cell transcription factors and even less is known about target
genes acted on by combinations of transcription factors. One
very useful approach to identify transcriptional regulatory
information is the dissection of enhancer regions that control
the expression of useful markers of progenitor cells. Such an
approach has been applied to Nestin, a widely used marker
for CNS progenitor cells (Dahlstrand et al., 1995; Lendahl et
al., 1990). Studies employing transgenic mice as well as
biochemical studies led to the identification of a small
region in intron 2 of Nestin as essential for expression in
CNS progenitor cells (Josephson et al., 1998; Lothian et al.,
1999; Yaworsky and Kappen, 1999; Zimmerman et al.,
1994). Proteins that bound to this region were identified as
the POU domain family of transcription factors, specifically
the Class III POU domain factors Brn-1 (Pou3f3), Brn-2
(Pou3f2, N-Oct3), and Tst-1/SCIP (Pou3f1, Oct6) (Josephson et al., 1998). This particular class of transcription factor
had already been partially described in terms of CNS
expression (Alvarez-Bolado et al., 1995; He et al., 1989).
These data strongly implied Class III POU domain factors
as regulators of CNS progenitor cell gene expression. Nestin
is also expressed in RPCs, although its expression has not
241
been well characterized nor has the role of Class III POU
domain factors in its regulation in the retina been addressed
(Ahmad et al., 1999; Sheedlo and Turner, 1996).
An understanding of Chx10 regulation in RPCs and
bipolar cells may provide new insights into transcription
regulation as well as lead to the identification of new genes
with roles in cell fate determination. We utilized chick
electroporation and bioinformatics tools to identify an
enhancer of Chx10 that is necessary for reporter activity
in RPCs. Biochemical analysis revealed that the binding
proteins are the Class III POU domain transcription factors,
Brn-2 and Tst-1/SCIP. These proteins also bind to the
characterized Nestin intron 2 enhancer, as does Chx10.
Analysis of Brn-2, Tst-1/SCIP, and Nestin early in development revealed Brn-2 and Nestin to be specifically expressed
in intermediate/late RPCs and the restriction of Nestin to
intermediate/late RPCs appeared to be mediated in part
through Chx10. Finally, we present findings from transgenic
mice that implicate Brn-2 binding to Chx10 enhancer
sequences in control of intermediate/late RPC expression
as well as possibly bipolar-specific expression of Chx10.
These data provide the first characterization of transcription
factor targets in intermediate/late RPCs and point to
important roles for Brn-2 and Chx10 in coordinating proper
cell fate determination in intermediate and late-born cell
types.
Materials and methods
Construction of Chx10 reporters
The initial 3Kb-AP reporter was constructed using a
genomic BamHI–SacII fragment isolated from a Chx10
BAC (Rowan and Cepko, 2004) and subcloned into pBluescript (Stratagene). An SV40 intervening sequence (IVS)
was isolated from pNASSh (Clontech) digested with EcoRI
and blunt cloned into the 3V SacII restriction site. The fulllength alkaline phosphatase (AP) reporter construct was
isolated from the SV40all plasmid (gift of S. Dymecki). All
other reporter constructs were modifications of the 3 Kb-AP
reporter or derivatives thereof. The Del50-AP reporter was
made by removing an internal XbaI–NheI fragment using an
XbaI partial digest. The 2.4 Kb-AP reporter was generated
by truncating the 3 Kb-AP reporter upstream of the NheI site.
Del1 Kb-AP was generated by removing an internal NheI–
XbaI fragment using an XbaI partial digest. 2.1 Kb-AP was
made by truncating the 3 Kb-AP reporter upstream of the
HindIII site. The 500 bp-AP reporter arose as a spontaneous
deletion from cloning steps to make 2.1 Kb-AP. It was fully
sequenced to reveal that only 500 bp of upstream Chx10
sequence remained. 500 bp-AP was used as a template to
generate the 500 bpD22-AP reporter. Site directed mutagenesis was performed using the Transformer site-directed
mutagenesis kit according to the manufacturer’s instructions
(Clontech) using the targeting oligonucleotide, 5V-P-GGC-
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GGGAGCTGCCAGCCTGGCCCTGGTGAT-3V. The 3
KbDel22-AP construct was made using the 500 bpD22-AP
reporter as a template and subcloning the SalI–AgeI
fragment from 3 Kb-AP reporter upstream of the 22 bp
deleted region. The BGZA-22 bp-lacZ construct was made
by multimerizing the 22 bp motif oligonucleotide (see
below) and cloning the multimer into the BGZA minimal
promoter vector (Timmer et al., 2001).
In ovo electroporation of Chx10 reporters
Electroporations were performed by injecting Chx10
reporter DNA along with pMiw-GFP expression vector
(Schulte et al., 1999) into the right optic vesicle of HH
stage 10–11 chick embryos as described previously
(Schulte et al., 1999). Following electroporation, embryos
were allowed to develop for an additional 24–48 h and
harvested. Embryos were assayed for successful electroporation by visualization of GFP using an Olympus IMT-2
inverted microscope or a Leica MZFLIII dissecting
microscope.
Alkaline phosphatase detection
Embryos or retinas were stained as whole-mounts for
alkaline phosphatase (AP) using standard methods (Fekete
and Cepko, 1993) The reaction was allowed to proceed from
15 min at room temperature to overnight at 48C until judged
complete and tissue was transferred to PBS. For cryosectioning, tissue was embedded in OCT (Tissue-Tek) or
gelatin (Sigma) and 20 Am sections were collected on
superfrost plus slides (VWR).
Bioinformatics
All sequences for motif analysis were identified from the
NCBI genome browser except the human Pax6 and quail
Pax6 sequences, which were obtained from accession
numbers AJ009905, AJ009907, AJ009906, and Z95332.
Sequences were input into AlignACE (Roth et al., 1998)
and run from the resident website: http://www.atlas.med.
harvard.edu/cgi-bin/alignace.pl using a 20 column alignment and 0.6 fractional background GC content. Motifs
were sorted based on MAP scores and uniqueness. Motifs
were input into Jalview (http://www2.ebi.ac.uk/~michele/
jalview/) to generate a graphical alignment of the motif.
Determination of transcription factor binding sites was
carried out using software designed to analyze the TRANSFAC database (Wingender et al., 2000). VISTA plots were
generated using the VISTA server (http://www.gsd.lbl.gov/
vista/index.shtml) (Mayor et al., 2000).
EMSA analysis
Retinal or cortical rat extracts were prepared by
hypotonic lysis of frozen tissue using the protocol of Chew
et al. (1999) except that the initial low speed centrifugation
step was removed. Therefore, nuclear extracts also contained some membrane proteins. Extracts were then frozen
and stored at 708C.
Oligonucleotide probes were designed as complementary single stranded oligonucleotides containing 4 bp 5V
overhangs at each end (in lower case). The following
oligonucleotides were used: 22 bp motif: 5V-agctCAGCTTTTTGGAATTCCTAATCGCTCCT-3V; Nestin FTPT4
(Josephson et al., 1998): 5V-agctGTGTGGACAAAAGGCAATAATTAGCATGAGAATCGGCCTC-3V; Chx10 consensus (Percin et al., 2000): 5-ctagATGCATAACTAATTAQ
GCTTAGGTTAG-3V. Complementary oligonucleotides
were annealed and end-labeled with [a-32P] dCTP as described previously (Josephson et al., 1998).
Binding reactions were performed as described previously (Josephson et al., 1998) using 2–4 Ag of nuclear
extract, 6–8 Ag of cytoplasmic extract, or 1 AL of
baculovirus lysate. The Chx10 baculovirus contained the
full-length Chx10 coding sequence and lysate was prepared
under nondenaturing conditions according to the manufacturer’s instructions (PharMingen) (A. Chen and C.L.
Cepko, unpublished). For supershifts, 1 AL of antibody
was added to the protein–DNA complex after 1 h and
incubated a further 30 min at room temperature. Antibodies
used for supershift assays were: guinea pig anti-Brn-1,
guinea pig anti-Brn-2 (both gifts of A. Ryan and M.G.
Rosenfeld), rabbit anti-Tst-1/SCIP (gift of B. Andersen and
M. Wegner), rabbit anti-Chx10 (polyclonal antisera produced against an N-terminal 139 amino acid peptide (A.
Chen and C. L. Cepko, unpublished), or a control rabbit
polyclonal antibody not recognizing any mouse proteins.
Binding reactions were electrophoresed at 100 V on a 6%
TBE precast gel (Novex, Invitrogen) and run in 0.5 TBE
(Ausubel et al., 2001) at 48C. Gels were dried and
visualized autoradiographically.
Western blot analysis
10 Ag of nuclear or cytoplasmic extract was denatured
in an SDS containing buffer, electrophoresed on a precast
4–12% Bis–Tris gel (NuPAGE, Invitrogen), and transferred
to a nitrocellulose filter. Membranes were blocked and
subsequently probed with 1:500 guinea pig anti-Brn-2,
1:500 guinea pig anti-Brn-1, or 1:500 rabbit anti-Tst-1/
SCIP. Antibodies were detected using 1:20,000 horseradish
peroxidase (HRP) anti-guinea pig or anti-rabbit (Amersham) and were developed with ECL detection reagents
(Amersham).
In situ hybridization
Section in situ hybridization was performed as previously
described (Murtaugh et al., 1999). Riboprobes labeled with
DIG were detected with NBT/BCIP (Sigma). Probes were as
follows: Chx10 (3VUTR, an MscI/XhoI fragment isolated
S. Rowan, C.L. Cepko / Developmental Biology 281 (2005) 240–255
from full-length mouse Chx10 (+19 amino acid splice form)
(A. Chen and C. Cepko, unpublished data)), Brn-2 (fulllength cDNA (gift from B. Wang and A. Bonni)), Tst-1/
SCIP (5V partial cDNA (gift from R. Pearse and M.G.
Rosenfeld)), and Nestin (EST-BE981380).
Immunohistochemistry
For antibody staining, cryosections were prepared and
stained as described previously (Chen and Cepko, 2002).
Antigen retrieval was performed using the Vector antigen
retrieval solution according to manufacturer’s instructions
(Vector). Primary antibodies used were 1:5000 rabbit antiChx10, 1:1000 guinea pig anti-Brn-2, 1:1000 rabbit antiTst-1/SCIP, and 1:500 mouse anti-Nestin (PharMingen). For
biotin amplification and histochemistry, the Vectastain ABC
kit followed by DAB detection was used (Vector). Antibody
staining of dissociated cells was performed as described
previously (Chen and Cepko, 2002).
Transgenic and mutant mice
Chx10 reporter transgenic mice were generated by
isolating the Chx10-AP insert from the vector DNA, usually
by digestion with EcoRV. Inserts were purified on gelpurification columns (Qiagen) and injected at a concentration of 4–5 ng/AL into male pronuclei of SJL/B6 fertilized
eggs using standard procedures (Nagy, 2003). Mice were
genotyped using PCR primers to the 3V UTR of AP: 5VGTGGTCCCCGCGTTGCTTCCTCTG-3V, 5V-GCGCGGGGTCTGGGTGCTTCTTTT-3V. 2.4 Kb-AP transgenic mice
were maintained on a mixed SJL/B6 and C57/B6 background. or J mice were obtained from Jackson Laboratories
and were maintained on a mixed background containing
FVB, 129/Sv, SJL/B6, and C57BL/6.
Results
Analysis of Chx10 reporter constructs by in ovo
electroporation
Previous studies have utilized in ovo electroporation to
assay reporter constructs made from mouse or chick
sequences (Itasaki et al., 1999; Timmer et al., 2001;
Uchikawa et al., 2003). A genomic DNA fragment from
the mouse Chx10 locus containing approximately 3 Kb of
sequence upstream of the Chx10 start methionine was
engineered into a reporter construct containing the fulllength human placental alkaline phosphatase (AP) gene.
Between the Chx10 sequences and the AP reporter, an
SV40-derived intervening sequence was inserted in order to
create a splicing event (see Fig. 2). This reporter, referred to
as the 3 Kb-AP reporter, was co-electroporated into chick
optic vesicles from HH stage 10–11 embryos with pMiwGFP, a constitutively expressing GFP reporter, as a
243
cotransfection marker. Embryos were allowed to develop
in ovo for 1–2 days following electroporation and successful
transfectants were screened for GFP expression in the eye
and assayed for reporter activity by whole-mount AP
staining.
Embryos successfully transfected with pMiw-GFP and 3
Kb-AP showed strong GFP and AP reporter activity in the
eye (Figs. 1A, B). In embryos where the separate developing layers of the retina and retinal pigmented epithelium
(RPE) could be discerned, AP reporter activity was
observed in cells in both layers (Fig. 1C). Sections of eyes
from successfully transfected chicks also showed AP
reporter activity in the retina and RPE (Figs. 1D, E). At
these stages, the majority of retinal cells are RPCs.
However, AP reporter activity usually was detected only
in a subset of these cells (Fig. 1E). AP reporter activity in
the RPE was surprising, as the RPE does not express Chx10
in mouse or chick (Chen and Cepko, 2000; Liu et al., 1994).
This ectopic activity may indicate a role for sequences
outside of the 3 Kb region in repression of Chx10 in the
RPE or may indicate a general non-specific activity of the 3
Kb-AP reporter. However, since structures outside of the
eye that were transfected, primarily the neural tube, did not
show significant AP reporter activity (Fig. 1E), it does not
appear to be generally non-specific expression. These data
show that sequences within the 3 Kb upstream region of
Chx10 can confer some of the endogenous Chx10 expression pattern.
In order to identify the region(s) upstream of Chx10
containing the RPC enhancer described above, a variety of
deletions within the 3 Kb-AP reporter were constructed
(Fig. 2). Some of these deletions corresponded with regions
that showed strong conservation between mouse and human
sequences (Fig. 2). These reporters were tested by in ovo
electroporation as described above. Deletions of the distal
part of the upstream sequences had little or no effect on the
ability to mediate enhancer activity in RPCs (Del50-AP, 2.4
Kb-AP, and 2.1 Kb-AP reporters). A construct with an
internal deletion, in which approximately 1 Kb of sequence
was removed, was still capable of activating AP reporter
activity in RPCs, but at a reduced level compared to the 3
Kb-AP construct. Since the 2.1 Kb-AP reporter was
indistinguishable from the 3 Kb-AP reporter, there appears
to be sequences between the HindIII and proximal XbaI
restriction enzyme sites that play a role in regulating levels
of transcription. This region also contains a block of
conservation between mouse and human Chx10 (Fig. 2).
A small proximal region upstream of Chx10 that only
contained approximately 500 bp of sequence was assayed,
and that region activated reporter activity similarly to Del1
Kb-AP (also see Fig. 3B). This sequence showed very high
conservation with human Chx10 sequences, in contrast to
much of the more distal upstream region. Its sufficiency in
directing reporter activity in RPCs suggested that sequences
within the proximal 500 bp region might constitute an RPC
enhancer.
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S. Rowan, C.L. Cepko / Developmental Biology 281 (2005) 240–255
Fig. 1. In ovo electroporation analysis of Chx10 3 Kb-AP reporter. (A–E) Chick embryos were co-electroporated with pMiw-GFP (as an electroporation marker)
and the Chx10 3 Kb-AP reporter. (A) GFP was detected in the eye of an intact HH stage 20 embryo. (B) The same embryo as in panel (A) was stained for AP in
whole-mount. (C) High magnification view of an HH stage 14 embryo stained for AP in whole-mount. (D, E) An HH stage 21 AP-stained chick embryo was
sectioned and visualized for GFP (D) or visualized by Nomarski illumination (E). l, lens; nt, neural tube; r, retina; rpe, retinal pigmented epithelium.
Identification of a 22 bp sequence required for RPC
enhancer activity
In order to identify candidate sequences in the 500 bp
upstream region of Chx10 that might confer RPC enhancer
activity, a bioinformatics approach was performed to find
sequences that might be conserved between Chx10 and other
genes expressed in RPCs. To facilitate the bioinformatic
analysis, only the immediate 500 bp upstream regions of
these genes (based on mapped transcriptional start sites)
were chosen for comparison with the 500 bp upstream of
Chx10. The genes selected for analysis were Cyclin D1,
Six3, Rax, and a Pax6 enhancer, all of which have been
shown previously to be expressed in RPCs (Furukawa et al.,
1997; Livesey et al., 2004; Oliver et al., 1995; Plaza et al.,
1999; Sicinski et al., 1995). The sequences were analyzed by
the AlignACE program, designed to identify motifs shared
among sequences (Roth et al., 1998). One particular motif of
interest, 22 bp in length, was shared by Chx10, Pax6, and
Cyclin D1 in multiple species and in at least two copies (Fig
3A). Three residues were invariant within all motifs and
seven other resides were conserved in at least 10/13 copies
of the motif. Only four residues showed poor conservation.
In order to determine whether the 22 bp motif identified
above contained critical sequences for the RPC enhancer,
one copy of it was deleted from the 500 bp-AP reporter.
This reporter was chosen for comparison since this was the
construct used for the initial identification of the enhancer,
and sequences upstream, especially within the 1 Kb deleted
region, may interact unpredictably with this sequence. The
more distal copy of the motif was deleted since the proximal
copy partially overlapped with the predicted TATA box of
Chx10. Fig. 3B shows an example of an embryo transfected
with the 500 bp-AP reporter compared to the 500 bpD22-AP
reporter lacking the motif. The 500 bp-AP reporter induced
reporter activity in regions of the retina and RPE that were
successfully transfected, but not the neural tube (Fig. 3B,
top). The 500 bpD22-AP reporter did successfully activate
reporter activity in the RPE and within a small region of the
neural tube, but not within the retina or the rest of the neural
tube (Fig. 3B, bottom). The ability of the reporter to mediate
ectopic expression indicated that the basal promoter was not
affected by the deletion. Although the reporter expression
seen in the RPE in this section was lower than for the 500
bp-AP reporter, other sections and independent electroporated embryos showed high AP reporter activity in the
RPE but not retina (data not shown). In some independent
electroporations, a few cells in the retina did express high
amounts of AP within a larger AP-negative, but successfully
transfected region. The frequency and amount of staining
resembled the occasional reporter activity in the neural tube.
In order to determine whether the 22 bp motif was
sufficient to function as an RPC enhancer, a construct was
made containing a multimer of the 22 bp sequence coupled to
the h-actin minimal promoter and h-galactosidase reporter
(BGZA-22 bp-lacZ). This construct was electroporated into
S. Rowan, C.L. Cepko / Developmental Biology 281 (2005) 240–255
245
Fig. 2. Map of Chx10 reporter constructs and upstream region. The illustration at the top shows a restriction enzyme map of the upstream region of Chx10
covering the region encompassed by the 3 Kb-AP reporter in red. The asterisk denotes the location of the start methionine of Chx10 (ATG) within the first exon
(boxed). Restriction sites shown are: B, BamHI; E, EcoRI; H, HindIII; N, NheI; S, SacII; X, XbaI. VISTA plots are shown below the restriction map of Chx10
showing the amount of conservation between mouse and human Chx10 (top VISTA plot) or mouse and chick Chx10 (bottom VISTA plot). Windows of 100 bp
were used in the analysis. Sequence blocks showing greater than 50% conservation are shaded in pink, and regions where sequence was unavailable for
comparison are shown shaded in red. The reporter constructs used in chick electroporation studies are shown below. The reporter cassette inserted downstream
of genomic sequences contained an SV40-derived intervening sequence (IVS, green box) followed by the full-length human placental alkaline phosphatase
gene (PLAP, black box). The 1 Kb region deleted in the Del1Kb-AP construct is colored in blue. Reporter activity in RPCs, as assayed by in ovo
electroporation, is summarized by plus signs to right of the construct: ++, strong AP staining; +, weak or moderate AP staining. Also shown is the number of
informative electroporations used for the analysis of each construct (N).
chick optic vesicles as described above but was not capable
of activating reporter activity (data not shown). Therefore,
other sequences upstream of Chx10 are required in combination with the 22 bp sequence to activate Chx10 expression.
Identification of Brn-2 and Tst-1/SCIP as 22 bp motif
binding proteins
Sequence analysis of the 22 bp motif indicated a number
of potential binding proteins, including the POU domain
containing homeoproteins Brn-2 and Tst-1/SCIP. As these
molecules were also implicated in regulation of Nestin
expression in CNS progenitor cells (Josephson et al., 1998),
electrophoretic mobility shift assays (EMSA) were per-
formed to determine binding activities on either the Chx10
22 bp motif or the well-characterized Nestin intron 2
enhancer region (FTPT4) in retinal extracts. Nuclear and
cytoplasmic extracts were made from rat P0 and P10 retinas
to test for binding. P0 rat retinas are enriched for RPCs and
postmitotic early-born cell types (Alexiades and Cepko,
1996) whereas P10 retinas contain only postmitotic cells.
Fig. 4A shows the results of such an analysis. A single band,
corresponding to at least one protein–DNA complex, was
observed following binding to the 22 bp motif, with strong
enrichment in P0 nuclear extracts (Fig. 4A, arrow). Multiple
bands were seen following binding to the Nestin FTPT4
sequence, including a band that migrated similarly to the
one observed for the 22 bp motif, which was highly
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Fig. 3. Identification and analysis of a 22 bp motif. (A) Sequence of the 22 bp motif as identified in the different sequences analyzed from quail (q), human (h),
mouse (m), or rat (r). Consensus strength of individual residues is shown below with higher bars representing stronger conservation. Colors correspond to
conserved residues (G—orange, A/C—blue, T—green). (B) Chick embryos were electroporated in ovo with pMiw-GFP and the 500 bp-AP reporter (top) or
500 bpD22-AP reporter (bottom). Sections from AP-stained embryos were visualized for GFP (left) or Nomarski illumination (right). l, lens; nt, neural tube; r,
retina; rpe, retinal pigmented epithelium.
enriched in P0 nuclear extracts. Faster and slower migrating
bands also were observed in P0 and P10 extracts. Crosscompetition experiments revealed that similar activities
bound to the Chx10 22 bp motif and Nestin FTPT4
sequence, with the exception of the fast migrating Nestin
FTPT4 band, which was not competed by cold Chx10 22 bp
motif (Fig. 4B). To determine whether POU domain factors
were expressed in the retinal extracts, Western blotting
analysis was performed to detect Brn-1, Brn-2, and Tst-1/
SCIP. P5 cortex extract, known to contain these three factors
(Josephson et al., 1998), was included as a positive control
(Fig. 4C). Antibodies to Brn-2 and Tst-1/SCIP both labeled
single bands that migrated at their predicted molecular
weights in P0, but not P10 nuclear retinal extract, with more
intense labeling of Brn-2 than Tst-1/SCIP in the retina.
In order to determine whether POU domain factors
were responsible for binding activities on the Chx10 22 bp
motif, P0 nuclear extract was pre-incubated with antibodies
to different POU domain factors as well as Chx10
antibody, both as a control and to examine the possibility
of autoregulation (Fig. 4D). Two different polyclonal Brn1 antibodies had no significant effect on band formation
(2nd antibody not shown). Conversely, a polyclonal
antibody against Brn-2 eliminated or supershifted most
of the band. The remaining band appeared to be faster
migrating. A polyclonal antibody against Tst-1/SCIP led to
a slight reduction in band formation, specifically in the
faster migrating region. Addition of both antibodies
together completely abrogated complex formation and/or
supershifted the band. A polyclonal antibody to Chx10 had
no effect on the band. This experiment was repeated using
P5 cortex extract, and bands were seen with all three
proteins, which were similarly affected by antibody
additions (Fig. 4D). The ratios of Brn-2 to Tst-1/SCIP in
S. Rowan, C.L. Cepko / Developmental Biology 281 (2005) 240–255
247
Fig. 4. Binding of the Chx10 22 bp motif or Nestin enhancer by POU domain factors. (A) Autoradiogram shows EMSA analysis of complexes bound to the
Chx10 22 bp motif (Lanes 1–4) or Nestin FTPT4 binding site (Lanes 5–8). Cytoplasmic (C) or nuclear (N) extracts from P0 (Lanes 1, 2, 5, 6) or P10 (Lanes 3,
4, 7, 8) rat retinas were complexed with the above binding sites. The arrow marks the specific complex enriched in P0 nuclear extract. (B) Autoradiogram
shows EMSA analysis of P0 retinal complexes bound to the Chx10 22 bp motif (Lanes 1–9) or Nestin FTPT4 binding site (Lanes 10–18) in the presence of
increasing quantities cold Chx10 22 bp motif or cold Nestin FTPT4 binding sites as indicated above gel. Cold binding sites were added in increasing amounts
from 10–33–100–333. The arrow marks the Chx10 22 bp binding complex. (C) Western blots of cytoplasmic (C) or nuclear (N) extracts from P0 rat
retina (Lanes 1, 2), P10 rat retina (Lanes 3, 4), or P5 rat cortex (Lanes 5, 6). Blots were reacted with anti-Brn-2 (top), anti-Tst-1/SCIP (middle), or anti-Brn-1
antibodies (bottom). Brn-1 was detected following Brn-2, and the Brn-1 specific band is labeled with an arrow. (D) Autoradiogram shows EMSA analysis of
complexes bound to the Chx10 22 bp motif from P0 retina extracts (Lanes 1–6) or P5 cortex extracts (Lanes 7–12) following addition of buffer or antibodies to
Brn-1, Brn-2, Tst-1/SCIP, Brn-2 and Tst-1/SCIP, or Chx10 as indicated. The Brn-2/Tst-1/SCIP complex is indicated by arrows. This autoradiogram was
exposed for a longer period of time than in panels (A) and (B) and hence some of the faster migrating complexes appear darker. (E) Autoradiogram shows
EMSA analysis of complexes bound to the Nestin FTPT4 binding site following addition of water, control rabbit polyclonal antibody, or Chx10 antibody as
indicated. The fast migrating complex affected by Chx10 antibody addition is marked with an arrow. (F). Autoradiogram shows EMSA analysis of complexes
bound to a Chx10 consensus binding site (Lanes 1–5), Chx10 22 bp motif (Lanes 6–8), or Nestin FTPT4 binding site (Lanes 9–11) from control baculovirus
lysates, Chx10-expressing baculovirus lysates, or P0 retina extracts, as indicated. Competition experiments using 10 excess (+) or 33 excess (++) cold
Nestin FTPT4 binding site are also indicated. The arrow marks the Chx10 complex.
the band varied between P0 retinal extract and P5 cortical
extract in a fashion that mimicked the apparent protein
level differences, as determined by Western blotting
(Fig. 4C).
Antibody addition experiments using the Nestin FTPT4
sequence also revealed Brn-2 and Tst-1/SCIP binding (data
not shown). Surprisingly, the anti-Chx10 control antibody
led to a loss of the faster migrating FTPT4 band, without
affecting the binding that led to the slower migrating bands
(Fig. 4E). To verify this result, baculovirus control or
Chx10-expressing lysates were added to the Chx10 22 bp
and Nestin FTPT4 binding sites, as well as to a characterized Chx10 consensus binding site (Percin et al., 2000).
Strong binding of Chx10 was observed on both the
consensus Chx10 binding site as well as the Nestin FTPT4
site, with only minimal binding to the Chx10 22 bp binding
site (Fig. 4F). This binding activity resulted in a band that
migrated similarly to the fast-migrating band observed with
P0 retinal extract and was competed from the Chx10
consensus binding site using cold Nestin FTPT4 binding
sites (Fig. 4F). Interestingly, a band was also formed on the
Chx10 consensus site that may correspond to the POU
domain factor binding as it migrated similarly and was
competed by cold Nestin FTPT4 (Fig. 4F). Sequence
analysis of both the Nestin FTPT4 and Chx10 consensus
binding sites revealed the presence of the core Chx10
binding site, TAATTAGC, which may then additionally
function as a POU domain factor binding site, depending
upon adjacent sequences.
Expression of Brn-2, Tst-1/SCIP, and Nestin in RPCs
The EMSA and Western blot analyses described above
suggest that Brn-2 and Tst-1/SCIP are regulators of Chx10
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and Nestin expression in RPCs. Co-expression of Brn-2
and/or Tst-1/SCIP with Chx10 would further support these
factors as positive regulators of Chx10 expression. In situ
hybridization for these genes was performed on sections
from E13.5, E16.5, and P0 retinas, which have RPCs of the
early, intermediate, and late stages of development (Fig.
5A). Additionally, antibody staining was performed at P0.
Chx10 was abundantly expressed in the outer neuroblastic
layer (ONBL), where RPCs reside, at all stages. Chx10
RNA peaked at E16.5 and decreased at P0, along with
protein levels (Fig. 5A and data not shown). Brn-2 RNA
levels were barely detectable at E13.5 and were concentrated in the inner neuroblastic layer (INBL), where earlyborn postmitotic cells begin to differentiate. However, Brn-2
also was weakly expressed in the ONBL. At E16.5, the
expression pattern reversed, with relatively higher levels of
Brn-2 RNA in the ONBL, and lower levels in the INBL.
Analysis of Brn-2 protein and RNA at P0 revealed
essentially the same distribution as Chx10, but Brn-2
protein and RNA could also be detected in the ganglion
Fig. 5. Analysis of gene expression patterns in RPCs. (A) Sections through the eyes of E13.5 embryos, E16.5 embryos, or P0 eyes were analyzed by in situ
hybridization for Chx10, Brn-2, Tst-1/SCIP, or Nestin RNA as indicated, or by immunohistochemistry for Chx10, Brn-2, Tst-1/SCIP, or Nestin protein as
indicated. (B) Sections through the eyes of E12.5 or E17.5 embryos were analyzed by in situ hybridization for Chx10 or Brn-2 RNA as indicated. (C) Sections
through the eyes of E14.5 or E17.5 or J /+ heterozygotes or or J /or J homozygotes were analyzed by in situ hybridization for Nestin RNA as indicated. All scale
bars are 100 Am, shown on left panel for each respective timepoint. Note scale bar differences between or J /+ heterozygotes or or J /or J homozygotes in panel
(C). onbl, outer neuroblastic layer; inbl, inner neuroblastic layer; gcl, ganglion cell layer.
S. Rowan, C.L. Cepko / Developmental Biology 281 (2005) 240–255
cell layer (GCL). Brn-2 protein expression in the GCL was
more prominent than RNA expression due to the nuclear
localization of Brn-2 protein. Tst-1/SCIP expression at
E13.5 resembled that of Brn-2, but high expression in the
INBL was maintained through E16.5. Tst-1/SCIP expression was detected in the ONBL at all stages examined;
however, this expression was weak and variable. At P0,
Tst-1/SCIP protein was preferentially expressed in the GCL
and INBL, although lower levels were detected through the
ONBL. Nestin expression was almost undetectable at
E13.5 but was expressed at higher levels at E16.5 in a
pattern resembling Brn-2. At P0, Nestin RNA was very
weakly detected in the ONBL, while protein was readily
detected. Antibody staining of dissociated cells revealed a
high level of Nestin protein in RPCs at P0 in cells coexpressing Chx10 and Brn-2 (Fig. S1 in the Appendix A).
Overall, at P0, 96.2% of Chx10-expressing cells also
expressed Brn-2 (n = 131), while 97.0% of Chx10expressing cells also expressed Nestin (n = 134). Conversely, 90.6% of Brn-2-expressing cells also expressed
Chx10 (n = 127), while 89.2% of Nestin-expressing cells
also expressed Chx10 (n = 130).
In order to more closely evaluate the relationship of
Chx10 and Brn-2 expression, in situ hybridization analysis
was carried out at E12.5 and E17.5 (Fig. 5B). Chx10 RNA
was expressed at high levels and throughout the E12.5
retina. In contrast, Brn-2 was only detected in the center of
the retina, and at a very low level. At E17.5, Chx10 was
expressed throughout the ONBL, as at E16.5, but the RNA
levels were lower. Brn-2 expression at E17.5 resembled
Chx10 as it was expressed at a lower level than at E16.5.
Weak expression in the inner part of the INBL was also
observed. Together, these data are consistent with a role for
Brn-2 in positive regulation of Chx10 expression in
intermediate and late, but not early, RPCs.
Regulation of Nestin by Chx10 in vivo
The EMSA analysis above indicated that Chx10 could
bind to Nestin regulatory elements and therefore might
regulate Nestin expression. To genetically study the
relationship between Nestin expression and Chx10 function, in situ hybridization analysis was performed for
Nestin in mice heterozygous or homozygous for a Chx10
null allele, or J (Fig. 5C). At E14.5, like E13.5, Nestin
expression was barely detected in wildtype retinas (data
not shown) or in or J /+ heterozygous retinas. Nestin RNA
levels were dramatically elevated in or J /or J retinas and
restricted to the ONBL. In situ hybridization analysis at a
later timepoint, E17.5, did not reveal any differences in
Nestin levels in heterozygous versus homozygous animals.
Brn-2 RNA levels were unchanged in or J /or J mutants at
both timepoints (data not shown). These data point to
Chx10 as a possible negative regulator of Nestin expression in early RPCs, but not in intermediate/late RPCs,
when Brn-2 levels are high.
249
Transgenic mouse analysis of Chx10 AP reporters in RPCs
As an independent means to verify the chick in ovo
electroporation results and test predictions about early
versus later RPC regulation of Chx10, transgenic mice
were generated for some of the reporters described in Fig. 2.
A transgenic mouse line was established for the 2.4 Kb-AP
reporter and reporter expression was characterized in RPCs
(Figs. 6A–E). At E11.5, AP reporter activity was detected in
the ventral neural tube beginning at the hindbrain level, in
some mesenchymal cells near the developing branchial
arches, and in the eye (Fig. 6A). The staining in the neural
tube was highly reminiscent of the normal pattern of Chx10
in V2 interneurons, whereas the mesenchymal staining
appeared to be ectopic. Sections through the eye revealed
light AP staining only in the center of the retina (Fig. 6B).
Analysis of the 2.4 Kb-AP transgenic mice at E12.5 (Fig.
6C) and E15.5 (Fig. 6D) revealed a pattern of staining in
RPCs that extended more peripherally, and at progressively
higher levels over time. By E15.5, the entire retina was APpositive, except for the most peripheral part, a non-neural
region known to express Chx10 (Rowan and Cepko, 2004;
Rowan et al., 2004). The amount of AP reporter activity
correlated with increasing levels of Brn-2 over time in RPCs
(see Fig. 5A), despite not showing strong spatial correlation.
2.4 Kb-AP stable transgenic mice were analyzed by
whole-mount AP staining at P0 and showed strong AP
staining in the retina (Fig. 6E). The AP activity was
variegated across the retina; a phenomenon observed in all
Chx10 reporter transgenic mice, including those derived
from a Chx10 BAC (Rowan and Cepko, 2004). Sectioning
of these retinas suggested that AP staining occurred in
RPCs, as AP-positive cells extended processes through the
radial dimension of the retina (data not shown). Transient
transgenic mice were generated from 3 Kb-AP reporters and
showed a similar pattern of AP staining in 5/13 independent
transgenic mice to that of the 2.4 Kb-AP transgenic mouse
line (Fig. 6F). To investigate the importance of the 22 bp
enhancer described above, transient transgenic mice were
generated that contained the 3 Kb-AP reporter lacking the
22 bp enhancer. Overall, nine independent transgenic mice
were analyzed for AP staining at P0 and none had AP
staining in RPCs (Fig. 6G).
Analysis of Brn-2 and Chx10 AP reporter constructs in
postnatal retinas
Since Brn-2 expression resembled Chx10 expression at
late embryonic timepoints and at P0, the expression of Brn2 and Chx10 was analyzed at two postnatal timepoints, P6
and P10 (Figs. 7A–F). Brn-2 RNA was detected variably
throughout the inner nuclear layer (INL) and in the GCL
(Figs. 7A, B) and Brn-2 protein at P10 was detected in part
of the INL and in ganglion cells (Fig. 7C). Within the INL,
Brn-2 appeared to be expressed in some, but not all, bipolar
and amacrine cells. Chx10 RNA (Figs. 7D, E) and protein
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Fig. 6. Transgenic mouse analysis of Chx10 reporters. (A–E) Chx10 2.4 Kb-AP stable transgenic mice were analyzed by AP staining. (A) An E11.5 embryo
was stained for AP in whole-mount. (B–D) Embryos were sectioned and subsequently AP-stained at E11.5 (B), E12.5 (C), or E15.5 (D). (E–G) P0 retinas
were stained for AP in whole-mount for mice stably transgenic for 2.4 Kb-AP (E), transiently transgenic for 3 Kb-AP (F), or transiently transgenic for 3
KbD22-AP (G).
(Fig. 7F) were detected only in the outer half of the INL in
bipolar cells, as would be expected for its known
expression. Expression of Tst-1/SCIP was also analyzed,
but it was only detectable in amacrine and ganglion cells,
consistent with its earlier pattern of expression (data not
shown).
The probable colocalization of Brn-2 and Chx10 in a
subset of developing bipolar cells suggested a role for Brn-2
in control of Chx10 expression in these subsets. To
investigate whether any of the Chx10 reporter constructs
described above contained regulatory elements that could
confer bipolar cell expression, transient transgenic mice
were generated for four of the reporter constructs (Figs. 7G,
H, K, L). Transient transgenic mice for the 3 Kb-AP (Fig.
7G) and 2.4 Kb-AP reporters (Fig. 7H) were analyzed at
P10 for AP staining in the retina. Both constructs (4/4 3 KbAP and 2/3 2.4 Kb-AP) directed high-level AP activity
throughout the outer half of the INL and the inner plexiform
layer (IPL), where bipolar cells have their axonal ramifications. Reporter activity was observed in many, but not all,
bipolar cells, similarly to Brn-2 expression. Stable transgenic 2.4 Kb-AP reporter mice also showed AP reporter
activity in bipolar cells. Reporter activity was analyzed in
immature bipolar cells at P6 (Fig. 7I) and in mature bipolar
cells in adult mice (Fig. 7J). The AP staining pattern
observed at P6 correlated with an early bipolar expression
pattern, in which immature processes ascend through the
outer nuclear layer (ONL) (Rowan and Cepko, 2004). In
adults, AP activity was only detected in what appeared to be
a single kind of cone bipolar cell, as defined morphologically (Ghosh et al., 2004).
In order to assess the importance of the 22 bp enhancer
described above, transient 3 KbD22-AP transgenic mice
were analyzed at postnatal timepoints. None of the transgenic mice analyzed showed reporter activity in bipolar cells
(0/4). Surprisingly, two independent transgenic mice did
have some reporter activity in non-bipolar cells. Fig. 7K
shows an example of a 3 KbD22-AP transgenic mouse that
induced reporter activity exclusively, although variably and
weakly, in photoreceptors. A different 3 KbD22-AP transgenic mouse mediated AP reporter activity exclusively in a
single kind of amacrine cell (data not shown). These results
and expression analysis above suggest that Brn-2 may be an
important regulator of Chx10 expression in bipolar cells. A
different deletion construct, Del1Kb-AP reporter, also failed
to direct AP reporter activity to bipolar cells or elsewhere in
the retina (0/4) (Fig. 7L), suggesting additional elements
controlling bipolar cell expression of Chx10.
Discussion
Analysis of Chx10 regulatory regions
We have identified an enhancer region, Chx10 enhancer
1 (CE1), of 500 bp proximal to the start site of transcription
S. Rowan, C.L. Cepko / Developmental Biology 281 (2005) 240–255
251
Fig. 7. Analysis of gene expression or Chx10 reporter activity in postnatal mouse retinas. (A–F) Sections from P6 retinas (A, D) or P10 retinas (B, C, E, F) were
analyzed by in situ hybridization for Brn-2 (A, B) or Chx10 RNA (D, E) or by immunohistochemistry for Brn-2 (C) or Chx10 protein (F). (G, H) P10 retinas
from mice transiently transgenic for 3 Kb-AP (G) or 2.4 Kb-AP (H) were AP-stained and sectioned. (I, J) Retinas from mice stably transgenic for 2.4 Kb-AP at
P6 (I) or Adult (J) were AP-stained and sectioned. (K, L) P14 retinas from mice transiently transgenic for 3 KbD22-AP (K) or Del1Kb-AP (L) were AP-stained
and sectioned. Scale bar (shown in panel (A)) is 100 Am for all panels. onl, outer nuclear layer; inl, inner nuclear layer; ipl, inner plexiform layer; gcl, ganglion
cell layer.
that directs Chx10 expression in a subset of RPCs as well as
in a subset of bipolar cells. Results from analysis of
transgenic mice expressing the Chx10 2.4 Kb-AP reporter,
which includes CE1, showed that reporter activity began
quite early, establishing CE1 as one of the only described
regulatory regions for gene expression in early RPCs. The
expression in the early RPCs was mosaic and the onset was
at a slightly later time than Chx10 RNA. These observations
imply the existence of an optic vesicle enhancer, as well as
an enhancer(s) that is required for other subsets of RPCs.
These enhancers appear to be absent from the 2.4 Kb
fragment. Since the expression of Chx10 occurs in most, if
not all RPCs, these data suggest that expression in RPCs,
even at one time, as well as over time, is due to multiple
regulatory elements. A similar finding has been reported for
Pax6 in RPCs (Griffin et al., 2002; Kammandel et al., 1999;
Plaza et al., 1999; Xu et al., 1999), suggesting complex
regulatory networks as well as heterogeneity within RPCs.
Our study also led to the identification of two proteins, Brn2 and Tst-1/SCIP, that bind to a 22 bp motif in the CE1 and
demonstrated that they are expressed in the correct temporal
pattern to regulate Chx10 within this subset of RPCs. Fate
mapping experiments may determine if different subsets of
Chx10-expressing RPCS, which may utilize different
enhancers, have equivalent potencies.
Our data point to the existence of multiple regulatory
regions not only for expression in RPCs, but also for bipolar
cells, and further suggest some overlap in the sequences that
regulate Chx10 in both RPCs and in bipolar cells. A 1 Kb
region further 5V to CE1, which was deleted in the Del1KbAP reporter (see Fig. 2), affects expression in the early
retina, as determined by chick electroporation. Interestingly,
deletion of the same 1 Kb region in transient transgenic
mice also led to loss of bipolar cell enhancer activity. The
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simple explanation for this finding is the presence of an
important bipolar cell enhancer within the deleted 1 Kb
region, although we have not eliminated the possibility that
expression was simply weakened in a non-specific manner
due to this deletion. A surprising result was that deletion of
the CE1 22 bp binding site for Brn-2/Tst-1/SCIP led to the
loss of bipolar cell enhancer activity. This finding suggests
that the regulation of Chx10 from the CE1 in RPCs and in
bipolar cells is related.
In keeping with the notion of multiple regulatory regions
directing expression of Chx10 in RPCs, we also saw mosaic
expression in bipolar cells using these reporters. The bipolar
cell enhancer(s) contained within the 2.4 Kb-AP and 3 KbAP reporters did not direct expression to all bipolar cells.
Furthermore, analysis of 2.4 Kb-AP stable transgenic mice
showed reporter activity in what appeared to be a single
kind of bipolar cell. Although this may be an integrationspecific effect, it demonstrates that specific subsets of
bipolar cells may express Chx10 at different levels under
different regulatory controls. Indeed, Chx10 expression
levels vary significantly among different bipolar cells
(Rowan and Cepko, 2004), suggesting heterogeneity in
their regulation. Conceivably, fine mapping of Chx10
bipolar cell enhancers may reveal how such regulation is
achieved and may point to other molecular differences
among types of bipolar cells.
While the locations of enhancers outside of CE1 remain
to be determined, a previous analysis using a Chx10 BAC
reporter mouse indicates that these enhancers must be
located within the BAC, as Chx10 BAC reporter activity
recapitulated the endogenous pattern of Chx10 in RPCs and
bipolar cells (Rowan and Cepko, 2004). This particular
Chx10 BAC contained approximately 55 Kb of sequence
upstream of the Chx10 start methionine and 22 Kb of
sequence downstream of the Chx10 polyadenylation
sequences.
Transcriptional changes in RPCs
The identification of CE1, which directs reporter activity
within a subset of RPCs, opens the possibility that Chx10
enhancer usage in RPCs mirrors changes in RPCs. Such
changes occur both temporally and spatially. One possibility
is exemplified by a preneurogenic to neurogenic transition
zone within the central–peripheral axis of the early retina.
Early central RPCs give rise to neurons, while early RPCs
that are more peripheral divide without giving rise to
neurons. The area where RPCs switch from preneurogenic
to neurogenic correlates with the boundary of expression of
the reporter activity in 2.4 Kb-AP transgenic mice, which
proceeds in a central to peripheral fashion from E11.5–
E15.5. Initiation of CE1 activity may be concomitant with
this change in neurogenic behavior. Chx10 expression levels
also change over time in RPCs, peaking at E16.5, before
decreasing again shortly before bipolar cell genesis. These
expression level changes may be controlled by CE1, which
showed similar temporal patterns of reporter activity. The
spatial and temporal changes in Chx10 expression, and
potentially enhancer usage, may mirror other changes in
RPCs affecting the kinds of neurons generated, the nature of
cell divisions, and/or the length of the cell cycle (Livesey
and Cepko, 2001).
Expression of Brn-2 in RPCs was undetectable during
the preneurogenic phase of RPCs and was present at very
low levels in RPCs that gave rise to the earliest-born cell
types. Brn-2 expression levels increased and reached a peak
at E16.5 and remained at moderate, albeit lower levels,
throughout later stages of development. The finding of
critical POU factor binding sites in the Chx10 22 bp
enhancer suggests that Brn-2 may be controlling these
stages of Chx10 expression. Chx10 reporter activity may
thus reveal the timing of Chx10 regulation by Brn-2,
although early CE1 reporter activity may be mediated by
other POU domain factors, including some that we have not
yet identified. The fact that the 3 KbD22-AP transgenic
mice did not activate reporter activity in E11.5 RPCs (data
not shown) confirms the importance of POU factor binding
in early CE1 activity. Since Tst-1/SCIP bound the 22 bp
enhancer in retinal extracts, it may contribute to regulation
of Chx10 function. However, Tst-1/SCIP expression in
RPCs did not show the temporal correlation with reporter
activity that Brn-2 did. Nonetheless, Tst-1/SCIP appears to
be expressed in what may be subsets of RPCs and warrants
further consideration.
Intriguingly, Brn-2 and Chx10 both bound to a key
Nestin enhancer and may coordinately regulate Nestin
expression. In general, Brn-2 expression and Nestin
expression overlapped greatly, suggesting direct regulation
of Nestin by Brn-2. However, Nestin expression was not
readily detectable when low levels of Brn-2 were observed.
This may be explained by the finding that Nestin was
upregulated in RPCs in Chx10 mutant mice and was
therefore likely directly repressed by Chx10. This is a
significant finding, as direct target genes of Chx10 in RPCs
or bipolar cells have not been described. The proposed
coordinate regulation of Nestin by an early-expressed
transcriptional repressor, Chx10, and a late-expressed transcriptional activator, Brn-2, allows for tight control over
gene changes in RPCs. Also intriguing is the finding that the
consensus Chx10 binding site found in the Nestin intron 2
enhancer contains a binding site for POU domain factors.
This suggests that a number of Chx10 target genes may also
be regulated by Brn-2 or some other POU domain factor.
What other genes might be controlled by Brn-2 and/or
Chx10? One expectation is that genes that control cell cycle
will be differentially expressed in RPCs over time. While
early RPCs divide relatively rapidly and generate new RPCs,
late RPCs transition to generating only two postmitotic
daughter cells (Turner and Cepko, 1987; Turner et al., 1990;
Young, 1985). This transition needs to be carefully controlled. Cyclin-dependent kinase inhibitors are used to
regulate RPC exit and two in particular, p27 and p57, are
S. Rowan, C.L. Cepko / Developmental Biology 281 (2005) 240–255
253
used to allow exit of different subsets of RPCs at different
points of the cell cycle (Dyer and Cepko, 2001). p57 is used
for cell cycle exit only in a subset of cells from E14.5–E17.5
(Dyer and Cepko, 2000). This period corresponds to the peak
expression period of Brn-2 and Chx10, allowing for the
possibility that p57 is regulated in part by these proteins.
Another cell cycle inhibitor, Gas1, is not expressed in early
RPCs but is expressed in a Brn-2/Chx10-like fashion in
intermediate and late RPCs (Rowan et al., 2004). Analysis of
the upstream region of Gas1 revealed the presence of
putative POU factor binding sites. Some of the cell cycle
inhibitor effects of p57 and Gas1 may be alleviated through
expression of Cyclin D1, which contained a 22 bp-like motif,
including a POU factor binding site. Cyclin D1 is also
expressed throughout late RPCs. Mice lacking Chx10 have
lower levels of Cyclin D1 and higher levels of p27,
suggesting both of these genes as targets, although likely
indirect ones, for Chx10 (Green et al., 2003).
The genesis of the embryonic CNS of Drosophila shows
some similarities with vertebrate retinal development.
Neurons are generated in a defined order from multipotent
neuroblasts. Doe and colleagues have elegantly demonstrated that the sequential expression of several transcription
factors is critical to this progression (Isshiki et al., 2001;
Pearson and Doe, 2004). These factors do not specify fate
per se. Rather, they define a temporal state of neuroblasts,
which then generate a variety of daughter cell types, with
each neuroblast generating the daughter cell types appropriate to its segment for that time in development. One of
the transcription factors that controls temporal fate in the
late stages of development is Pdm1, a POU domain
transcription factor highly related to Brn-2. While much
needs to be done to determine if this mechanism of temporal
control is used by the vertebrate retina, our data are at least
consistent with Brn-2 playing a role in temporal control of
retinogenesis.
Genetic analysis of POU domain transcription factors in
CNS development
Acknowledgments
The different members of Class III POU domain transcription factors have been implicated in many processes
during nervous system development. Brn-2 knockout mice
have deficiencies in hypothalamic, supraoptic, and paraventricular nuclei but not other locations where Brn-2 is
expressed, suggesting possible redundancies with other POU
domain proteins (Nakai et al., 1995; Schonemann et al.,
1995; Schreiber et al., 1997). Indeed, studies have pointed to
redundant functions for Brn-1 and Brn-2 in cortical development (McEvilly et al., 2002; Sugitani et al., 2002), Brn-2 and
Tst-1/SCIP in Schwann cells (Jaegle et al., 2003) and Brn-1,
Brn-2, and Tst-1/SCIP in oligodendrocytes (Schreiber et al.,
1997). All class III POU domain transcription factors have
shown similar DNA binding specificities but slightly varying
transactivation potential (Schreiber et al., 1997).
A full understanding of the role for POU domain
transcription factors in Chx10 expression and retinal
development may then require compound mutant analyses.
Tst-1/SCIP appears to be co-expressed with Brn-2, at least
in subsets of RPCs, and these factors may show functional
redundancy. Mice lacking Brn-2 or Tst-1/SCIP die perinatally and retinas have never been analyzed. Given that
phenotypes are likely to be restricted to intermediate and
late-born cell types, careful analysis of late development
stage and/or mature retinas may be necessary. Double
knockout mice have not been generated, and genetic
analysis may require generation of double conditionally
mutant animals. These analyses are critical to determine
what role, if any, there is for Brn-2 and/or other POU factors
in regulation of Chx10 expression. Alternative methods,
such as in ovo electroporation of putative dominantnegative Brn-2 constructs in chick, have not satisfactorily
addressed the POU factor requirement for Chx10 expression
(S. Rowan and C.L. Cepko, unpublished data).
We would like to thank S. Dymecki, R. Pearse, M.G.
Rosenfeld, B. Wang, and A. Bonni for plasmids; A. Ryan,
M.G. Rosenfeld, B. Andersen, and M. Wegner for antibodies; A. Chen for Chx10 baculovirus lysates; and J.
Trimarchi for the Nestin riboprobe. Y. Grad and R. Livesey
provided very helpful assistance in bioinformatic analysis,
A. Abney performed the pronuclear injections, and A. Shaw
assisted with animal husbandry. Finally, we are grateful to
R. Livesey, R. Pearse, and J. Trimarchi for critical review of
the manuscript, and the members of the Cepko and Tabin
laboratories for encouragement and helpful discussions. S.
Rowan was a predoctoral fellow of the Howard Hughes
Medical Institute during a portion of these studies. C.L.
Cepko is an investigator of the Howard Hughes Medical
Institute. This work was also supported by NIH EY08064.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.ydbio.2005.
02.023.
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