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First published online 12 December 2007
doi: 10.1242/dev.009688
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1 Department of Biochemistry and Molecular Biology, The University of Texas M.
D. Anderson Cancer Center, Houston, TX 77030, USA.
2 Graduate Training Program in Genes and Development, The University of Texas
Graduate School of Biomedical Sciences at Houston, Houston, TX 77030,
USA.
3 Developmental Biology Program, Sloan-Kettering Institute, New York, NY 10021,
USA.
* Author for correspondence (e-mail: whklein{at}mdanderson.org)
Accepted 19 October 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Eomesodermin, T-box transcription factors, Pou4f2, Retinal ganglion cells, Optic nerve development, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the mouse, differentiating RGCs can first be visualized with molecular
markers as early as embryonic day 11.5 (E11.5) when dividing retinal
progenitor cells (RPCs) cease mitosis and migrate basally from the neuroblast
side of the retinal epithelium towards the vitreal side to the emerging
ganglion cell layer (GCL) (Young,
1985a
; Young,
1985b). We and others have investigated the mechanisms controlling
RGC fate specification and differentiation, and by doing so, have identified
key regulatory factors that are essential for these events to occur normally
(for reviews, see Mu and Klein,
2004; Mu and Klein,
2008; Cayouette et al.,
2006). In particular, we have proposed a gene regulatory network
model for RGC development featuring a hierarchical cascade of transcription
factors that ultimately controls the expression of terminal downstream
effector genes (Mu et al.,
2005). Two transcription factors occupy key nodes in the RGC gene
regulatory network: the proneural bHLH transcription factor Math5 (Atoh7 -
Mouse Genome Informatics) and the class IV Pou domain-containing transcription
factor Pou4f2 (also called Brn3b). Math5 is responsible for endowing RPCs with
the competence to acquire a RGC fate (Brown
et al., 2001; Wang et al.,
2001), whereas Pou4f2 is positioned genetically downstream of
Math5 and is essential for RGC differentiation, cell survival, neurite
outgrowth and axon pathfinding (Erkman et
al., 1996; Erkman et al.,
2000; Gan et al.,
1996; Gan et al.,
1999; Wang et al.,
2000). Notably, RGCs that lack Pou4f2 are still able to
differentiate, albeit abnormally, and the expression of many RGC genes is
refractory to the absence of Pou4f2 (Mu et
al., 2004). This suggests that pathways operate in parallel along
with Pou4f2 to control RGC differentiation
(Mu et al., 2004;
Mu et al., 2005).
For our RGC gene regulatory network model to be meaningful, connections
must be made between individual upstream regulatory genes such as
Pou4f2 to terminal downstream genes such as those that control axon
projections. Unfortunately, with the exception of Pou4f1
(Trieu et al., 1999), little
is known about regulatory genes that might be direct targets of Pou4f2. We
therefore sought to determine whether any genes encoding transcription factors
downstream of Pou4f2 are direct Pou4f2 targets, and whether these
transcription factors mediate the roles played by Pou4f2 in regulating genes
involved in terminal differentiation events. We focused on the eomesodermin
(Eomes) gene for the following reasons. Eomes (also referred
to as Tbr2) belongs to the Tbr1/Eomes/Tbet subfamily of
genes (Naiche et al., 2005)
that were initially found to play essential roles during trophoblast and
mesoderm development in mice (Russ et al.,
2000; Strumpf et al.,
2005). Eomes is also expressed in the developing central
nervous system and has been implicated in the development of the human central
nervous system; a homozygous breakpoint mutation in a Moroccan family silences
Eomes and leads to microcephaly
(Baala et al., 2007). Recent
studies have also indicated that Eomes is a component of a pathway that
regulates glutamatergic neurogenesis in the cerebral cortex and cerebellum in
mouse development (Bulfone et al.,
1999; Hevner et al.,
2006; Quinn et al.,
2007). Of particular interest from the standpoint of our studies,
Eomes was also found to be expressed in the GCL of the retina
(http://www.scripps.edu/cb/friedlander/gene_expression/),
prompting us to hypothesize that Eomes plays a role in RGC
differentiation.
In this study, we extend the initial findings on Eomes in the developing retina by providing evidence that Eomes is a direct target of Pou4f2, and by demonstrating a role for Eomes in RGC differentiation and optic nerve development. We also discovered a novel role for Eomes: that it regulates myelin ensheathment in the developing optic nerve.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Eomesflox/flox homozygous mice were obtained by
interbreeding against the C57BL/6J background.
Eomesflox/flox mice were viable and fertile.
A CMV-Cre transgenic line was used to generate
Eomes+/
flox in
one-cell zygotes (Su et al.,
2002). We then interbred
Eomes+/flox mice to
recover
Eomesflox/flox
embryos, which displayed phenotypes identical to those observed in
germline-generated Eomes-/- embryos
(Russ et al., 2000). To delete
exon 3 in the developing retina, Eomes+/flox mice were
bred to the Six3-Cre transgenic line, which expresses Cre in
neural progenitor cells in the retina
(Furuta et al., 2000). The
resulting Eomes+/flox;Six3-Cre males were bred to
Eomesflox/flox females to generate
Eomesflox/flox;Six3-Cre embryos and neonates in which exon
3 was specifically deleted in the retina
(Eomesflox/flox;Six3-Cre).
Three PCR primers were used simultaneously to distinguish wild-type, floxed and deleted Eomes alleles (see Fig. 6C): Em15, 5'-CATGATTAAGGAAGGCTGGATGCAC-3'; Em16, 5'-AAGGAGACAGCCTTCACCCAGGC-3'; and Em18, 5'-GACTGAGAAGTAAGAGGGTCAGCAG-3'. PCR primers used to genotype the Six3-Cre transgene were Cre01, 5'-AACGAGTGATGAGGTTCGCAAGAAC-3', and Cre02, 5'-CGCTATTTTCCATGAGTGAACGAACC-3'.
To generate embryos carrying the Eomes-enhancer transgene, a 2.8
kb region from base pairs -1182 to -3918 (translational start site, +1) of
Eomes was subcloned into a plasmid containing the
hsp68-lacZ-pA reporter gene
(Kothary et al., 1989). The
Eomes-enhancer transgene was used to generate transgenic embryos,
which were collected at the desired times.
The US Public Health Service Policy on Humane Care and Use of Laboratory Animals was followed in all experiments using mice, and the M. D. Anderson Institutional Animal Care and Use Committee approved all animal protocols used in these experiments.
Histology studies, in situ hybridization, immunohistochemistry and X-gal staining
Embryos and eyes dissected from embryos or animals were fixed,
paraffin-embedded and sectioned into 7 µm or 12 µm slices for
immunohistochemistry studies or in situ hybridization, respectively
(Mu et al., 2004). After
de-waxing and rehydration, the sections were stained with Hematoxylin and
Eosin for histology studies. In situ hybridization was performed as described
by Mu et al. (Mu et al.,
2004).
For the immunohistochemistry studies, sections were placed in a microwave
oven at 600 watts in 10 mM sodium citrate for 18 minutes to expose the antigen
epitopes. Microwave-treated sections were then incubated with primary
antibodies. A tyromide signal amplification kit (TSA biotin system,
PerkinElmer) was used in conjunction with a NovaRed kit (Vector Labs), to
detect Eomes protein expression by color, and the immunostained sections were
counterstained with Methyl Green. To detect Eomes expression using indirect
immunofluorescence, we used the TSA plus fluorescence kit to optimize the
signal intensity. For double-immunofluorescence labeling, Eomes expression was
detected as described above and the expression of all other proteins was
detected using Alexa-conjugated secondary antibodies (Invitrogen). The primary
antibodies were anti-BrdU (Upstate, 1/4), anti-Brn3b/Pou4f2 (Santa Cruz,
1/200), anti-GFP (Invitrogen, 1/1000), anti-NFL (Invitrogen, 1/250),
anti-syntaxin (Chemicon, 1/1000), anti-Tbr1 (Chemicon, 1/1000) and
anti-Tbr2/Eomes (Chemicon, 1/1000). HRP-conjugated secondary antibody for
tyromide signal amplification was obtained from Jackson ImmunoResearch.
Flat-mount retinas were prepared as described by Xiang et al.
(Xiang et al., 1995).
TUNEL assays and BrdU labeling
TUNEL assays on embryonic retinas were performed using an in situ cell
death detection kit (Roche Applied Science) following the manufacturer's
instructions. Three paraffin-embedded sections were used for data analysis.
For pulse labeling with BrdU, 100 µg of BrdU (Upstate) per gram of body
weight was injected intraperitoneally into pregnant females 1 hour before
euthanization. Embryos were then processed as described previously by Fu et
al. (Fu et al., 2006).
Chromatin immunoprecipitation and electrophoretic mobility shift assays
E15.5 retinas were isolated for chromatin immunoprecipitation (ChIP) assays
following the procedures described by Wells and Farnham
(Wells and Farnham, 2002) and
Kiyama and Klein (Kiyama and Klein,
2007). Two microliters of the immunoprecipitated chromatin DNA and
input genomic DNA were used for PCR amplification with the following primer
sets: Eo3b007, 5'-GACCAACTTGCCACAAAAAACCC-3' and Eo3b008,
5'-CTGAACAGGCTTGCTGCATGCTC-3'; or TTN E4F,
5'-TTGCAGCAACCACTCTTGTC-3' and TTN E4R,
5'-GCATGATGGGAGAGGACCTA-3'. PCR amplifications were performed for
30 cycles. The amplified products were resolved by 6% polyacrylamide gel
electrophoresis and visualized by staining with GelStar nucleic acid gel stain
(CAMBREX).
Electrophoretic mobility shift assays (EMSAs) were performed as described
by Mu et al. (Mu et al.,
2004). Oligonucleotides for the wild-type and mutated site A (see
Fig. 4A) were: wild-type,
5'-GGGAGTGCTGGTGACCCGTTAATGTTGGAATGTTTTCCTA-3' and mutant,
5'-GGGAGTGCTGGTGACCCGAAAAAGTTGGAATGTTTTCCTA-3'. Pou4f2 protein was
synthesized in vitro using a transcription/translation system (Promega), and 2
µl of the transcription/translation reaction mixture was used for each EMSA
reaction.
Cell cultures, DNA transfections, and lacZ reporter gene assays
U2OS cells were cultured in 10% Dulbecco's modified Eagle's medium and 10%
fetal bovine serum at 37°C in a 5% CO2 atmosphere. For the
Eo2.8k (AmBm)-HSP68p-LacZ-pA plasmid, the core Pou4f2
binding site A was changed from 5'-GTTAATGTT-3' to
5'-GAAAAAGTT-3' and site B was changed from
5'-ATTAATGAG-3' to 5'-AAAAATGTG-3' using the
Quikchange site-directed mutagenesis kit (Stratagene). Full-length Pou4f2 cDNA
was subcloned into pIRES-hrGFP-1a (Stratagene) for transactivation
experiments. Transfections were carried out on cover slides placed in six-well
culture plates using FuGene HD (Roche) following the manufacturer's
instructions. Cells were co-transfected with 0.5 µg of the Pou4f2
expression construct or the empty expression vector and 0.5 µg of the
reporter plasmid. X-gal staining was performed 36 hours after transfection.
Transfection efficiency was determined by visually inspecting the number of
GFP-expressing cells for each experiment. All experiments were
performed in triplicate.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
(Fig. 1B). To determine whether
Eomes is expressed at stages earlier than E14.5, we performed
immunohistochemistry with anti-Eomes antibody in sections obtained at E13.5.
Although Eomes protein was readily detected in the forebrain at this stage, it
was not detected in the retina (Fig.
1C).
Because the GCL consists of displaced amacrine cells as well as RGCs, it
was possible that Eomes was expressed in either both or one of these
neuronal cells types. To test these possibilities, we performed
immunohistochemistry in sections of wild-type and Math5-/-
retinas obtained at E16.5. Retinas of Math5-/- embryos
lack RGCs and in their place are increased numbers of displaced amacrine cells
(Wang et al., 2001). Although
wild-type retinas strongly expressed Eomes in the GCL, Eomes
expression in Math5-/- retinas was virtually undetectable
(Fig. 1D,E). These results
demonstrated that RGCs in the GCL are the major site of Eomes
expression from E14.5 to E16.5, a time when newly forming RGCs are populating
the GCL.
Pou4f2 is one of the earliest markers of RGC differentiation
(Gan et al., 1999) and is
expressed in newly forming RGCs within the NBL and in differentiated RGCs that
have migrated to the GCL (Gan et al.,
1999). By comparing Pou4f2 and Eomes protein expression, we
determined how much the expression of the two transcription factors overlapped
and their potential relationship to each other. Although Eomes expression was
not detected in retinas at E13.5 (Fig.
1C), Pou4f2 was already expressed at high levels at this time
(Gan et al., 1999). By E14.5,
Eomes expression was apparent and coincided with the existence of a subset of
Pou4f2-expressing cells within the GCL
(Fig. 1F-H). However, many
Pou4f2-expressing cells, particularly those still migrating to the GCL, did
not express Eomes. In addition, although Pou4f2 was expressed in RGCs
throughout the GCL, Eomes was not expressed in the peripheral-most region
(Fig. 1H). This would be in
keeping with the fact that neuronal differentiation begins at the center and
advances to the periphery of the retina. At E16.5, expression of Pou4f2 was
largely confined to the GCL and many, but not all, of the Pou4f2-expressing
cells also expressed Eomes (Fig.
1I-K). These results suggested that RGCs increasingly express
Eomes as they migrate to the GCL and undergo terminal differentiation.
Weaker expression of Eomes within the NBL at E14.5 and E16.5 did not
overlap with the expression of Pou4f2 and persisted throughout retinogenesis.
As cells within the NBL are a mixture of mitotically active RPCs and newly
formed postmitotic cells that have left the cell cycle and committed to a
retinal cell fate (Le et al.,
2006), we determined whether Eomes was expressed in mitotic cells
by pulse-labeling S-phase cells of the E18.0 retinas with BrdU and comparing
the relative distributions of the S-phase cells and Eomes-expressing cells. We
detected little, if any, overlap between these two cell populations
(Fig. 2A-C), suggesting that
Eomes-expressing cells are mainly postmitotic.
We also observed that retinal Eomes expression persisted into postnatal life. In particular, Eomes was observed to be strongly expressed in the GCL and in the innermost region of the emerging inner nuclear layer (INL) of newborn mice (P0) (Fig. 2D). Weaker expression was observed throughout the NBL (Fig. 2D). However, Eomes expression was largely absent in the GCL but persisted in the INL and NBL of retinas of newly born Math5-/- mice (Fig. 2E). Because amacrine cells are the major cell type in the innermost region of the INL, it is possible that the Eomes-expressing cells were amacrine cells and that the Eomes-expressing cells in the NBL were amacrine cell progenitors.
At P12, Eomes expression was detected in a subpopulation of cells in the GCL, where its expression co-localized with that of the RGC marker neurofilament light subunit (NFL) (Fig. 2F). At P30, Eomes expression in the INL co-localized with that of the pan-amacrine cell marker syntaxin (Fig. 2G) but not of other amacrine cell markers calbindin and parvalbumin (data not shown). A few Eomes-expressing cells in the INL were found to be colocalized with the amacrine marker calretinin (data not shown). These results suggested that Eomes-expressing cells in the INL constituted a distinct subpopulation of amacrine cells.
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These results indicated that Eomes is genetically downstream of
Pou4f2 but they did not show whether Pou4f2 is directly involved in regulating
Eomes transcription. We therefore determined whether conserved
noncoding sequences containing Pou4f2 consensus DNA-binding sites
[5'-(A/G)TTAATGAG(C/T)-3'; Xiang et al.
(Xiang et al., 1995)] could be
identified within the Eomes locus. We focused our search on a 20 kb
stretch of genomic DNA that encompasses the Eomes gene along with 9.5
kb of DNA upstream of the Eomes translational initiation codon and
4.1 kb of DNA downstream of the Eomes translational termination
codon. By comparing human, mouse, rat and dog genomes, we identified several
blocks of conserved noncoding sequences that were located within the upstream
and downstream DNA sequences flanking Eomes
(Fig. 3C). Furthermore, two
putative Pou4f2-binding sites were identified in two closely separated
regions. One site (site B, Fig.
3C,D) began at base pair -2937 (5'-ATTAATGAG-3') and
its sequence precisely matched that of the consensus Pou4f2-binding site. The
other site (site A, Fig. 3C,D)
began at base pair -3854 (5'-GTTAATGTT-3') and differed from
another Pou4f2-binding site by one base pair. However, Pou4f2-binding site B
was not conserved in the corresponding position in human, rat and dog genomes
(Fig. 3D). By contrast,
Pou4f2-binding site A was located within a highly conserved 192 bp region
(Fig. 3D), and this site was
present in all four vertebrate genomes with the exception of one mismatched
nucleotide in the dog genome.
To determine whether the conserved upstream regions containing the
Pou4f2-binding sites were capable of directing expression in the developing
retina, we constructed a transgene containing a 2.8 kb fragment that included
both Pou4f2-binding sites fused to an hsp68-promoter-lacZ-pA reporter
gene (Fig. 3E). The resultant
transgenic E14.5 embryos expressed lacZ in retinas and limbs, a
previously known region of Eomes expression
(Russ et al., 2000)
(Fig. 3E). In control
experiments, a transgene construct with disabling mutations in sites A and B
did not express in either the limb or retina (data not shown). In the
wild-type construct, lacZ was expressed in a narrow strip within the
inner part of the retina just above the GCL
(Fig. 3F).
β-Galactosidase-stained retinal sections co-stained with anti-Eomes
antibody, showed that most of the lacZ-expressing cells did not
co-express the endogenous Eomes protein (compare Fig.
3A with
3F). A possible explanation for
the lack of overlap is that the lacZ-expressing cells represent newly
forming RGCs in the process of migrating to the GCL. To determine whether this
was the case, we performed immunohistochemistry analysis of
β-galactosidase-stained retinal sections from E14.5 transgenic embryos
using anti-Brn3b (Pou4f2) antibody. From these experiments, it was clear that
most of the lacZ-expressing cells also expressed Pou4f2, although
Pou4f2 expression was present in many cells that were not expressing the
transgene (Fig. 3G). These
results demonstrated that the 2.8 kb fragment is sufficient to direct
transgene expression in a subpopulation of RGCs and that this subpopulation
also expresses Pou4f2. However, the transgene appeared to be expressed at
earlier stages in RGC differentiation than the stages when endogenous Eomes is
expressed. The reason why transgene and Eomes expression do not correlate is
uncertain, but it suggests that crucial cis-regulatory elements are
missing in the 2.8 kb region. Consistent with this, a 225 kb Bac transgenic
mouse line containing Eomes fused to an eGFP reporter (Bac
Eomes::GFP) (Kwon and Hadjantonakis,
2007) expresses GFP in the GCL of E14.5 and E16.5 retinas
in a manner similar to that of endogenous Eomes expression
(Fig. 3H-K).
To determine whether either Pou4f2-binding sites A or B within the 2.8 kb fragment bind to Pou4f2, we used EMSA to detect Pou4f2 binding in vitro and ChIP to detect Pou4f2 binding in vivo. EMSA was performed using an in vitro-synthesized full-length Pou4f2 protein and an oligonucleotide probe containing the putative Pou4f2-binding site A within the conserved 192 bp region. We detected a specific Pou4f2-DNA complex but no complex was formed with transcription-translation lysates that lacked the Pou4f2 template (Fig. 4A). The formation of the Pou4f2-DNA complex was inhibited when excess homologous unlabeled oligonucleotide competitor but not when excess oligonucleotide competitor containing a mutated Pou4f2 site was added. Addition of the anti-Pou4f2/Brn3b antibody also blocked complex formation (Fig. 4A). Notably, an oligonucleotide competitor containing the Pou4f2-binding site B did not interfere with complex formation, suggesting that this nonconserved sequence was not a bona fide Pou4f2-binding site.
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To provide further evidence that Pou4f2 is a transcriptional regulator of Eomes, we performed transient transactivation assays in U2OS cells using the 2.8 kb fragment-lacZ reporter gene construct containing Pou4f2-binding sites A and B (see Fig. 3E). A control construct, Eo2.8K (AmBm)-HSP68p-LacZ-pA, was generated in which binding sites A and B were mutated. The number of cells expressing lacZ was sharply increased when the wild-type construct was co-transfected with the Pou4f2 expression plasmid (Fig. 5A,B), with little increase in the number of cells with the mutated construct (Fig. 5C,D). These results demonstrate that Pou4f2 is able to activate transcription from the 2.8 kb fragment and suggest that Pou4f2-binding to site A, and to a lesser extent site B, enhances Eomes transcription in RGCs.
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|
flox/flox retinasflox/flox
mice at P24 showed a slight reduction in the thickness of the GCL compared
with Eomes+/flox controls, whereas the INL and ONL were
normal (Fig. 7A,B). This
suggests that in the absence of Eomes, the RGCs are reduced in number
and that they possess other defects as well.
To visualize the RGC axons, we prepared flat mounts of retinas that were
immunohistochemically stained with anti-NFL antibody. In
Eomesflox/flox
P30 retinas, we observed 
30% reduction in the number of RGC axons in both
the central and peripheral regions of the retina when compared with
Eomes+/flox retinas
(Fig. 7C-F). The density of the
axon bundles was also notably reduced, and their orientation towards the optic
disk appeared abnormal (arrowheads, Fig.
7F). The reduction in the number of RGCs and the defects
associated with RGC axons associated with
Eomesflox/flox
retinas were similar to those identified in Pou4f2-/-
retinas, although the overall effects appeared less severe in
Eomesflox/flox
retinas.
As RGCs undergo enhanced apoptosis between E15.5 and E18.5 in
Pou4f2-/- retinas (Gan
et al., 1999), we performed a TUNEL analysis of sectioned retinas
from Eomes+/flox and
Eomesflox/flox
embryos and postnatal mice to look for differences in apoptosis patterns
between the two. Although there was no significant enhancement of apoptosis
through E16.5 (data not shown), at E18.5 we detected a dramatic increase in
apoptotic cells in
Eomesflox/flox
retinas in both the GCL and NBL with respect to
Eomes+/flox retinas (arrowheads,
Fig. 7G-J). At P1, P6 and P12,
the number of apoptotic cells was consistently higher in
Eomesflox/flox
retinas than in Eomes+/flox controls, but the differences
were less dramatic than they were at E18.
(Fig. 7I,J). Apoptotic cells
were also observed in the GCL, INL and ONL. We did not observe significant
differences between
Eomesflox/flox
retinas and Eomes+/flox controls after P12. These results
suggest that the reduced number of RGCs that we observed in the retinas of P30
Eomesflox/flox
mice resulted from the enhanced apoptosis of RGCs within the GCL between E18
and P12. However, the reason for increased cell death in the retinal NBL, INL
and ONL of embryos and postnatal
Eomesflox/flox
mice is uncertain. Some of the apoptotic cells might represent the
Eomes-positive amacrine cells and their progenitors that weakly expressed
Eomes in the NBL at earlier stages.
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flox/flox
and Eomes+/flox mice, and examined their ultrastructure
(Wang et al., 2000). In a
section about 0.5 cm from the optic disk, a region where mature RGC axons are
well myelinated and ensheathed, low-magnification TEM showed that the area of
Eomesflox/flox
optic nerves was 30% smaller than that of Eomes+/flox
optic nerves (Fig. 8A,B),
consistent with the finding that RGC numbers were reduced by 30%. At
higher magnifications, we observed striking differences between
Eomesflox/flox
and Eomes+/flox optic nerves
(Fig. 8C,D). In particular, the
axons in the optic nerves of Eomes+/flox mice were
ensheathed with a well-organized, compact myelinated layer. However, only a
few axons in the optic nerves of
Eomesflox/flox
mice were myelinated and where present, myelin ensheathment was thinner,
disorganized and loosely packed (Fig.
8C, parts iii-vi, Fig.
8D, parts iii-vi). Moreover, in the optic nerves of
Eomesflox/flox
mice, we observed neurites that were smaller in diameter than those of normal
axons and that contained large numbers of microtubules rather than the
neurofilaments normally associated with RGC axons in the optic nerve
(arrowheads, Fig. 8D, part vi).
This finding was highly reminiscent of the abnormalities we observed in the
optic nerves of Pou4f2-/- mice reported earlier
(Wang et al., 2000). Because
we had not in that study determined whether myelination was abnormal in
Pou4f2-/- optic nerves, we performed a similar TEM
analysis in Pou4f2-/- mice. We found, indeed, that there
were similar defects in myelination and ensheathment in
Pou4f2-/- mice (data not shown), providing additional
evidence that Eomes mediates the functions of Pou4f2 in RGC and optic nerve
development. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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flox/flox
retinas indicated that Eomes plays roles similar to but more
restricted than that of Pou4f2 in RGC differentiation, cell survival
and optic nerve development. Overall, our results argue strongly for our
hypothesis that Eomes is positioned immediately downstream of
Pou4f2 and is a mediator, at least in part, of Pou4f2 function.
Eomes, therefore, appears to be an important transcription factor that
connects Pou4f2 to the expression of genes associated with terminal
differentiation, particularly those involved in axon outgrowth and optic nerve
development.
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flox/flox
retinas, suggesting that Eomes regulates its own expression (C.-A.M. and
W.H.K., unpublished).
Eomes in RGC differentiation and retinal development
Major defects were observed in the retinas of
Eomesflox/flox
mice. This included reduced numbers of RGC, abnormal RGC axon outgrowth and an
aberrant and disorganized myelin sheath surrounding RGC axons. The massive
cell death in the GCL associated with the loss of Eomes indicates that Eomes
is required for the survival of at least some RGCs. Presumably, the absence of
Eomes results in abnormal differentiation, which in turn triggers apoptosis.
The increased number of apoptotic cells in other retinal layers might be
associated with the weak expression of Eomes observed in non-RGCs or
might be an indirect consequence of abnormal levels of apoptotic cells within
the GCL.
One of the striking retinal defects in
Eomesflox/flox
embryos is the disorganized and loosely packed myelin sheaths surrounding RGC
axons in the optic nerve. Axon ensheathment is essential for the normal
propagation of membrane potential, and when defective, results in slow and
continuous depolarization along the axon
(Waxman et al., 1995). It is
therefore likely that the propagation of visual signals into the brain is
severely compromised in
Eomesflox/flox
mice. Because axon outgrowth and myelination require changes in the cell
adhesion properties of axons (Bartsch,
2003), the abnormal ensheathment possibly has its source in
defective cell adhesion. Many members of the T-box transcription factor
family, including Eomes, are involved in regulating genes whose products are
important for cell adhesion (Strumpf et
al., 2005; Inman and Downs,
2006). Eomes acting downstream of Pou4f2 might therefore regulate
the genes whose products are required for substrate adhesion.
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;
Sowden et al., 2001). In
addition, Tbx12 and Tbx20 are expressed in the developing
retina and Tbx12 is required for the formation of retinal cells and the
organization of retinal layers (Carson et
al., 2000; Carson et al.,
2004; Kraus et al.,
2001).
A conserved DNA-binding T-domain is found in all T-box transcription
factors (Kispert and Herrmann,
1993; Bruneau et al.,
2001; Conlon et al.,
2001; Paxton et al.,
2002; Wang et al.,
2004) and a consensus T-box element is believed to be the binding
site for all members of the T-box transcription factor family. Thus, it is
possible that other T-box factors partially compensate for the loss of Eomes.
To examine whether the absence of Eomes resulted in an upregulation of other
T-box genes, which would be indicative of compensation, we determined the
expression patterns of eight T-box genes in Eomes+/flox
and
Eomesflox/flox
retinas. We were able to detect retinal expression of only Trb1 and
Tbx5, and no significant differences in expression were observed
between Eomes+/flox and
Eomesflox/flox
retinas (see Fig. S1 in the supplementary material). It therefore seems likely
that Eomes has evolved a specialized function in RGC development and is
positioned in the RGC gene regulatory hierarchy downstream of Math5 and
Pou4f2.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/2/271/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Baala, L., Briault, S., Etchevers, H. C., Laumonnier, F., Natiq, A., Amiel, J., Boddaert, N., Picard, C., Sbiti, A., Asermouh, A. et al. (2007). Homozygous silencing of T-box transcription factor EOMES leads to microcephaly with ploymicrogyria and corpus callosum agenesis. Nat. Genet. 39,454 -456.[CrossRef][Medline]
Bartsch, U. (2003). Neural CAMS and their role in the development and organization of myelin sheaths. Front. Biosci. 8,d477 -d490.[Medline]
Brown, N. L., Patel, S., Brzezinski, J. and Glaser, T.
(2001). Math5 is required for retinal ganglion cell and optic
nerve formation. Development
128,2497
-2508.
Bruneau, B. G., Nemer, G., Schmitt, J. P., Charron, F., Robitaille, L., Caron, S., Conner, D. A., Gessler, M., Nemer, M., Seidman, C. E. et al. (2001). A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106,709 -721.[CrossRef][Medline]
Bulfone, A., Martinez, S., Marigo, V., Campanella, M., Basile, A., Quaderi, N., Gattuso, C., Rubenstein, J. L. and Ballabio, A. (1999). Expression pattern of the Tbr2 (Eomesodermin) gene during mouse and chick brain development. Mech. Dev. 84,133 -138.[CrossRef][Medline]
Carson, C. T., Kinzler, E. R. and Parr, B. A. (2000). Tbx12, a novel T-box gene, is expressed during early stages of heart and retinal development. Mech. Dev. 96,137 -140.[CrossRef][Medline]
Carson, C. T., Pagratis, M. and Parr, B. A. (2004). Tbx12 regulates eye development in Xenopus embryos. Biochem. Biophys. Res. Commun. 318,485 -489.[CrossRef][Medline]
Cayouette, M., Poggi, L. and Harris, W. A. (2006). Lineage in the vertebrate retina. Trends Neurosci. 29,563 -570.[CrossRef][Medline]
Conlon, F. L., Fairclough, L., Price, B. M., Casey, E. S. and
Smith, J. C. (2001). Determinants of T box protein
specificity. Development
128,3749
-3758.
Erkman, L., McEvilly, R. J., Luo, L., Ryan, A. K., Hooshmand, F., O'Connell, S. M., Keithley, E. M., Rapaport, D. H., Ryan, A. F. and Rosenfeld, M. G. (1996). Role of transcription factors Brn-3.1 and Brn-3.2 in auditory and visual system development. Nature 381,603 -606.[CrossRef][Medline]
Erkman, L., Yates, P. A., McLaughlin, T., McEvilly, R. J., Whisenhunt, T., O'Connell, S. M., Krones, A. I., Kirby, M. A., Rapaport, D. H., Bermingham, J. R. et al. (2000). A POU domain transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system. Neuron 28,779 -792.[CrossRef][Medline]
Farley, F. W., Soriano, P., Steffen, L. S. and Dymecki, S. M. (2000). Widespread recombinase expression using FLPeR (flipper) mice. Genesis 28,106 -110.[CrossRef][Medline]
Fu, X., Sun, H., Klein, W. H. and Mu, X. (2006). Beta-catenin is essential for lamination but not neurogenesis in mouse retinal development. Dev. Biol. 299,424 -437.[CrossRef][Medline]
Furuta, Y., Lagutin, O., Hogan, B. L. and Oliver, G. C. (2000). Retina- and ventral forebrain-specific Cre recombinase activity in transgenic mice. Genesis 26,130 -132.[CrossRef][Medline]
Gan, L., Xiang, M., Zhou, L., Wagner, D. S., Klein, W. H. and
Nathans, J. (1996). POU domain factor Brn-3b is required for
the development of a large set of retinal ganglion cells. Proc.
Natl. Acad. Sci. USA 93,3920
-3925.
Gan, L., Wang, S. W., Huang, Z. and Klein, W. H. (1999). POU domain factor Brn-3b is essential for retinal ganglion cell differentiation and survival but not for initial cell fate specification. Dev. Biol. 210,469 -480.[CrossRef][Medline]
Hevner, R. F., Hodge, R. D., Daza, R. A. and Englund, C. (2006). Transcription factors in glutamaergic neurogenesis: conserved programs in neocortex, cerebellum, and adult hippocampus. Neurosci. Res. 55,223 -233.[CrossRef][Medline]
Inman, K. E. and Downs, K. M. (2006). Brachyury
is required for elongation and vasculogenesis in the murine allantois.
Development 133,2947
-2959.
Kispert, A. and Herrmann, B. G. (1993). The Brachyury gene encodes a novel DNA binding protein. EMBO J. 12,3211 -3220.[Medline]
Kiyama, T. and Klein, W. H. (2007). SpGataE, a Strongylocentrotus purpuratus ortholog of mammalian Gata4/5/6: protein expression, interaction with putative target gene spec2a, and identification of friend of Gata factor SpFog1. Dev. Genes Evol. 217,651 -663.[CrossRef][Medline]
Kothary, R., Clapoff, S., Darling, S., Perry, M. D., Moran, L.
A. and Rossant, J. (1989). Inducible expression of an
hsp68-lacZ hybrid gene in transgenic mice. Development
105,707
-714.
Kraus, F., Haenig, B. and Kispert, A. (2001). Cloning and expression analysis of the mouse T-box gene tbx20. Mech. Dev. 100,87 -91.[CrossRef][Medline]
Kwon, G. S. and Hadjantonakis, A. K. (2007). Eomes::GFP - a tool for live Imaging cells of the trophoblast, primitive streak, and telencephalon in the mouse embryo. Genesis 45,208 -217.[CrossRef][Medline]
Le, T. T., Wroblewski, E., Patel, S., Riesenberg, A. N. and Brown, N. L. (2006). Math5 is required for both early retinal neuron differentiation and cell cycle progression. Dev. Biol. 295,764 -778.[CrossRef][Medline]
Mu, X. and Klein, W. H. (2004). A gene regulatory hierarchy for retinal ganglion cell specification and differentiation. Semin. Cell Dev. Biol. 15,115 -123.[CrossRef][Medline]
Mu, X. and Klein, W. H. (2008). Gene regulatory networks and the development of retinal ganglion cells. In Eye, Retina, and Visual System of the Mouse (ed. L. M. Chalupa and R. W. Williams), pp.?? Cambridge, MA: MIT Press. In press.
Mu, X., Beremand, P. D., Zhao, S., Pershad, R., Sun, H., Scarpa,
A., Liang, S., Thomas, T. L. and Klein, W. H. (2004).
Discrete gene sets depend on POU domain transcription factor
Brn4b/Brn-3.2/POU4f2 for their expression in the mouse embryonic retina.
Development 131,1197
-1210.
Mu, X., Fu, X., Sun, H., Beremand, P. D., Thomas, T. L. and Klein, W. H. (2005). A gene network downstream of transcription factor math5 regulates retinal progenitor cell competence and ganglion cell fate. Dev. Biol. 280,467 -481.[CrossRef][Medline]
Naiche, L. A., Harrelson, Z., Kelly, R. G. and Papaioannou, V. E. (2005). T-box genes in vertebrate development. Annu. Rev. Genet. 39,219 -239.[CrossRef][Medline]
Paxton, C., Zhao, H., Chin, Y., Langner, K. and Reecy, J. (2002). Murine Tbx2 contains domains that activate and repress gene transcription. Gene 283,117 -124.[CrossRef][Medline]
Pflugfelder, G. O., Roth, H., Poeck, B., Kerscher, S., Schwarz,
H., Jonschker, B. and Heisenberg, M. (1992). The lethal
(1)optomotor-blind gene of Drosophila melanogaster is a major organizer of
optic lobe development: isolation and characterization of the gene.
Proc. Natl. Acad. Sci. USA
89,1199
-1203.
Quinn, J. C., Molinck, M., Martynoga, B. S., Zaki, P. A., Faedo, A., Bulfone, A., Hevner, R. F., West, J. D. and Price, D. J. (2007). Pax6 controls cerebral cortical cell number by regulating exit from the cell cycle and specifies cortical cell identity by a cell autonomous mechanism. Dev. Biol. 302, 50-65.[CrossRef][Medline]
Russ, A. P., Wattler, S., Colledge, W. H., Aparicio, S. A., Carlton, M. B., Pearce, J. J., Barton, S. C., Surani, M. A., Ryan, K., Nehls, M. C. et al. (2000). Eomesodermin is required for mouse trophoblast development and mesoderm formation. Nature 404, 95-99.[CrossRef][Medline]
Sowden, J. C., Holt, J. K., Meins, M., Smith, H. K. and
Bhattacharya, S. S. (2001). Expression of Drosophila
omb-related T-box genes in the developing human and mouse neural retina.
Invest. Ophthalmol. Vis. Sci.
42,3095
-3102.
Strumpf, D., Mao, C. A., Yamanaka, Y., Ralston, A.,
Chawengsaksophak, K., Beck, F. and Rossant, J. (2005). Cdx2
is required for correct cell fate specification and differentiation of
trophectoderm in the mouse blastocyst. Development
132,2093
-2102.
Su, H., Mills, A. A., Wang, X. and Bradley, A. (2002). A targeted X-linked CMV-Cre line. Genesis 32,187 -188.[CrossRef][Medline]
Trieu, M., Rhee, J. M., Fedtsova, N. and Turner, E. E.
(1999). Autoregulatory sequences are revealed by complex
stability screening of the mouse brn-3.0 locus. J.
Neurosci. 19,6549
-6958.
Wang, S. W., Gan, L., Martin, S. E. and Klein, W. H. (2000). Abnormal polarization and axon outgrowth in retinal ganglion cells lacking the POU-domain transcription factor Brn-3b. Mol. Cell. Neurosci. 16,141 -156.[CrossRef][Medline]
Wang, S. W., Kim, B. S., Ding, K., Wang, H., Sun, D., Johnson,
R. L., Klein, W. H. and Gan, L. (2001). Requirement for math5
in the development of retinal ganglion cells. Genes
Dev. 15,24
-29.
Wang, T. F., Ding, C. N., Wang, G. S., Luo, S. C., Lin, Y. L., Ruan, T., Rubenstein, J. L. and Heueh, Y. P. (2004). Identification of Tbr-1/CASK complex target genes in neurons. J. Neurochem. 91,1483 -1492.[CrossRef][Medline]
Waxman, S. G., Kocsis, J. D. and Black, J. A. (1995). Pathophysiology of demyelinated axons. In The Axon: Structure, Function and Pathophysiology (ed. S. G. Waxman, J. D. Kocsis and P. K. Stys), pp. 438-461. New York: Oxford University Press.
Wells, J. and Farnham, P. J. (2002). Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation. Methods 26, 48-56.[CrossRef][Medline]
Xiang, M., Zhou, L., Macke, J. P., Yoshioka, T., Hendry, S. H., Eddy, R. L., Shows, T. B. and Nathans, J. (1995). The Brn-3 family of POU domain factors: primary structure, binding specificity, and expression in subsets of retinal ganglion cells and somatosensory neurons. J. Neurosci. 15,4762 -4785.[Abstract]
Young, R. W. (1985a). Cell differentiation in the retina of the mouse. Anat. Rev. 212,199 -205.[CrossRef]
Young, R. W. (1985b). Cell proliferation during postnatal development of the retina in the mouse. Brain Res. 353,229 -239.[Medline]
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