PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Invest Ophthalmol Vis Sci. Author manuscript; available in PMC Feb 1, 2009.
Published in final edited form as:
PMCID: PMC2615067
NIHMSID: NIHMS74629

Characterization of a transient TCF/LEF-responsive progenitor population in the embryonic mouse retina

Abstract

Purpose

High mobility group (HMG) transcription factors of the T-cell-specific transcription factor/lymphoid enhancer binding factor (TCF/LEF) family are a class of intrinsic regulators that are dynamically expressed in the embryonic mouse retina. Activation of TCF/LEFs is a hallmark of the Wnt/β-catenin pathway, however, the requirement for Wnt/β-catenin and noncanonical Wnt signaling during mammalian retinal development remains unclear. Our goal was to characterize more fully a TCF/LEF-responsive retinal progenitor population in the mouse embryo, and to correlate this with Wnt/β-catenin signaling.

Methods

TCF/LEF activation was analyzed in the TOPgal reporter mouse (TOP: TCF optimal promoter) at embryonic ages and compared to Axin2 mRNA expression, an endogenous readout of Wnt/β-catenin signaling. Reporter expression was also examined in embryos with a retina-specific deletion of the/β-catenin gene (Ctnnb1), using Six3-Cre transgenic mice. Finally, the extent to which TOPgal cells coexpress cell cycle proteins, basic helix-loop–helix (bHLH) transcription factors and other retinal cell type markers was tested by double-immunohistochemistry.

Results

TOPgal reporter activation occurs transiently in a subpopulation of embryonic retinal progenitor cells. Axin2 is not expressed in the central retina, and TOPgal reporter expression persists in the absence of β-catenin. Although a proportion of TOPgal-labeled cells are proliferative, most coexpress the cyclin-dependent kinase inhibitor p27/Kip1.

Conclusions

TOPgal cells give rise to the four earliest cell types: ganglion cells, amacrines, horizontals and photoreceptors. TCF/LEF activation in the central retina does not correlate with Wnt/β-catenin signaling, pointing to an alternate role for this transcription factor family during retinal development.

Introduction

The neural retina develops from ventral forebrain neuroepithelium, and when mature, is comprised of seven major types of neurons and glia. These retinal cell types are generated in an evolutionary conserved order with ganglion cells formed first, then cones, horizontals, amacrines and rods, with bipolars and Müller glia generated last.15 Retinal progenitors are generally multipotent; however, over time they exhibit both lineage and competence restrictions such that fewer and fewer distinct cell types arise as development proceeds.611 The competence of retinal progenitors is regulated by both extracellular and intrinsic factors.1114 The intrinsic factors have so far fallen into main two protein classes, either basic helix-loop-helix (bHLH) or homeobox transcription factors. In particular, the bHLH factors regulate the development of particular retinal cell types, as they do throughout the vertebrate nervous system.11,14,15 For example, one bHLH factor, Math5, is required for ganglion cell formation.1620 while another, NeuroD, is necessary for amacrine, S cone and rod photoreceptor cell genesis.2123 Interestingly, different bHLH factors are further segregated by their expression at distinct stages of the mitotic cell cycle, with Ngn2 and Mash1 expressed by many S phase retinal progenitors24, and Math5 present in newly postmitotic, transitional cells.25,26 Because the expression patterns and functions of these intrinsic factors are insufficient to explain how all retinal cell types develop, their integration with other pathways is essential to understand retinal neurogenesis at the molecular level.

We and others observed activity of the HMG transcription factors TCF/LEF in retinal progenitors in the mouse embryo, consistent with endogenous TCF/LEF mRNA expression.2729 TCF/LEFs (TCF1/TCF7, LEF1, TCF3/TCF711, TCF4/TCF712) are best known for mediating Wnt (wingless-type MMTV integration site family) signaling through interaction with the co-activator β-catenin.3032 Upon Wnt binding to the Frizzled receptor, stabilized β-catenin translocates to the nucleus where it interacts with TCF/LEFs, to activate the transcription of target genes. Previous studies in Xenopus suggested that Wnt/β-catenin signaling is critical for early retinal development, where it controls neural competence of retinal progenitor cells by regulating Sox2 function.33 However, in the mammalian eye, the role of Wnt/β-catenin signaling in retinal progenitors is less clear. Some aspects of TCF/LEF activity in the developing retina directly correlate with Wnt/β-catenin signaling, for example, during ciliary body and iris formation.3438 However, conditional deletion of β-catenin in the central embryonic retina only resulted in abnormal lamination, with no obvious defects in retinal progenitor proliferation or cell fate specification.39,40 In addition, direct modulation of LEF function in either chick or mouse retinal experiments did not affect progenitor proliferation or differentiation.38,39 Therefore, Wnt/β-catenin signaling appears largely dispensable during embryonic retinal neurogenesis.

Here we characterize TCF/LEF-responsive retinal cells in greater depth during mouse embryonic development. Transgenic constructs with multimerized TCF/LEF binding sites upstream of a minimal promoter that drive expression of a reporter are commonly used as a readout of activated TCF/LEF-mediated transcription. At least four different mouse TCF/LEF reporter lines have been described.4145 We analyzed the TOPgal reporter generated by Elaine Fuchs and colleagues44, and demonstrate TCF/LEF activity within retinal progenitors that differentiate as ganglion cells, cone photoreceptors, amacrines or horizontal cells. Surprisingly, this reporter is active in the absence of β-catenin suggesting that TCF/LEF transcription factors work independent from the Wnt/β-catenin pathway during retinal neurogenesis.

Material and Methods

Animals

TOPgal mice 44 were crossed with Ctnnb1 mice containing an allele of β-catenin with exons 2–6 of the gene flanked by loxP sites (termed floxed β-catenin in this paper).46 A separate stock of mice carrying the Six3-Cre transgene (Tg(Six3-cre)69Frty) was crossed with a β-catenindel allele.46,47 For our experiments, Six3-Cre; β-catenin del/+ mice were crossed to those homozygous for both the TOPgal transgene and floxed β-catenin (β-cateninFL/FL). In the resulting embryonic litters, Six3-Cre;TOPgal;β-catenin del/FL embryos (termed β-catenin mutant embryos) were compared to control littermates containing one wild type allele of β-catenin or no Six-Cre transgene. Genotyping for floxed β-catenin, β-catenin del and the Six3-Cre was performed as described.46,47 TOPgal genotyping by PCR used the primers: 5′ cgatgaatccagaaaagcgg 3′ (forward); 5′ gcttgggtggagaggctatt 3′ (reverse) and 35 cycles with an annealing temperature of 62°C within a standard protocol. The day of the observed plug was designated embryonic day 0.5. Animal experiments were performed according to the guidelines of the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the University of Utah and Children’s Hospital Research Foundation Institutional Animal Care and Use Committees.

Detection of X-Gal Activity

Tissue was fixed with 4% PFA for 10–20 min depending on the age. Cryostat sections (14–16 μm) were incubated with X-Gal substrate for 8–15 hours, post-fixed and mounted in Fluoromount G (Southern Biotech). The pattern of β-galactosidase activity was confirmed by immunodetection of β-galactosidase protein expression (see below). Images were taken using Nomarski optics with an Olympus BX51 microscope and processed using Photoshop CSS.

In situ hybridization

Whole mount in situ hybridization on separate eyes was performed as previously described48,49 using digoxygenin-labeled antisense and sense riboprobes for Axin250 and an antisense probe for Math5.19

Immunohistochemistry and BrdU pulse labeling

Embryonic tissue was fixed for 45–60 min with 4% paraformaldehyde (PFA) in PBS. For BrdU pulse labeling, pregnant mice were injected as described in Le et al., 2006. Cryostat sections (10–12 μm) were processed for antibody labeling as previously described.51 Primary antibodies used are: rat anti-β-galactosidase (1:750–1000, a gift from Tom Glaser), mouse anti-p27/Kip1 (1:100, BD Transduction), rabbit anti-Ki67 (1:1000, Vector), rabbit anti-phosphorylated histone H3 (1:1000, Upstate), mouse anti-PCNA (1:1000, DakoCytomation), rat anti-BrdU (1:200, Serotec), rabbit anti-β-catenin (1:4000, Sigma), rabbit anti-Hes1 (1:1000, Brown lab), rabbit anti-Ngn2 (1:1000, a gift from Masato Nakufuku), goat anti-Brn3b (1:50, Santa Cruz), rabbit anti-Otx2 (1:2500, Chemicon), rabbit anti-Olig2 (1:1000, a gift from Masato Nakufuku), rabbit anti-RXRγ (1:200, Santa Cruz), rabbit anti-Ptfla (1:800, a gift from Helena Edlund). Secondary antibodies were directly conjugated with Alexa Fluor 488, Alexa Fluor 568 or Alexa 594 (1:1000–2000, Molecular Probes) or indirectly with biotin (horse; 1:200, Vector or rodent; 1:200, Jackson) and strepavidin-conjugated Texas Red (1:200, Jackson Immunologicals). Nuclei were counterstained with 4,6-dimidino-2-phenylindole (DAPI). PCNA labeling was performed after antigen retrieval by immersion of slides in hot citric acid buffer for 30–40 min following anti-β-galactosidase detection. Anti-BrdU labeling was performed after antigen retrieval in 0.2M HCl/0.5% Triton XI00 for 1 hour. At least four β-catenin mutant and control embryos were analyzed for β-catenin and β-galactosidase expression.

Cell Counting

Right and left eyes of three TOPgal transgenic animals were analyzed using two coronal, nonadjacent 10–12 μm sections of the central retina. Images were taken using Olympus BX51 epifluorescence and Olympus Confocal microscope FV1000 and subsequently processed using ImageJ and Photoshop CS3; or using an Axioplan2 epifluorescent microscope equipped with Zeiss deconvolution, Axiovision software and processed with Photoshop 7. For each marker, the percentage of double-positive cells per total number of β-galactosidase-positive cells was determined in each retinal section, from a minimum of three independent TOPgal embryos per age.

Results

The TOPgal reporter is transiently active in the embryonic mouse retina

In this study, we used TOPgal transgenic mice to define which retinal cells have TCF/LEF activity during embryonic development. This transgene contains three TCF/LEF consensus binding sites and the c-fos minimal promoter driving β-galactosidase expression.44,52 At E9.5, β-galactosidase expression is first observed in the dorsal optic vesicle, consistent with other TCF/LEF reporters (not shown and 29,43,53). At E10.5, the TOPgal reporter is also activated in the dorsal retinal pigment epithelium (RPE) of the optic cup, dorsal optic stalk and periocular mesenchyme, while no activation is observed in the retina (Fig. 1A). At E11.5, scattered cells in the neural retina initiate reporter expression and the number of these cells is obviously increased by E13.5 (Fig. 1B, C). Starting at E14, the spatial localization of the TOPgal (TOPGal+) expressing population changes; as these cells accumulate near the ventricular/proliferative zone of the retina, adjacent to the RPE where M-phase progenitors and photoreceptor precursors reside (Fig. 1D, shown here at E15.5). At E16.5, the number of TOPGal+ cells decreases substantially and is barely detectable by E17.5 (Fig. 1E, F). No TOPgal+ cells were present in the postnatal and adult central retina (not shown). These results indicate that activation of the TCF/LEF reporter occurs transiently in a subpopulation of embryonic retinal cells. Consistent with the activity of other TCF/LEF reporters, we also observed transient expression in the ciliary body, iris and RPE between birth and postnatal day 30 (not shown).5,4,28,29

Fig. 1
Transient TCF/LEF activation in the embryonic retina of the TOPgal reporter mouse

TOPgal activity in the central mouse retina is independent of Wnt/β-catenin signaling

We observed TOPgal reporter activity in the developing retina that is substantially different from TCF/LEF activity reported by other labs using different transgenic lines. To determine how well our TOPgal activity reports Wnt/β-catenin signaling activity, we examined the mRNA expression of the scaffold protein Axin2, which is a universal readout and antagonist for Wnt/β-catenin signaling. Axin2 mRNA is detectable at E13.5 in the RPE, periocular mesenchyme and presumptive ciliary body and iris, but, neither the antisense (Fig. 2A) nor sense (Fig. 2B) probes showed any expression in the central retina, where Math5 mRNA is easily observed (Fig. 2C).

Fig. 2
Expression of Axin2 in the embryonic mouse eye at E13.5

The absence of Axin2 expression in the central retina suggests that either Wnt/β-catenin signaling may not be active, or Axin2 expression may not reflect such signaling in this tissue. To examine this question further, we examined embryonic retinas in which the β-catenin gene was conditionally inactivated by Six3-Cre.40,47 We confirmed that Six3-Cre-mediated recombination at E14.5 is detectable in the optic stalk, in the central retina and in some cells of the peripheral retina (S.F and M.L.V.; not shown). Normally, β-catenin protein is present throughout the retina, including within TOPgal+ cells (Fig. 3A–F).40 In β-catenin mutant embryos, we observed variable β-catenin deletion in large patches in the central retina, along with lamination defects consistent with other studies (Fig. 3H, K).40,55 Surprisingly, we also observed the persistence of TOPgal+ cells in regions lacking β-catenin expression (Fig. 3D–I). This TCF/LEF activity, suggests that TCF/LEF may act independently of Wnt/β-catenin signaling during embryonic retinal development.

Fig. 3
TOPgal reporter activity independent of β-catenin expression

The TOPgal expression is present in both proliferating and postmitotic retinal progenitors

To define TOPgal+ retinal cells more fully, we next examined the cell cycle status of these cells by performing antibody double-labeling for β-galactosidase and cell cycle markers (Fig. 4 and Table 1). To understand if TCF/LEF activity is confined to a particular stage of the cell cycle, we quantified the proportion of TOPgal+ cells that coexpress proliferating cell nuclear antigen (PCNA), Ki67, BrdU, phosphohistone H3 and the cyclin-dependent kinase inhibitor p27/Kip1. The markers PCNA and Ki67 are expressed in all phases of the cell cycle56. Our analysis shows that 53% (E13.5) and 56% (E15.5) of the TOPgal+population coexpress PCNA (Fig. 4A–C, Table 1). Likewise, we observed 69% (E13.5) and 63% (E15.5) coexpression of TOPgal with Ki67 (Fig. 4D–F, Table 1). Next we found that there is essentially no overlap of TOPgal expression with BrdU (Table 1) and only 7% of TOPgal+ cells coexpress the M phase marker phosphohistone H3 (Fig. 4G–I, Table 1).57 However, more than 76% of TOPgal+ cells coexpress the CKI p27/Kip1 at E13.5 and E15.5 (Fig. J–L, Table 1). These observations indicate that TCF/LEF-responsive cells are largely nonproliferative, with the greatest degree of overlap occurring with p27/Kip1 expression. This suggests that TCF/LEF is most active when retinal progenitors are transitioning out of the cell cycle to differentiate.

Fig. 4
TCF/LEF activation occurs in retinal progenitors in different stages of the cell cycle
Table 1
Percentage of β-galactosidase-positive cells co-expressing the following markers

TOPgal reporter expression is present in multiple early retinal cell types

Next we wished to correlate TOPgal+ cells with markers of neuronal specification and differentiation. First we compared β-galactosidase expression to that of Math5 and NeuroD, two bHLH factors known to be expressed by a number of retinal cells at E13.5 and E15.5.19,22,58 However, only rare TOPgal+” cells were found to coexpress either Math5 or NeuroD in TOPgal retinal sections (not shown). Next we tested the bHLH factor Neurogenin2 (Ngn2), whose lineage contributes to all seven retinal cell types, but in which an obvious role in cell type specification has not yet been determined.24 We found that at E13.5, but not at E15.5, a subset of TOPgal cells was also Ngn2+. The double positive cohort represented 26% of the TOPgal+ population (Fig 5A–C, Table 1). Then, expression of the bHLH factor Olig2 was tested for the extent to which it overlaps with the TOPgal-expressing cells. In the mouse retina, Olig2 initiates expression at E13 in undifferentiated progenitors and is present postnatally in ganglion cells, amacrines, horizontals, bipolar neurons and Müller glia.59,60 Interestingly, we found substantial coexpression of β-galactosidase and Olig2 at both E13.5 and E15.5, 64% and 57% respectively (Fig. 5D–F, Table 1). Finally, no overlap of the TOPgal reporter was seen with the bHLH factor Hes1 (Fig. 5G–I), which is expressed in proliferating progenitors and acts as a transcriptional repressor to regulate the timing of neuronal differentiation.6163 Thus, when combined with the high numbers of TOPgal-p27Kip1 coexpressing cells our data suggest that TCF/LEF activity may act within retinal progenitors as they exit the cell cycle.

Fig. 5
Overlap of TOPgal reporter expression with transcription factors expressed in retinal progenitor cells at E13.5

TCF/LEF activity is present in neurons or neuronal precursors generated during the embryonic period

Our observations suggest that TOPgal+ cells largely represent the postmitotic transitional cells of separate neuronal lineages. Therefore, we predicted that TOPgal reporter expression would be present in nascent ganglion cells, photoreceptors, amacrines and horizontal cells. The paired-type homeobox transcription factor Otx2 controls maturation of photoreceptor and bipolar cells in the rodent retina.6467 Otx2 is coexpressed in many TOPgal+ cells: 59% at E13.5 and 67% at E15.5 (Fig. 6A–C, Table 1). To determine independently that TCF/LEF activity is present in photoreceptor precursors, we looked in the outer retina for coexpression of β-galactosidase with RXRγ, a nuclear hormone receptor essential for fine-tuning the differentiation of cone subpopulations (arrowheads in Fig. 6D–F).6870 In the embryonic retina, RXRγ also labels ganglion cells and we observed β-galactosidase- RXRγ double positive inner retinal cells as well (arrows in Fig. 6D–F). To quantify the percentage of TOPgal+ ganglion cells, we compared β-galactosidase and Brn3b coexpression71,72 and found 30% of E12.5 TOPgal+ cells also express Brn3b (Table 1 and data not shown). Finally, to examine early amacrine and horizontal neurons, we compared β-galactosidase expression to that of Ptf1a, a bHLH factor expressed specifically by amacrine and horizontal precursors.73,74 Between 20% (E13.5) and 23% (E15.5) of the TOPgal+ cells co-express Ptf1a indicating that the TOPgal+ cells also contribute to amacrine and horizontal cell fates (Fig. 6G–I, Table 1).

Fig. 6
TCF/LEF is active in embryonic cone photoreceptors, ganglion, amacrine and horizontal cells

Discussion

Here we define more fully the transient TCF/LEF-responsive population of retinal progenitors that contribute to the four earliest retinal cell types: ganglion cells, amacrines, horizontals and cone photoreceptors. We conclude that TCF/LEF reporter activity does not require Wnt/β-catenin signaling, because a) Axin2 is not expressed in the central retina, and b) TOPgal expression persists in the absence of functional β-catenin. Our results point to a possible regulatory role for TCF/LEFs during early mammalian retinal neurogenesis, which correlates with the cell cycle exit of these early-generated cell types.

TCF/LEFs encode different protein isoforms and, depending on the interaction with other cofactors, they can activate or repress target gene transcription.3032,75 For reviews, see TCF/LEFs promote a variety of processes when activated by the Wnt/β-catenin pathway, such as embryonic patterning, regeneration, stem cell renewal or differentiation in both embryonic and adult tissues, and deregulated activity can lead to tumor formation. For reviews, see 79,80 Substantial progress has been made in elucidating the interaction of β-catenin with TCF/LEFs, however, previous reports show that other coregulators can interact with TCF/LEFs to promote transcription in the absence of β-catenin. LEF1 can transactivate the T-cell receptor α enhancer in a complex with the co-activators ALY and AML-1, which does not require the β-catenin interaction domain of LEF-1.81,82 Similarly, a LEF1 version lacking the β-catenin binding domain activates transcription of the Xenopus homeobox gene twin by interacting with effectors of the TGFβ/Activin signals, Smad2, 3 and 4.83 Our findings also suggest that not all TCF/LEF functions are dependent on Wnt/β-catenin signaling. In the developing retina, LEF1 and TCF3 expression is present in the central retina between E12.5 and E14.5 while TCF1 and TCF4 show weak expression at E14.5. However, targeted inactivation of TCF1, LEF1 and TCF4 do not appear to cause eye defects, which may be due to functional redundancy8490. Mutations in both TCF1 and LEF1 result in severe developmental defects and lethality around E10, which has so far prevented further analyses of retinal neurogenesis.85 Thus, loss of function-studies where multiple TCF/LEF genes are simultaneously deleted during retinal development are required to elucidate the role of TCF/LEFs in this tissue.

Surprisingly, our results strongly suggest that Wnt/β-catenin signaling is not active in the embryonic retina in mouse, despite TCF/LEF reporter activation. TOPgal activation in the embryonic mouse retina is not artifactual, since endogenous TCF/LEF and TOPgal reporter expression overlap and an independently generated reporter shows a very similar expression pattern in the embryonic mouse retina (TCF/LEF line).28,29 For example, this TCF/LEF reporter is expressed in apical embryonic retinal cells that express CRX, a transcription factor expressed in photoreceptor precursors, consistent with TOPgal-Otx2 coexpression in our study. However, ectopic activation of Wnt/β-catenin suppresses CRX expression indicating that this pathway must be inactive in committed precursors to ensure proper development of photoreceptors.37 Data presented here agree with this notion. Why the previously characterized TCF/LEF reporter shows persistent activity in embryonic and adult ganglion and amacrine cells is unclear.37 Since we observed TOPgal activity in embryonic ganglion cells and putative amacrine precursors, it is possible that the activity levels and perdurance of these different reporters vary, since not all transgenic constructs are equivalent and undoubtedly reside in different insertion sites throughout the mouse genome.

To provide context for the TCF/LEF reporter activity during retinal development, we compared TOPgal expression to that of transcription factors promoting retinal cell fates, mitotic cell cycle and differentiation markers for the four main embryonic retinal cell types. Together these data show that TCF/LEF reporter activity is not confined to a single retinal lineage. Among the bHLH factors examined, very little TOPgal expression was seen in Math5+ or NeuroD+ cells, but more extensive in early Ngn2+ cells. However, the highest coincidence occurred in Olig2+ retinal cells. Intriguingly, there is extensive TOPgal activity in both Olig2+ and Otx2+ cohorts, which are complementary. Because very few Math5-TOPgal double positive cells were observed, yet a subset of Brn3b+ cells express TOPgal, the onset of TCF-LEF reporter activity is consistent with retinal progenitor progression towards differentiation. In support of this idea, we found many more TOPgal+ cells express p27/Kip1 than any of the other cell cycle markers examined. Therefore, we propose that TCF/LEF activity assists in some aspect of retinal progenitor transition into a postmitotic precursor. The integrated expression of TCF/LEF proteins with bHLH and homeobox factors appears to be one means by which the complexity of retinal cell type specification may occur. Future studies will address the combinatorial functions of these pathways.

Acknowledgments

We are grateful to Elaine Fuchs, Yasuhide Furuta, Gabrielle Kardon and Charles Murtaugh for providing TOPGAL, Six3-Cre, floxed β-catenin and β-catenindel mice, respectively. We thank Ben Atkins, Alyssa van Bibber, Annie Chen, Amy Eggers, Kim Howes, Ashley Riesenberg, the Levine lab and specifically Gaurav Das for technical help. We are grateful to Masato Nakafuku for the gift of Ngn2 and Olig2 antibodies and Valerie Wallace for sharing experimental data before publication. We thank Ed Levine and Rich Dorsky for helpful comments and critical reading of the manuscript.

Supported by NIH grants, EY14954 (SF, MLV) and EY13612 (NLB), NIH Core Grant, EYO14800, as well as an unrestricted grant from Research to Prevent Blindness, Inc., to the Department of Ophthalmology, University of Utah (SF).

References

1. Rapaport DH, Wong LL, Wood ED, Yasumura D, LaVail MM. Timing and topography of cell genesis in the rat retina. J Comp Neurol. 2004;474:304–324. [PubMed]
2. Sidman RJ. Histogenesis of mouse retina studied with thymidine-H3. In: Smelser GK, editor. Structure of the eye. Academic Press; New York: 1961. pp. 487–506.
3. Young RW. Cell proliferation during postnatal development of the retina in the mouse. Brain Res. 1985;353:229–239. [PubMed]
4. Young RW. Cell differentiation in the retina of the mouse. Anat Rec. 1985;212:199–205. [PubMed]
5. Carter-Dawson LD, LaVail MM. Rods and cones in the mouse retina. II. Autoradiographic analysis of cell generation using tritiated thymidine. J Comp Neurol. 1979;188:263–272. [PubMed]
6. Wetts R, Fraser SE. Multipotent precursors can give rise to all major cell types of the frog retina. Science. 1988;239:1142–1145. [PubMed]
7. Holt CE, Bertsch TW, Ellis HM, Harris WA. Cellular determination in the Xenopus retina is independent of lineage and birth date. Neuron. 1988;1:15–26. [PubMed]
8. Turner DL, Cepko CL. A common progenitor for neurons and glia persists in rat retina late in development. Nature. 1987;328:131–136. [PubMed]
9. Turner DL, Snyder EY, Cepko CL. Lineage-independent determination of cell type in the embryonic mouse retina. Neuron. 1990;4:833–845. [PubMed]
10. Cepko CL, Austin CP, Yang X, Alexiades M, Ezzeddine D. Cell fate determination in the vertebrate retina. Proc Natl Acad Sci U S A. 1996;93:589–595. [PubMed]
11. Livesey FJ, Cepko CL. Vertebrate neural cell-fate determination: lessons from the retina. Nat Rev Neuro sci. 2001;2:109–118. [PubMed]
12. Fuhrmann S, Chow L, Reh TA. Molecular control of cell diversification in the vertebrate retina. Results Probl Cell Differ. 2000;31:69–91. [PubMed]
13. Yang XJ. Roles of cell-extrinsic growth factors in vertebrate eye pattern formation and retinogenesis. Semin Cell Dev Biol. 2004;15:91–103. [PubMed]
14. Vetter ML, Brown NL. The role of basic helix-loop-helix genes in vertebrate retinogenesis. Semin Cell Dev Biol. 2001;12:491–498. [PubMed]
15. Hatakeyama J, Kageyama R. Retinal cell fate determination and bHLH factors. Semin Cell Dev Biol. 2004;15:83–89. [PubMed]
16. Kanekar S, Perron M, Dorsky R, et al. Xath5 participates in a network of bHLH genes in the developing Xenopus retina. Neuron. 1997;19:981–994. [PubMed]
17. Liu W, Mo Z, Xiang M. The Ath5 proneural genes function upstream of Brn3 POU domain transcription factor genes to promote retinal ganglion cell development. Proc Natl Acad Sci U S A. 2001;98:1649–1654. [PubMed]
18. Kay JN, Finger-Baier KC, Roeser T, Staub W, Baier H. Retinal ganglion cell genesis requires lakritz, a Zebrafish atonal Homolog. Neuron. 2001;30:725–736. [PubMed]
19. Brown NL, Kanekar S, Vetter ML, Tucker PK, Gemza DL, Glaser T. Math5 encodes a murine basic helix-loop-helix transcription factor expressed during early stages of retinal neurogenesis. Development. 1998;125:4821–4833. [PubMed]
20. Wang SW, Kim BS, Ding K, et al. Requirement for math5 in the development of retinal ganglion cells. Genes Dev. 2001;15:24–29. [PubMed]
21. Liu H, Etter P, Hayes S, et al. NeuroD1 regulates expression of thyroid hormone receptor 2 and cone opsins in the developing mouse retina. J Neurosci. 2008;28:749–756. [PubMed]
22. Morrow EM, Furukawa T, Lee JE, Cepko CL. NeuroD regulates multiple functions in the developing neural retina in rodent. Development. 1999;126:23–36. [PubMed]
23. Yan RT, Wang SZ. Requirement of neuroD for photoreceptor formation in the chick retina. Invest Ophthalmol Vis Sci. 2004;45:48–58. [PMC free article] [PubMed]
24. Ma W, Wang SZ. The final fates of neurogenin2-expressing cells include all major neuron types in the mouse retina. Mol Cell Neurosci. 2006;31:463–469. [PMC free article] [PubMed]
25. Le TT, Wroblewski E, Patel S, Riesenberg AN, Brown NL. Math5 is required for both early retinal neuron differentiation and cell cycle progression. Dev Biol. 2006;295:764–778. [PubMed]
26. Dyer MA, Bremner R. The search for the retinoblastoma cell of origin. Nat Rev Cancer. 2005;5:91–101. [PubMed]
27. Smith AN, Miller LA, Song N, Taketo MM, Lang RA. The duality of beta-catenin function: a requirement in lens morphogenesis and signaling suppression of lens fate in periocular ectoderm. Dev Biol. 2005;285:477–489. [PubMed]
28. Liu H, Mohamed O, Dufort D, Wallace VA. Characterization of Wnt signaling components and activation of the Wnt canonical pathway in the murine retina. Dev Dyn. 2003;227:323–334. [PubMed]
29. Liu H, Thurig S, Mohamed O, Dufort D, Wallace VA. Mapping canonical Wnt signaling in the developing and adult retina. Invest Ophthalmol Vis Sci. 2006;47:5088–5097. [PubMed]
30. Behrens J, von Kries JP, Kuhl M, et al. Functional interaction of beta-catenin with the transcription factor LEF-1. Nature. 1996;382:638–642. [PubMed]
31. Molenaar M, van de Wetering M, Oosterwegel M, et al. XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell. 1996;86:391–399. [PubMed]
32. van de Wetering M, Cavallo R, Dooijes D, et al. Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF. Cell. 1997;88:789–799. [PubMed]
33. Van Raay TJ, Moore KB, lordanova I, et al. Frizzled 5 signaling governs the neural potential of progenitors in the developing Xenopus retina. Neuron. 2005;46:23–36. [PubMed]
34. Fuhrmann S. Wnt signaling in eye organogenesis. Organogenesis. 2008;4 In Press. [PMC free article] [PubMed]
35. Kubo F, Nakagawa S. Wnt signaling in retinal stem cells and regeneration. Develop Growth Differ. 2008;50:245–251. [PubMed]
36. Kubo F, Takeichi M, Nakagawa S. Wnt2b controls retinal cell differentiation at the ciliary marginal zone. Development. 2003;130:587–598. [PubMed]
37. Liu H, Xu S, Wang Y, et al. Ciliary margin transdifferentiation from neural retina is controlled by canonical Wnt signaling. Dev Biol. 2007;308:54–67. [PubMed]
38. Cho SH, Cepko CL. Wnt2b/beta-catenin-mediated canonical Wnt signaling determines the peripheral fates of the chick eye. Development. 2006;133:3167–3177. [PubMed]
39. Ouchi Y, Tabata Y, Arai K, Watanabe S. Negative regulation of retinal-neurite extension by beta-catenin signaling pathway. J Cell Sci. 2005;118:4473–4483. [PubMed]
40. Fu X, Sun H, Klein WH, Mu X. Beta-catenin is essential for lamination but not neurogenesis in mouse retinal development. Dev Biol. 2006;299:424–437. [PMC free article] [PubMed]
41. Moriyama A, Kii I, Sunabori T, et al. GFP transgenic mice reveal active canonical Wnt signal in neonatal brain and in adult liver and spleen. Genesis. 2007;45:90–100. [PubMed]
42. Mohamed OA, Clarke HJ, Dufort D. Beta-catenin signaling marks the prospective site of primitive streak formation in the mouse embryo. Dev Dyn. 2004;231:416–424. [PubMed]
43. Maretto S, Cordenonsi M, Dupont S, et al. Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors. Proc Natl Acad Sci U S A. 2003;100:3299–3304. [PubMed]
44. DasGupta R, Fuchs E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development. 1999;126:4557–4568. [PubMed]
45. Barolo S. Transgenic Wnt/TCF pathway reporters: all you need is Lef? Oncogene. 2006;25:7505–7511. [PubMed]
46. Brault V, Moore R, Kutsch S, et al. Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development. Development. 2001;128:1253–1264. [PubMed]
47. Furuta Y, Lagutin O, Hogan BL, Oliver GC. Retina- and ventral forebrain-specific Cre recombinase activity in transgenic mice. Genesis. 2000;26:130–132. [PubMed]
48. Fuhrmann S, Levine EM, Reh TA. Extraocular mesenchyme patterns the optic vesicle during early eye development in the embryonic chick. Development. 2000;127:4599–4609. [PubMed]
49. Henrique D, Adam J, Myat A, Chitnis A, Lewis J, Ish-Horowicz D. Expression of a Delta homologue in prospective neurons in the chick. Nature. 1995;375:787–790. [PubMed]
50. Zeng L, Fagotto F, Zhang T, et al. The mouse Fused locus encodes Axin, an inhibitor of the Wnt signaling pathway that regulates embryonic axis formation. Cell. 1997;90:181–192. [PubMed]
51. Philips GT, Stair CN, Young Lee H, et al. Precocious retinal neurons: Pax6 controls timing of differentiation and determination of cell type. Dev Biol. 2005;279:308–321. [PubMed]
52. Korinek V, Barker N, Willert K, et al. Two members of the Tcf family implicated in Wnt/beta-catenin signaling during embryogenesis in the mouse. Mol Cell Biol. 1998;18:1248–1256. [PMC free article] [PubMed]
53. Burns CJ, Zhang J, Brown EC, et al. Investigation of Frizzled-5 during embryonic neural development in mouse. Developmental Dynamics. 2008 In Press. [PMC free article] [PubMed]
54. Westenskow P, Fuhrmann S. unpublished observations, will be described elsewhere. 2008
55. Elshatory Y, Everhart D, Deng M, Xie X, Barlow RB, Gan L. Islet-1 controls the differentiation of retinal bipolar and cholinergic amacrine cells. J Neurosci. 2007;27:12707–12720. [PMC free article] [PubMed]
56. Barton KM, Levine EM. Expression patterns and cell cycle profiles of PCNA, MCM6, cyclin D1, cyclin A2, cyclin B1, and phosphorylated histone H3 in the developing mouse retina. Dev Dyn. 2008;237:672–682. [PubMed]
57. Prigent C, Dimitrov S. Phosphorylation of serine 10 in histone H3, what for? J Cell Sci. 2003;116:3677–3685. [PubMed]
58. Pennesi ME, Cho JH, Yang Z, et al. BETA2/NeuroD1 null mice: a new model for transcription factor-dependent photoreceptor degeneration. J Neurosci. 2003;23:453–461. [PubMed]
59. Shibasaki K, Takebayashi H, Ikenaka K, Feng L, Gan L. Expression of the basic helix-loop-factor Olig2 in the developing retina: Olig2 as a new marker for retinal progenitors and late-born cells. Gene Expr Patterns. 2007;7:57–65. [PubMed]
60. Nakamura K, Harada C, Namekata K, Harada T. Expression of olig2 in retinal progenitor cells. Neuroreport. 2006;17:345–349. [PubMed]
61. Tomita K, Ishibashi M, Nakahara K, et al. Mammalian hairy and Enhancer of split homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis. Neuron. 1996;16:723–734. [PubMed]
62. Hatakeyama J, Bessho Y, Katoh K, et al. Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation. Development. 2004;131:5539–5550. [PubMed]
63. Lee HY, Wroblewski E, Philips GT, et al. Multiple requirements for Hes 1 during early eye formation. Dev Biol. 2005;284:464–478. [PubMed]
64. Baas D, Bumsted KM, Martinez JA, Vaccarino FM, Wikler KC, Barnstable CJ. The subcellular localization of Otx2 is cell-type specific and developmentally regulated in the mouse retina. Brain Res Mol Brain Res. 2000;78:26–37. [PubMed]
65. Viczian AS, Vignali R, Zuber ME, Barsacchi G, Harris WA. XOtx5b and XOtx2 regulate photoreceptor and bipolar fates in the Xenopus retina. Development. 2003;130:1281–1294. [PubMed]
66. Koike C, Nishida A, Ueno S, et al. Functional roles of Otx2 transcription factor in postnatal mouse retinal development. Mol Cell Biol. 2007;27:8318–8329. [PMC free article] [PubMed]
67. Nishida A, Furukawa A, Koike C, et al. Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development. Nat Neurosci. 2003;6:1255–1263. [PubMed]
68. Roberts MR, Hendrickson A, McGuire CR, Reh TA. Retinoid X receptor (gamma) is necessary to establish the S-opsin gradient in cone photoreceptors of the developing mouse retina. Invest Ophthalmol Vis Sci. 2005;46:2897–2904. [PubMed]
69. Roberts MR, Srinivas M, Forrest D, Morreale de Escobar G, Reh TA. Making the gradient: thyroid hormone regulates cone opsin expression in the developing mouse retina. Proc Natl Acad Sci U S A. 2006;103:6218–6223. [PubMed]
70. Mori M, Ghyselinck NB, Chambon P, Mark M. Systematic immunolocalization of retinoid receptors in developing and adult mouse eyes. Invest Ophthalmol Vis Sci. 2001;42:1312–1318. [PubMed]
71. Gan L, Xiang M, Zhou L, Wagner DS, Klein WH, Nathans J. POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells. Proc Natl Acad Sci U S A. 1996;93:3920–3925. [PubMed]
72. Xiang M, Zhou L, Peng YW, Eddy RL, Shows TB, Nathans J. Brn-3b: a POU domain gene expressed in a subset of retinal ganglion cells. Neuron. 1993;11:689–701. [PubMed]
73. Fujitani Y, Fujitani S, Luo H, et al. Ptf1a determines horizontal and amacrine cell fates during mouse retinal development. Development. 2006;133:4439–4450. [PubMed]
74. Nakhai H, Sel S, Favor J, et al. Ptf1a is essential for the differentiation of GABAergic and glycinergic amacrine cells and horizontal cells in the mouse retina. Development. 2007;134:1151–1160. [PubMed]
75. Brannon M, Brown JD, Bates R, Kimelman D, Moon RT. XCtBP is a XTcf-3 co-repressor with roles throughout Xenopus development. Development. 1999;126:3159–3170. [PubMed]
76. Hoppler S, Kavanagh CL. Wnt signalling: variety at the core. J Cell Sci. 2007;120:385–393. [PubMed]
77. Willert K, Jones KA. Wnt signaling: is the party in the nucleus? Genes Dev. 2006;20:1394–1404. [PubMed]
78. Brantjes H, Roose J, van De Wetering M, Clevers H. All Tcf HMG box transcription factors interact with Groucho-related co-repressors. Nucleic Acids Res. 2001;29:1410–1419. [PMC free article] [PubMed]
79. Arce L, Yokoyama NN, Waterman ML. Diversity of LEF/TCF action in development and disease. Oncogene. 2006;25:7492–7504. [PubMed]
80. Yi F, Merrill BJ. Stem cells and TCF proteins: a role for beta-catenin--independent functions. Stem Cell Rev. 2007;3:39–48. [PubMed]
81. Hsu SC, Galceran J, Grosschedl R. Modulation of transcriptional regulation by LEF-1 in response to Wnt-1 signaling and association with beta-catenin. Mol Cell Biol. 1998;18:4807–4818. [PMC free article] [PubMed]
82. Carlsson P, Waterman ML, Jones KA. The hLEF/TCF-1 alpha HMG protein contains a context-dependent transcriptional activation domain that induces the TCR alpha enhancer in T cells. Genes Dev. 1993;7:2418–2430. [PubMed]
83. Labbe E, Letamendia A, Attisano L. Association of Smads with lymphoid enhancer binding factor 1/T cell-specific factor mediates cooperative signaling by the transforming growth factor-beta and wnt pathways. Proc Natl Acad Sci U S A. 2000;97:8358–8363. [PubMed]
84. van Genderen C, Okamura RM, Farinas I, et al. Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient mice. Genes Dev. 1994;8:2691–2703. [PubMed]
85. Galceran J, Farinas I, Depew MJ, Clevers H, Grosschedl R. Wnt3a−/−-like phenotype and limb deficiency in Lef1(−/−)Tcf1(−/−) mice. Genes Dev. 1999;13:709–717. [PubMed]
86. Galceran J, Miyashita-Lin EM, Devaney E, Rubenstein JL, Grosschedl R. Hippocampus development and generation of dentate gyrus granule cells is regulated by LEF1. Development. 2000;127:469–482. [PubMed]
87. Korinek V, Barker N, Moerer P, et al. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nat Genet. 1998;19:379–383. [PubMed]
88. Okamura RM, Sigvardsson M, Galceran J, Verbeek S, Clevers H, Grosschedl R. Redundant regulation of T cell differentiation and TCRalpha gene expression by the transcription factors LEF-1 and TCF-1. Immunity. 1998;8:11–20. [PubMed]
89. Verbeek S, Izon D, Hofhuis F, et al. An HMG-box-containing T-cell factor required for thymocyte differentiation. Nature. 1995;374:70–74. [PubMed]
90. Gregorieff A, Grosschedl R, Clevers H. Hindgut defects and transformation of the gastrointestinal tract in Tcf4(−/−)/Tcf1(−/−) embryos. Embo J. 2004;23:1825–1833. [PubMed]