Mapping Canonical Wnt Signaling in the Developing and Adult Retina

Abstract

purpose. The role of the Wnt[b]/β-catenin–dependent pathway (canonical Wnt pathway) in the context of retinal development and homeostasis is largely unknown. This study was undertaken to characterize activation of the Wnt canonical pathway and its relevance to cell type populations in the developing and adult retina.

methods. Tissue from TCF/Lef-LacZ (T-cell–specific transcription factor/lymphoid enhancer–binding factor) transgenic mice was used for monitoring the activation of the canonical Wnt pathway. Lithium (Li⁺) treatment was applied to induce ectopic activation of the TCF/Lef-LacZ reporter gene in retinal explants. Gene expression and retinal cell types were examined by in situ hybridization (ISH) or by immunohistochemistry (IHC).

results. On Li⁺ treatment, ectopic expression of the TCF/Lef-LacZ reporter gene was rapidly and dramatically induced in retinal explants. The pattern of TCF/Lef-LacZ reporter gene expression was dynamic throughout retinal development and in the adult retina. There was a distinctive expression pattern in each cellular layer, in the developing ciliary margin (CM), and the prospective ciliary epithelium. In the mature retina, the TCF/Lef-LacZ reporter gene was expressed in subsets of retinal ganglion cells (RGCs) and amacrine cells. The expression of the four TCF/Lef transcription factors overlapped with activation of the TCF/Lef-LacZ reporter.

conclusions. The TCF/Lef-LacZ transgene is a faithful reporter of canonical Wnt signaling in the retina. The pattern of TCF/Lef-LacZ reporter gene activation and of TCF/Lef transcription factor expression suggests that activation of the canonical Wnt pathway is developmental-stage dependent and is spatially modulated. Our findings also imply the involvement of this pathway in the specification and/or generation of ciliary epithelium, cellular differentiation, axon guidance, and connectivity to targets in the central nervous system and in the maintenance or function of specific retinal neurons in the adult.

Wnts are secreted glycoproteins that are involved in various developmental and cell biological processes during embryogenesis and organogenesis, such as brain patterning and axis formation. ^{1 2 3 4 5 6} There are several different signaling cascades activated by Wnts, including the β-catenin–dependent pathway (canonical Wnt pathway), the planar cell polarity pathway, the Wnt/Ca²⁺ pathway, and a pathway that regulates spindle orientation and asymmetric cell division. ^{7 8 9} β-Catenin is an important component of cell adhesion through its association with E-cadherin and α-catenin at the plasma membrane and it is an essential mediator of canonical Wnt signaling. ^{10 11} In the canonical Wnt pathway, Wnt binding of its receptors, members of the Frizzled superfamily, and the coreceptors LRP5/6, ¹² results in the stabilization of cytoplasmic β-catenin, which then binds TCF/Lef (T-cell–specific transcription factor and lymphoid enhancer–binding factor) family of transcription factors, ^{13 14} to activate TCF-dependent gene transcription, and primarily regulates cell fate determination during development. In the absence of Wnt signaling, a protein complex containing glycogen synthesis kinase (GSK)-3β, axin, ^{15 16} and adenomatous polyposis coli (APC) phosphorylates β-catenin, resulting in the ubiquitination and subsequent degradation of β-catenin through the proteosome-mediated system. ^{6 17 18} The activity of GSK-3β can be inhibited pharmacologically with Li⁺, ¹⁹ leading to stabilization of β-catenin and transcriptional activation of target genes. ^{20 21 22 23 24}  

TCF/Lef transcription factors have been identified in several vertebrate species, including frog, fish, and chicken. ^{25 26 27 28 29 30} Four members of the TCF/Lef family have been identified in mammals, including Tcf1, Lef1, Tcf3, and Tcf4. ^{31 32 33} Generally, TCF/Lef transcription factors contain an N-terminal β-catenin interaction domain and a C-terminal DNA-binding domain, the high-mobility group (HMG box). ^{31 32 33 34} There is an additional domain, termed the Groucho binding domain, located upstream of the HMG box encoded by an alternatively spliced exon in some of the TCF/Lef transcription factors. ^{35 36} A TCF/Lef-responsive element, the TCF/Lef consensus motif (CCTTTGATC), has been identified in Wnt target genes ^{31 37} and has been used to drive reporter transgene expression in mice and fish. ^{38 39 40 41 42} Expression patterns of TCF/Lef transcription factors have been reported in zebrafish, frog, ^{29 43} and chick retinas ⁴⁴ ; however, a comprehensive analysis of TCF/Lef expression in the mouse retina is still lacking. 

In a previous study, we examined expression of Wnt pathway components and the TCF/Lef-LacZ reporter gene in the murine eye. ⁴⁵ In this reporter line, LacZ expression is under the control of the Hsp68 minimum promoter and six copies of TCF/Lef responsive element ⁴⁰ ; thus, detection of β-gal activity indicates the location of TCF/Lef-dependent gene expression within a tissue. We observed activation of the TCF/Lef-LacZ reporter gene in the ciliary margin (CM) and the neuroblast layer of the developing retina. Activation of a Lef1/β-catenin–dependent reporter gene in transgenic fish was also described in the retinal pigmented epithelium (RPE), CM, and lens. ⁴² Despite the findings in these studies, the timing of Wnt canonical pathway activation and its relevance to retinal cellular types during retinal development and in the adult retina remains unclear. 

Recently, Wnt signaling has been implicated in the formation of the vertebrate eye field and proliferation and differentiation within the lens and retina. Moreover, mutations in genes encoding Wnt pathway components have been shown to be associated with various ocular diseases. ⁴⁶ To gain additional insight into the role of canonical Wnt signaling in the context of mammalian retinal development and homeostasis, we compared the temporal and spatial activation of this signaling pathway in the developing and adult retina of TCF/Lef-LacZ reporter mice, with the expression of TCF/Lef transcription factors and cell-type–specific markers. 

Materials and Methods

Animals

Generation of the TCF/Lef-LacZ reporter transgenic mouse line was performed as described elsewhere ⁴⁰ and was maintained on a CD1 background. Tissues were obtained from time-mated homozygous or heterozygous TCF/Lef-LacZ mice crossed to CD1 females, and the day of vaginal plugging was considered embryonic day (E)0.5 (E0.5). All animal-related experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were permitted by University of Ottawa Animal Care and Veterinary Service. 

Retinal Explant Culture

Retinal explants from E14.5 mice were established by removing the RPE and placing the globe with lens side down on a polycarbonate filter (pore size: 0.8 μm; Nucleopore, Pleasanton, CA). The globe was opened at the optic nerve head, and the pieces of the retina (still attached to the lens) were flattened to the filter. Globes were dissected in MEM (Sigma-Aldrich, St. Louis, MO). Explants were cultured in serum-free conditions, as described previously ⁴⁷ and were treated with 20 mM LiCl or NaCl as the control. 

Detection of β-Gal Activity

Preparation and X-gal staining of postnatal day (P)7 and adult tissues was performed as described previously. ^{45 48} After X-gal staining, slides were rinsed in PBS for 30 minutes and mounted or processed for IHC. Preparation and X-gal staining of embryonic material was essentially the same, with the following modifications: Whole heads or dissected eyeballs were fixed in 4% paraformaldehyde for various lengths of time: 5 (E9.5–E10.5), 10 (E11.5–E13.5), 15 (E14.5–E15.5) or 15–20 (dissected eyeballs of E16 and older tissue) minutes. Lens were left intact, and the tissues were processed as described previously. ^{45 48}  

In Situ Hybridization

Tissues were prepared and processed for ISH, as described previously ^{45 48} with the following DIG-labeled antisense riboprobes: Crx (a kind gift from Connie Cepko, Harvard Medical School, Boston, MA); Otx1 and Otx2 (a kind gift from Masayo Takahashi, Kyoto University, Kyoto, Japan); Tcf1, Lef1, Tcf3, and Tcf4 (a kind gift from Johannes Meeldijk, University of Utrecht, The Netherlands); and Wnt2b (a kind gift from Lise Zakin, Howard Hughes Medical Institute, University of California, Los Angeles). 

Immunohistochemistry

IHC was performed according to the protocol for fluorescence detection or DAB reaction (3,3′-diaminobenzidine) described previously, ^{49 50} with the following primary antibodies: rabbit anti-β-gal (Invitrogen-Molecular Probes, Eugene, OR), mouse monoclonal anti-Brn3A (Clone 14A6; Santa Cruz Biotechnology, Santa Cruz, CA), goat polyclonal anti-Brn3B (Clone C-13; Santa Cruz Biotechnology), mouse monoclonal anti-calbindin (Sigma-Aldrich), goat anti-Chx10 (a kind gift from Rod Bremner, University of Toronto, Toronto, ON, Canada), rabbit polyclonal anti-phospho-histone H3 (anti-phosphoH3; Upstate Biotechnology, Lake Placid, NY), mouse monoclonal anti-Pax6 (Developmental Studies Hybridoma Bank, Iowa City, IA). 

Results

Induction of TCF/Lef-LacZ Reporter Gene Activity in Retinal Explants

We showed previously that reporter gene expression in the TCF/Lef-LacZ transgenic line could be detected in the developing embryonic eye. ⁴⁵ To confirm that the TCF/Lef-LacZ reporter gene expression accurately reflects activation of the canonical Wnt pathway, we treated retinal explants dissected from the TCF/Lef-LacZ reporter embryos with Li⁺, a well-known agonist of the Wnt pathway. 

Explants were stained to detect β-gal activity at different time points after Li⁺ treatment. In control explants, β-gal reporter activity was gradually downregulated after 1 day in vitro compared with the intensity of β-gal staining in vivo, especially in the CM area (compare Figs 1A 1C 1Ewith Figs. 3D 3E ). In contrast, Li⁺ treatment led to a significant up regulation of β-gal activity throughout the neural retina by 15 hours, reaching a maximum level at ∼24 hours and decreasing by 48 hours (Fig. 1) . These findings indicate that TCF/Lef-LacZ transgene is a faithful reporter of canonical Wnt signaling in the retina and that maintenance of canonical Wnt signaling, especially in the CM, is dependent on signals that are lost on transfer of the retina to culture. 

Activation of Canonical Wnt Signaling in the Developing Retina

Canonical Wnt signaling is activated in retinal development in various model systems ^{26 45} ; however, the timing of the canonical Wnt pathway activation at early stages in mammalian retinal development has not been reported. We took advantage of TCF/Lef-LacZ reporter mice to explore this question and characterized the expression of the reporter gene in the retina from the optic vesicle stage E9.5 to P0. Eye cup development in the mouse begins at E8 to 8.5 as an evagination (optic vesicle) originating from the neuroepithelium of the anterior neural plate. At E9.5, activation of the TCF/Lef-LacZ reporter gene was observed in a subset of neuroepithelial cells in the dorsonasal side of the optic vesicle (Fig. 2A ; Table 1 ). This β-gal⁺ region is contained within the region that will give rise to the CM and peripheral RPE, as defined by the expression of the CM/RPE marker Otx1 (Fig. 2E) . ^{51 52} At this stage, the onset of the reporter gene expression in the dorsal optic vesicle mirrors the pattern of Wnt2b expression (Fig. 2C) . This asymmetric pattern of Wnt2b expression in the dorsal eye has been reported in chick and mouse retina. ^{53 54} At later developmental stages, the TCF/Lef-LacZ reporter gene and Wnt2b expression are also induced in the ventral eye. ⁴⁵ At E10 the optic vesicle invaginates to form a bilayered optic cup, with the inner layer developing as neural retina, the outer layer developing as the RPE, and the rim of the eye cup as the CM. The distal CM gives rise to the iris, and the proximal CM gives rise to the two-layered ciliary epithelium, which consists of the outer pigmented ciliary epithelium (PCE) and the inner nonpigmented ciliary epithelium (nPCE). In the peripheral retina, from E11.5 to E15.5, the TCF/Lef-LacZ reporter gene was activated in the CM. From E16.5 onward, expression of this reporter gene was dramatically upregulated in the prospective nPCE and downregulated in the prospective PCE (Fig. 3 , Tables 1 2 ). 

TCF/Lef-LacZ reporter gene expression was induced in a discreet patch of cells in the neural retina at E11.5, and from by E12.5 onward reporter gene expression was predominantly segregated in the inner retina (RGC layer) and the apical side of the neuroblast layer (Fig. 3 , Tables 1 2 ). The expression of the reporter gene at the apical side of the neuroblast layer was dramatically downregulated after E18.5 (Fig. 3 , Table 2 ). Activation of the TCF/Lef-LacZ reporter gene in the inner neuroblast layer (between the RGC and apical side of the neuroblast layer) was detected from E14.5 until E17.5 and downregulated by birth (Fig. 3 , Tables 1 2 ). The reporter gene expression was also detected at a high level in the lens anterior epithelium from E11.5 to E14.5; but it was downregulated from E15.5 onward (Fig. 3 , Table 1 ). The dynamic expression of the TCF/Lef-LacZ reporter gene in several distinct regions of the retina during development suggests that canonical Wnt signaling plays a role in the development of multiple retinal cell types. 

Identification of TCF/Lef-LacZ Reporter⁺ Cells in the Outer Neuroblast Layer of the Developing Retina

From E13.5 to E18.5, activation of TCF/Lef-LacZ reporter gene was detected in an apical pattern in the outer neuroblast layer, a region that contains differentiating photoreceptors, marked by the expression of the homeobox transcription factors Crx and Otx2, but also mitotic progenitors. Crx is expressed soon after a cell is fated to the rod or cone photoreceptor lineage and is essential for terminal differentiation of photoreceptor cells. ^{55 56} Otx2 shares a high homology at the amino acid level with Crx and is known to compensate for the function of Crx in photoreceptor cell fate determination and differentiation. ^{57 58 59} To identify the TCF/Lef-LacZ reporter-positive cells in this region, we compared β-gal activity with ISH for Crx and Otx2 and IHC for phosphoH3, which marks mitotic cells, on serial sections of E16.5 retina. In both the central and peripheral retina, expression of the TCF/Lef-LacZ reporter gene was detected in a domain that was several cell diameters wide in the outer neuroblast layer, similar to the Crx expression pattern (Figs. 4A 4C) . Otx2 was expressed in a wider area (Fig. 4B) , whereas mitotic cells (anti-phosphoH3⁺) were restricted to a narrow, one-to-two-cell-wide stripe of cells adjacent to the RPE (Fig. 4D) . Double staining for β-gal and phosphoH3 revealed that most of the β-gal–positive cells were not mitotic (Fig. 4E 4F) . Therefore, we conclude that the majority of the apically located TCF/Lef-LacZ reporter-positive cells at this stage likely correspond to immature photoreceptor precursors. 

Dynamic Expression of TCF/Lef Family Factors in the Developing Retina

The expression pattern of the TCF/Lef transcription factors in the mammalian retina is largely unexplored. Because the TCF/Lef-LacZ reporter gene is regulated by consensus TCF/Lef sequences, we compared the expression of TCF/Lef factors with TCF/Lef-LacZ reporter activity. The expression of the four TCF/Lef transcription factors was examined throughout the dorsal–ventral axis of the retina; no difference was detected in the dorsal versus ventral sections (data not shown). We observed a similar expression pattern of Tcf1 and Lef1 in the murine retina, consistent with previous reports showing a high degree of overlap in expression pattern and function of these two genes in fish retina and during murine embryogenesis. ^{29 60} Both Tcf1 and Lef1 mRNAs were detected in the optic vesicle at E9.5, with Lef1 expressed at a higher level than Tcf1 in the proximal and intermediate optic vesicle that will give rise to the CM and RPE (Figs. 5A 5B ; Table 1 ). From E12.5 to E14.5, both of Tcf1 and Lef1 were expressed diffusely in the neuroblast layer and at higher level in both prospective PCE and nPCE of the CM (Figs. 6A 6B 6C 6D ; Table 1 ). However, we also detected differences in the expression pattern of these two genes. Tcf1 was expressed in the optic nerve head and lens anterior epithelium (Fig. 6B ; Table 1 ), whereas Lef1 expression was detected in the periocular mesenchyme at E12.5 (Fig. 6C) . Moreover, Lef1 expression was observed in an asymmetric pattern in the differentiating RGC layer, with a higher level in the temporal retina at E14.5 (Fig. 6D , Figs. 7A 7B ; Table 1 ). Consistent with the possibility that there is an asymmetric distribution of canonical Wnt signaling in the developing retina, we found the TCF/Lef-LacZ reporter gene activity in a similar asymmetric pattern in the RGC layer from E14.5 to E17.5 (Figs. 7C 7D 7E 7F) . 

Tcf3 expression was observed throughout the optic vesicle at E9.5 (Fig. 5C ; Table 1 ), in the entire neuroblast layer and at a higher level in the optic nerve head at E12.5 and E14.5, but was absent from the differentiating RGC layer (Figs. 6E 6F ; Table 1 ). Tcf3 transcripts were also detected in the periocular mesenchyme and in the lens anterior epithelium at the developmental stages examined (Figs. 6E 6F ; Table 1 ). Tcf4 mRNA was detected at a low level in the optic vesicle at E9.5, and was difficult to detect after E12.5 (Figs. 5D 6G 6H ; Table 1 ). We observed a high level of Tcf4 expression in the dorsal thalamus of the adjacent sections (data not shown), which was consistent with a previous study, ³³ proving that the ISH result is reliable. 

Activation of the TCF/Lef-LacZ Reporter Gene in the Postnatal and Adult Retina

Because some Wnts and Wnt signaling components are expressed in the postnatal and adult murine retina, ^{45 61 62 63} we asked whether and how canonical Wnt signaling is activated at these stages. 

In the P7 and adult retina TCF/Lef-LacZ reporter activity was detected primarily in the nPCE, the RGC and inner nuclear layer (INL; Fig. 8 ; Table 2 ). Note that the TCF/Lef-LacZ reporter gene activity in the adult ciliary epithelium was dramatically downregulated compared with the ciliary epithelium at P7, suggesting that canonical Wnt signaling is attenuated in this region of the adult eye. In the INL, the expression pattern of the TCF/Lef-LacZ reporter gene was detected at a level much higher than that in the inner neuroblast layer of the late-stage embryos (compare Figs. 8B 8Dwith Figs. 3F 3G 3H 3I ). Moreover, in contrast to our observation that TCF/Lef-LacZ reporter gene activity was gradually downregulated in the CM of retinal explants, its expression was initiated in the INL of the E14.5 explants after 10 days in culture (data not shown), which corresponds to high-level TCF/Lef-LacZ reporter gene activation in the postnatal retina. Although the source of canonical Wnt signaling in the CM may reside in the RPE, which is removed in the culture, the retinal environment in the explants appears to be permissive for the induction of high levels of TCF/Lef-LacZ reporter gene expression in at least a subset of cells within the explants. 

The identity of TCF/Lef-LacZ reporter⁺ cells was determined by position within the nuclear layers and double staining with β-gal and cell-type–specific markers. The analyses were performed on postnatal (P7) and adult retinas, with similar results. In the RGC layer, β-gal⁺ cells were identified as subsets of RGC cells (Brn3A⁺ or Brn3B⁺) ^{64 65} (Fig. 9) . In the INL, β-gal⁺ cells were subsets of amacrine cells (Pax6⁺ and calbindin⁺), ^{66 67 68 69} but not bipolar (Chx10⁺) ⁷⁰ or horizontal (calbindin⁺) cells ^{68 71} (Fig. 9) , implying that canonical Wnt signaling is likely to be involved in the maintenance or function of RGC and amacrine cells. 

Discussion

The expression patterns of the TCF/Lef-LacZ reporter transgene and TCF/Lef transcription factors in the developing and adult murine retina was investigated. Canonical Wnt pathway activation was dynamic throughout retinal development, suggesting that it plays a role in multiple aspects of retinal development and homeostasis, including the development of the CM and the ciliary epithelium, cell fate specification and/or differentiation, axon guidance, and retinal neuron homeostasis. 

The CM is a unique region in the developing eye. Although it is neuroepithelium derived, it is not fated to give rise to retinal neurons. Instead, the proximal part differentiates as the ciliary epithelium overlying the ciliary body and the distal part differentiates as the iris. A striking feature of our analysis is that the TCF/Lef-LacZ reporter transgene expression was maintained in the CM region of the developing eye from the optic vesicle stage to adulthood. This observation is consistent with a similar reporter analysis in transgenic zebrafish where the expression of a TOPdGFP reporter gene was documented in the CM in the early embryonic stages, ⁴² and suggests a conserved role for TCF/Lef-dependent canonical Wnt signaling in specification and/or formation of this region of the eye. One Wnt gene, Wnt2b (formerly known as Wnt13), has been reported to be expressed at the tip of the CM and in the RPE cells overlying the CM. ^{44 45} In several studies on chicken retina, cWnt2b was reported to induce accumulation of β-catenin, ⁴⁴ a feature of canonical Wnt signaling, and to maintain the proliferation of progenitors and inhibit neuronal differentiation of progenitors. ⁷² In the present study, Wnt2b and TCF/Lef-LacZ reporter gene expression is induced in an identical pattern in the dorsal retinal at the optic vesicle stage, suggesting that Wnt2b could be the relevant Wnt signal that is activating the TCF/Lef-LacZ reporter gene in the mouse. 

The topographic projection of RGC axons to their major midbrain target, the superior colliculus, is regulated by region-specific differences in RGC responsiveness to graded distribution of guidance cues expressed in the target. ⁷³ The differential responsiveness of RGCs to the guidance cues is controlled through graded gene expression along the dorsal–ventral and nasal–temporal axes of the retina. ^{73 74} The asymmetric expression of A-class Ephrin proteins along the nasal–temporal axis of the retina and the EphA receptor tyrosine kinase along the anterior–posterior axis of the target is part of the mechanism that establishes this topographical map. ^{75 76} Our observation that the TCF/Lef-LacZ reporter gene and Lef1 expression are asymmetrically expressed in the RGC layer in the temporal retina suggests that canonical Wnt pathway activation plays a role in RGC axon guidance. Although the target genes that may be controlled by TCF signaling in this context remain speculative, it is interesting to note that EphB/EphrinB expression in the intestinal epithelium is regulated by β-catenin/Tcf ^{77 78} and that graded RGC responsiveness to Wnt3 has been shown to play a role in retinotectal mapping in the chick visual system. ⁷⁹  

We report the first spatial and temporal expression patterns of Tcf1, Lef1, Tcf3, and Tcf4 in the developing mouse retina. Our observation of the expression of Tcf1 in the CM of the embryonic mouse retina is consistent with a previous report showing the expression of Tcf1 in the frog eye ⁴³ and Tcf7 (the zebrafish orthologue of Tcf1) in the retinal margin in fish ²⁹ ; however, we did not detect dorsal retina localization of Tcf1, as reported in fish. These observations suggest that the expression of Tcf1 in the retina is highly conserved, but in variable patterns between species. Expression of Lef1 in the CM is conserved between mouse and chick, ⁴⁴ whereas the asymmetric distribution of Lef1 mRNA in the RGC layer has not been reported previously. The detection of high levels of Tcf3 expression in the developing retina contradicts previous reports that Tcf3 is undetectable after E10.5 ³³ and is consistent with other studies showing that Tcf3 is expressed in the embryonic brain of the mouse at later stages. ^{80 81} Moreover, the headless (the zebrafish orthologue of Tcf3) mutant is characterized by a slight reduction in eye size in fish, ²⁷ suggesting an important role of Tcf3 during eye development; however, the roles of Tcf3 and other TCF/Lef transcription factors in the mouse retina remain to be explored. 

The TCF/Lef-LacZ transgenic mouse line used in this study was designed to report the TCF/Lef-dependent gene expression. Despite the Li⁺ sensitivity of this reporter, will it faithfully report all canonical Wnt/β-catenin signaling? It is possible that in some instances the activity of the transgenic reporter gene will not mirror the expression of endogenous Wnt target genes, thereby underestimating the extent and location of canonical Wnt pathway activation in various tissues. In other situations, activation of this reporter transgene could overestimate the fraction of TCF/Lef activity that is directly downstream of Wnt/β-catenin signaling. Several non-Wnt ligands have been reported to be able to trigger the canonical Wnt cascade, such as secreted Frizzled related protein 1 (Sfrp1) ⁸² ; Dkk2, a member of the Dickkopf family of secreted Wnt antagonists ⁸³ ; Wise, a secreted molecule ⁸⁴ ; and Norrin, the protein product of the Norrie disease gene. ⁸⁵ In particular, Norrin and Wnt receptor, Frizled-4 function as a ligand-receptor pair, controlling vascular development in the retina and inner ear. ⁸⁵ Nonetheless, the present study systematically mapped the ocular domains and cellular types in which TCF/Lef-dependent gene expression is activated, suggesting multiple roles of this pathway in various developmental processes and homeostasis in the murine retina, which will surely benefit further exploration of Wnt function in the context of mammalian retina and may yield important insights into novel therapeutic approaches to treat or prevent congenital eye diseases. 

 Supported by funding from the Canadian Institutes of Health Research and the Stem Cell Network of Canada. HL is a recipient of a studentship from the Foundation Fighting Blindness Canada and a scholarship from the University of Ottawa.

 Submitted for publication April 10, 2006; revised June 20 and July 28, 2006; accepted September 22, 2006.

 Disclosure: H. Liu, None; S. Thurig, None; O. Mohamed, None; D. Dufort, None; V.A. Wallace, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Activation of the TCF/Lef-LacZ reporter transgene in response to Li⁺ treatment in E14.5 retinal explants. Cross-sections of explants in control media (A, C, E, G) or in the presence of Li⁺ (B, D, F, H) for 3, 15, 24, or 48 hours were stained with X-gal to detect β-gal activity. (F) Dashed line: lens; triangle: upregulation of TCF/Lef-LacZ reporter gene activity at the lens equator. (D, F) Arrows: upregulation of reporter activity in the NR after 15 or 24 hours of treatment; (D, F, H) arrowheads: upregulation of reporter activity in the CM. CM, ciliary margin; NR, neural retina. Scale bar, 100 μm.

Figure 2.

 

Differential expression pattern of the TCF/Lef-LacZ reporter gene, Wnt2b, and Otx1 along the dorsal–ventral axis of the optic vesicle. Horizontal sections of the eye at E9.5 were stained with X-gal to detect β-gal activity (A, B) or were processed for ISH for Wnt2b (C, D) and Otx1 (E, F). n—t: nasal–temporal axis; dashed line: rim of the forebrain (A). Detection of TCF/Lef-LacZ reporter activity at the dorsal–nasal retina. (A, arrowhead) corresponds to the location of Wnt2b mRNA (C, arrow) and the CM/RPE marker Otx1 in the dorsal (E, open triangle) retina. Note that the TCF/Lef-LacZ reporter gene and Wnt2b was not expressed in the ventral optic vesicle at this stage (B, D, filled triangle). Scale bar, 50 μm.

Table 1.

 

Comparison of TCF/Lef-LacZ Reporter Gene Activation and Expression of TCF/Lef Transcription Factors during Ocular Development from E9.5 to E14.5

	OV (E9.5)*			CM	RGC	NB	LAE	ONH
		D	V
Reporter	+	−	+	+ (E12.5 to E14.5)	+	+	−
Tcf1	+	+	+	−	+	+	+
Lef1	+	+	+	+ (E14.5)	+	−	−
Tcf3	+	+	+	−	+	+	+
Tcf4	+	+	−	−	−/+	−	−

 CM, ciliary margin; D/V, dorsal/ventral; LAE, lens anterior epithelium; NB, neuroblast layer; ONH, optic nerve head; OV, optic vesicle; RGC, retinal ganglion cell layer.

*  Embryonic ages in parentheses indicate that reporter activity or gene expression in this region was restricted to this developmental time point.

Figure 3.

 

Expression of the TCF/Lef-LacZ reporter gene in the developing eye. (A–I) Horizontal sections of eyes from TCF/Lef-LacZ transgenic embryos at E9.5, E11.5, E12.5, E14.5, E15.5, E16.5, E17.5, E18.5, and P0 were stained with X-gal to detect β-gal activity. (A) n—t: nasal–temporal axis. (B, D, E, H, I, insets) Enlarged views of the boxed areas. (D, I, dashed lines) Boundaries between the prospective PCE (P) and the nPCE (nP) of the CM. Activation of the TCF/Lef-LacZ reporter gene was detected in the prospective CM (A, arrowhead), the CM, and the prospective CE (B–I, arrowheads). It was also detected in the central NR at E11.5 (B, ), the apical side of the NB layer from E12.5 to E17.5 (E, arrow), the inner NB layer from E14.5 to E17.5 (E, curved arrow) and the RGC layer from E14.5 to P0 (H, open triangle). Note that activation of the reporter gene in the PCE (P) of the CM was downregulated at P0 (compare enlarged view in D with that in I). CE, ciliary epithelium; CM, ciliary margin; NB, neuroblast; nPCE, non-pigmented ciliary epithelium; NR, neural retina; PCE, pigmented ciliary epithelium; RGC, retinal ganglion cell. Scale bars: 100 μm.

Table 2.

 

Activation of TCF/Lef-LacZ Reporter Gene in the Developing Retina from E15.5 to P7 and in the Adult Retina

CM/CE			RGC	INB/INL	ANB
PCE	nPCE
+ (−At P7 and adult)*	+	+	+ (−from E18.5 to P0)	+ (E15.5 to E18.5)

 ANB, apical neuroblast layer; CM/CE, ciliary margin/ciliary epithelium; INB/INL, inner neuroblast/inner nuclear layer; nPCE, nonpigmented ciliary epithelium; PCE, pigmented ciliary epithelium; RGC, retinal ganglion cell layer.

*  Ages in parentheses indicate that reporter activity in this region was excluded (−) or restricted to this developmental time point.

Figure 4.

 

Analysis of the TCF/Lef-LacZ reporter⁺ cells located in the apical neuroblast layer. (A–F) Horizontal sections of the eye at E16.5 were processed for ISH for Crx (A) or Otx2 (B), stained with X-gal for β-gal activity (C, blue), stained for IHC with anti-pH3 (D, red), or double stained for β-gal activity (E, blue) and pH3 (E, brown). Hoechst staining was used to reveal nuclei (D, blue). (F) Higher-magnification view of boxed area in (E). Brackets: overlapping expression pattern of Crx (A), Otx2 (B), and TCF/Lef-LacZ reporter activity (C) in the apical NB; triangle: narrow stripe of pH3⁺ cells adjacent to the RPE (D). A few cells were double labeled for TCF/Lef-LacZ reporter and anti-pH3 (F, arrowhead), whereas most of the TCF/Lef-LacZ reporter⁺ cells were not colabeled with anti-pH3 (F, arrows). NB, neuroblast layer; pH3, phosphoH3; RGC, retinal ganglion cell layer; RPE, retinal pigment epithelium. Scale bars: (A–D) 50 μm; (E) 100 μm; (F) 25 μm.

Figure 5.

 

Expression pattern of TCF/Lef transcription factors in the optic vesicle. (A–D) ISH of horizontal sections of the eye at E9.5 for Tcf1 (A), Lef1 (B), Tcf3 (C), and Tcf4 (D). (A) n—t: nasal–temporal axis; dashed line: rim of forebrain. The expression of Lef1 was detected in the nasal and proximal side of the optic vesicle (B, arrowhead and arrow). Note the diffuse expression of Tcf1 and high level of Tcf3 throughout the optic vesicle (A, C). (D, open triangle) Low level of Tcf4 expression in the temporal side of the optic vesicle. Scale bar, 50 μm.

Figure 6.

 

Expression pattern of TCF/Lef transcription factors in the developing eye. ISH of horizontal sections of the eye at E12.5 (A, C, E, G) or E14.5 (B, D, F, H) for Tcf1 (A, B), Lef1 (C, D), Tcf3 (E, F), and Tcf4 (G, H). n—t: nasal–temporal axis; insets: enlarged views corresponding to the boxed areas in (B), (D), and (F). (B, D, F, dashed lines) Boundaries between the prospective PCE (P) and nPCE (nP) of the CM. Tcf1 and Lef1 were expressed in the CM (A–D, arrowheads). Expression of Tcf1 and Tcf3 was also detected in the optic nerve head (B, E, F, triangles). A higher level of Lef1 expression was detected at the temporal retina at E14.5 (D, arrow). Tcf3 was expressed in the CM and throughout the NR (E, F); in contrast, expression of Tcf4 was difficult to detect at E12.5 and E14.5 (G, H). CM, ciliary margin; nPCE, nonpigmented ciliary epithelium; NR, neural retina; PCE, pigmented ciliary epithelium. Scale bars,100 μm.

Figure 7.

 

Asymmetric expression of Lef1 and the TCF/Lef-LacZ reporter gene in the temporal retina of the developing eye. (A–F) Horizontal sections of the eye at E14.5 (A–D) and E17.5 (E, F) were processed for ISH for Lef1 (A, B) or stained with X-gal (C–F) to detect β-gal activity. Higher level of Lef1 expression (A, arrows) and TCF/Lef-LacZ reporter activity (C, E, arrows) were detected in the temporal retina. Scale bar: 50 μm.

Figure 8.

 

Activation of the TCF/Lef-LacZ reporter gene in postnatal and adult eyes. Horizontal sections of eyes at P7 (A, B, E, F) and an adult eye (C, D) were stained with X-gal to detect β-gal activity. (E) Enlarged view of boxed area in (A); (F) corresponding region from a pigmented retina shown for easier distinction between the PCE and nPCE. (A, C, E, F, arrowheads) Activation of the TCF/Lef-LacZ reporter gene in the nPCE; (E, F) reporter gene expression was not detected in the PCE (arrows). TCF/Lef-LacZ reporter gene is also activated in the RGC (B, D, arrows) and the INL (B, D, open triangles) in the central NR. CE, ciliary epithelium; INL, inner nuclear layer; nPCE, nonpigmented ciliary epithelium; NR, neural retina; ONL, outer nuclear layer; PCE, pigmented ciliary epithelium; RGC, retinal ganglion cell layer. Scale bars: (A–D) 50 μm, (E, F) 10 μm.

Figure 9.

 

Cell-type identification of the TCF/Lef-LacZ reporter⁺ cells in the adult retina. Horizontal sections of adult eyes were stained with Hoechst to reveal nuclei (A, B), processed for IHC for β-gal (D, E, green), stained for Pax6 or calbindin (D, E, red), or double stained for β-gal activity (C, F, blue) and Brn3A+3B (C, brown) or Chx10 (F, brown) with DAB. (D, E, arrows) Colocalization of anti-Pax6 or calbindin with TCF/Lef-LacZ reporter gene expression in the INL, marking amacrine cells. (C, arrowheads) Colocalization of anti-Brn3A and -3B with TCF/Lef-LacZ reporter gene expression in the RGC layer, marking RGC cells. Note that the horizontal (E, open triangle) and bipolar (F, Chx10⁺) cells did not coexpress the TCF/Lef-LacZ reporter gene. INL, inner nuclear layer; ONL, outer nuclear layer; RGC, retinal ganglion cell layer. Scale bar, 25 μm.

The authors thank Sabine Fuhrmann for technical advice on β-gal detection in embryonic tissues.