Negative regulation of retinal-neurite extension by ß-catenin signaling pathway

Yasuo Ouchi, Yoko Tabata, Ken-ichi Arai and Sumiko Watanabe*

Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

* Author for correspondence (e-mail: sumiko{at}ims.u-tokyo.ac.jp)

Accepted 7 July 2005


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 Summary
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 Materials and Methods
 Results
 Discussion
 References
 
Although there have been many studies on the regulation of neurite extension in mouse brain, such a mechanism in neural retina has remained to be clarified. To delineate the role of Wnt signaling in retinal development, we used a retrovirus-vector-mediated expression system to express various mutants forms of Wnt signaling members in E17.5 mouse retinal explant cultures, which are an excellent system to examine retinal development in vitro. Expression of constitutively active ß-catenin or Lef-1 in the retinal cells resulted in failure of neurite extension, suggesting that ß-catenin negatively regulates neurite extension in the retina through Lef-1 transcriptional activity. However, proliferation and differentiation of retinal cells into mature retinal cells such as rod-photoreceptor cells and Muller glia cells were not affected by perturbation of the Wnt-Lef-1 pathway. As in retinal cells, activation of ß-catenin-Lef-1 signaling inhibited NGF-induced neurite extension in PC12 cells without affecting their proliferation. Interestingly, the Wnt-Lef-1 signaling pathway suppressed neurite extension without affecting Mek-1 signal activity, which is known to promote neurite extension. We found that MAPK was activated in retinal explant cultures, but that perturbation of MAPK signals did not affect neurite extension. Taken together, our data suggest that the Wnt pathway functions in proper neurite extension by opposing positive signals for promotion of neurite extension that are distinct from those of the MAPK pathway.

Key words: Retina, PC12, Wnt, ß-catenin, Neurite extension


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mature neural retina consists of six types of neurons and 1 type of glia. Because of the periodically arranged nucleolus and cell processes of retinal cells, a three-layered structure is visible in the neural retina. The outer nuclear layer (ONL) contains rod and cone photoreceptors; the inner nuclear layer (INL) contains bipolar, horizontal and amacrine interneurons and Muller glia cells; and the ganglion cell layer (GCL) contains ganglion cells and displaced amacrine cells. In addition, there exist an outer plexiform layer (OPL) and inner plexiform layer (IPL) consisting of cell processes of retinal cells. These seven types of cells are assumed to be differentiated from common precursors in a histogenetic order (Turner and Cepko, 1987Go). Differentiation of the neural retina can be roughly categorized into at least three steps: early differentiation, migration and neurite extension (Cepko, 1999Go; Harris, 1997Go). Various molecules such as transcription factors and neurotropic factors have been reported to play important roles in retinal cell differentiation (Jean et al., 1998Go). However, the precise roles of these molecules in each step, especially the mechanisms regulating neurite extension and formation of the neural retina network in the plexiform layers, are not yet well understood.

Members of the Wnt gene family encode secreted glycoproteins that play important roles in the proliferation and maintenance of various tissues (Cadigan and Nusse, 1997Go). In the central nervous system, Wnt molecules have multiple functions such as axonal remodeling and synaptic differentiation (Hall et al., 2000Go), and axon guidance in the spinal cord (Lyuksyutova et al., 2003Go). Recent reports showed promotion of differentiation of neural stem cell and neural crest stem cells by Wnt signaling (Hirabayashi et al., 2004Go; Lee et al., 2004Go). The frizzled gene family encodes seven-transmembrane-domain cell surface receptors that bind Wnts by the extracellular cysteine-rich domain (CRD) of the former (Nusse, 2003Go). Signaling of the canonical Wnt pathway transmitted through Frizzled results in the stabilization of cytoplasmic ß-catenin by inhibiting glycogen synthase kinase (GSK)-3ß function (Nusse, 2003Go). Then the stabilized ß-catenin is translocated into the nucleus and associates with HMG-box transcription factors called TCF/Lef-1 family proteins (Eastman and Grosschedl, 1999Go). Wnt signaling is modulated in the extracellular environment by antagonistic activities of members of the secreted-Frizzled-related protein (SFRP) family. SFRP members contain a CRD region that binds to Wnts, thus preventing Wnts from interacting with Frizzleds (Jones and Jomary, 2002Go). ß-Catenin also plays roles in stabilizing the actin cytoskeleton and mediating cell adhesion and morphogenesis as a component of the cadherin/catenin complex (Ben-Ze'ev and Geiger, 1998Go). Although the role of ß-catenin in the cadherin/catenin complex in cell morphogenesis has been extensively studied, that of ß-catenin-Lef-1 signaling in cell morphogenesis is less well understood.

The function of the Wnt canonical pathway in retinal development has been well documented in Drosophila (Tresman and Rubin, 1995Go). In Drosophila eye development, wingless, the Drosophila counterpart of the vertebrate Wnt-1, is expressed in the dorsal and ventral edges of the eye disc (Tio et al., 1996Go). Wingless inhibits morphogenetic furrow movement and keeps the eye disc undifferentiated (Tresman and Rubin, 1995Go). In some vertebrates, such as fish, frogs and chickens, the peripheral region of the retina called the ciliary marginal zone (CMZ) supplies newly produced retinal cells (Perron and Harris, 2000Go). A study using chickens showed that Wnt-2b, which is expressed in the marginal region of chicken retina, plays important roles in the maintenance and proliferation of retinal progenitor cells (Nakagawa et al., 2003Go). However, it is still not clear whether immature progenitor cells are localized in the CMZ or not in the mammalian retina. Furthermore, the role of Wnt in mammalian retinal development was largely unknown.

In this study, we sought to clarify the roles of the Wnt signaling pathway in mammalian neural retina development by expressing various mutants of signaling molecules in mouse retinal explant cultures. We found that Wnt-ß-catenin negatively regulated neurite extension in the mouse retina through Lef-1 transcriptional activities. Further studies using PC12 cells revealed that ß-catenin-Lef-1 suppressed neurite extension without affecting activation of the MAPK pathway, which enhances neurite extension in PC12 cells. Furthermore, we could not find any evidence of a role for MAPK in process extension in retinal explant cultures.


    Materials and Methods
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 Materials and Methods
 Results
 Discussion
 References
 
Plasmid constructions, reagents, and production of retroviruses
pMX-EGFP was constructed by insertion of EGFP into HindIII/NotI sites of pMX, a retrovirus vector. A constitutively active form of mouse ß-catenin (caß-catenin) cDNA was kindly provided by A. Nagafuchi (Kumamoto University, Japan), and its coding region was subcloned into StuI/XhoI sites of the pMX retrovirus vector containing IRES-EGFP (internal ribosomal entry site-enhanced green fluorescent protein), pMX-MCS-IRES-EGFP. Constitutively active Lef-1 (caLEF) cDNA, which was kindly provided by R. Grosschedl (University of Munich, Germany), was subcloned into SacII/SnaBI sites of pMX-MCS-IRES-EGFP. Retrovirus vector containing dominant-negative Lef-1 (dnLEF) (pMX-dnLEF-IRES-EGFP) was made by subcloning of dnLEF1 from RCAS-dnLEF1, a kind gift from M. Kengaku (RIKEN, Wako, Japan), into XhoI/NotI sites of pMX-MCS-IRES-EGFP. TCF-BP-EGFP was constructed from 7 tandem repeats of the TCF binding site from TCF-BP-Luc (Ueda et al., 2001Go), which was kindly provided by T. Shimotono (University of Kyoto, Japan) into pEGFP-1 (Clonetech). c-fos minimal promoter was cloned by PCR from mouse genomic DNA and inserted between the TCF binding site and the EGFP gene. PD98059 was purchased from New England BioLabs. Anti-phospho MAPK and MAPK antibodies were from Cell Signaling Technology and Santa Cruz Biotechnology, respectively. Western blotting was carried out as previously described (Watanabe et al., 2000Go).

Transient retrovirus packaging cell line PLAT-E (Morita et al., 2000Go) was transfected with retrovirus vectors containing various genes by using FuGene6 transfection reagent (Roche) according to the manufacturer's instructions. Two days after transfection, cell supernatants containing retrovirus were harvested and concentrated by centrifugation in a centrifugal filter device (Millipore). Retroviral infection of the retinal explants was carried out as described previously (Tabata et al., 2004Go). For PC12 cells, cells were incubated for 24 hours in DMEM containing 10% horse serum (HS) and 1 µl of retrovirus solution.

Retinal explant culture and cell culture
Retinal explant cultures were prepared as previously described (Tabata et al., 2004Go). Briefly, the neural retinas of E17.5 ICR mice without pigmented epithelium were placed on Millicell chamber filters (Millipore, diameter 30 mm, pore size 0.4 mm) in a 6-well culture plate, with the ganglion cell layer facing upwards. Each well contained 1 ml of explant culture medium (50% MEM with Hepes, 25% Hank's solution, 25% heat-inactivated HS, 200 mM L-glutamine, and 5.75 mg/ml glucose, penicillin and streptomycin). Explants were cultured at 34°C in 5% CO2, and the medium was changed every other day. In some cases, the explants were infected with retroviruses and cultured for three days. Then cells were harvested and treated with trypsin (0.25%) for disaggregation, replated into 8-well chamber slides (BD Falcon) coated with ornithine and fibronectine, and then cultured a further 11 days in DMEM/F12 supplemented with 1% FCS, N2 and penicillin and streptomycin. Cells were then fixed with 4% PFA and immunostained with appropriate antibodies.

Rat pheochromocytoma (PC12) cells (Tischler and Greene, 1976Go) were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% HS (HS-DMEM), penicillin and streptomycin. For neurite extension assays, PC12 cells (5x103 cells/well) were plated on poly-D-lysine-coated 4-well chamber slides (Falcon) and continued to be cultured in 1% HS-DMEM supplemented with 50 ng/ml of mouse 2.5s NGF (Sigma). The medium was changed every 2 or 3 days.

Immunohistochemical analysis
Immunohistochemistry was carried out as described earlier (Tabata et al., 2004Go). Briefly, retinal explants were fixed with 4% paraformaldehyde (PFA) and soaked in 30% sucrose. Then the samples were frozen-sectioned (10 µm thick) in OCT compound (Miles) and pre-incubated in a blocking solution. For immunohistochemistry of PC12, the cells were fixed with 4% PFA, 0.1% glutalaldehyde, and 0.1% Triton X-100 for 40 minutes on ice. The samples were preincubated with a blocking solution and incubated with the appropriate antibody solutions. The primary antibodies were visualized by using anti-rabbit IgG-Alexa Fluor 488 or anti-mouse IgG-Alexa Fluor 546 (Molecular Probes) as secondary antibodies. Primary antibodies used were anti-GFP polyclonal antibody (Clontech Laboratories), anti-Rho4D2 (kindly provided by R. S. Molday, The University of British Columbia, Vancouver, Canada), anti-glutamine synthetase (Chemicon), and anti-ßIII tubullin (BABCO). All samples were sealed with VectaShield (Vector Laboratories) containing DAPI for nuclear staining.

BrdU labeling and detection
Proliferation was analyzed by incorporation of BrdU. Retinal explants were incubated with 5 µM BrdU for 24 hours at 2, 6 or 13 days after retrovirus infection, harvested, and fixed with 4% PFA. The samples were embedded in OCT compound and frozen-sectioned. Then the sections were treated with 1 U/µl of DNAse (Takara) in PBS for 1 hour at 37°C, and the incorporated BrdU was visualized immunohistochemically by using anti-BrdU monoclonal antibody (Roche) and appropriate secondary antibody.

Luciferase assay
PC12 cells (1x105 cells/well) were plated in a 24-well culture plates 1 day before transfection with various combinations of plasmid DNAs, using Fugene6 transfection reagent (Roche). After 2 days in culture, the cells were harvested and lysed in 20 µl of 250 mM Tris-HCl, pH 7.4, by three cycles of freezing and thawing. Protein concentration was determined by using a BCA protein assay kit (Pierce). Luciferase activity toward a luciferase assay substrate (Promega) was measured with a luminometer (model LB9501, Berthold Lumat Co. Ltd).


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ß-catenin-Lef-1 signal pathway regulates process extension by retinal cells in retinal explants
To examine the role of the Wnt-ß-catenin-Lef-1 signal pathway in murine retinal development, we introduced constitutively active or dominant-negative forms of molecules of the ß-catenin-LEF-1 signaling pathway into mouse retinal explant cultures by using a replication-incompetent retrovirus vector. Before examining the effects of perturbation of Wnt signaling, we examined activation of the Wnt signaling pathway in explant culture by introduction of TCF-binding-motif-dependent EGFP activation. We introduced the plasmid containing tandem repeats of the TCF binding motif followed by c-fos gene minimal promoter and EGFP into E17-mice-derived neural retina and the retina was cultured as explant. EGFP can be strongly observed after 2 days of culture, and completely deceased after 1 week of culture, suggesting that the Wnt signaling pathway is activated in early stages of neural retina development in the explant culture (data not shown). We then examined the role of the Wnt-ß-catenin-Lef-1 signal pathway in retinal development. Since the retrovirus infects only mitotic cells, retinal precursor cells are assumed to be the major targets for gene transfer. We prepared retrovirus containing EGFP and constitutively active (ca) ß-catenin, which has a mutation in its N-terminal phosphorylation site recognized by GSK-3ß; ca Lef-1 (caLEF), consisting of the C-terminal transcriptional activation domain of human ß-catenin (amino acids 696-781) fused to the N-terminus of the C-terminal half of mouse Lef-1 (Hsu et al., 1998Go); or dominant-negative (dn) Lef-1 (dnLEF), which lacks the N-terminal ß-catenin-binding domain. The E17.5 neural retina, which is in the early stage of retinal development, consists of ganglion cells and blastic layers and was used for explant culture (Tabata et al., 2004Go). The explants were infected with the virus at the beginning of culture, and cultured for 2 weeks. The explanted retinal cells differentiate during culture and the localization and morphology of virus-infected cells were determined by monitoring EGFP-positive cells. Since the EGFP is distributed into the cell processes as well as in cell bodies, neurite extension can be clearly observed by EGFP expression (Fig. 1A). When the control EGFP virus was used to infect the retinal explants, all infected cells extended neurites, and about 80% of their cell bodies were located in the ONL and 20%, in the INL (Fig. 1A,B). By contrast, none of the retinal cells expressing caß-catenin-EGFP extended their processes (Fig. 1A); and nearly 50% of the EGFP-positive cells had their cell bodies in the INL, suggesting that sub-layer localization of the cells was also perturbed. Similarly, about 70% of the retinal cells expressing caLEF-EGFP failed to extend processes, and the remaining 30% slightly extended thick process-like structures (Fig. 1A). Interestingly, the sub-layer localization of caLEF-EGFP-expressing cells was like that in the control EGFP-expressing cells (Fig. 1B), indicating that the abnormal localization of cells induced by constitutively active ß-catenin may be caused by some mechanism other than a Lef-1-activation-dependent one. Conversely, the process extension of dnLEF-EGFP-expressing retinal cells was enhanced, as indicated by their bushy processes in the inner plexiform layer (IPL; Fig. 1A). However, sub-retinal localization of dnLEF-EGFP-expressing cells was indistinguishable from that of EGFP-expressing cells (Fig. 1A,B), supporting the idea that Lef-1 activity does not affect the subretinal localization of retinal cells. To examine these observation more quantitatively, we measured neurite length by dissociation culture. We infected virus into retinal explants as described above, and the explants were disaggregated with trypsin and replated onto chamber slides. After 2 weeks of culture, cells were immunostained with anti-GFP and anti-glutamate synthetase (GS), which is a Muller glia cell marker, and neurite extension of GFP-positive/GS-negative neural cells was examined and measured for quantitative analysis (Fig. 1C). We found that the population of cells expressing either caß-catenin-EGFP or caLEF-EGFP, which failed to extend neurites, was much larger than that of control EGFP-expressing cells. In addition, cells expressing caß-catenin-EGFP or caLEF-EGFP had a shorter average length and those expressing dnLEF-EGFP had a longer average length than the average length of control cells (Fig. 1D). However, statistical significance was observed only with caß-catenin-EGFP because of relatively high variation of values among independent experiments. We then examined the statistical significance of the results by comparing populations of cells by dividing them into 2 groups: 1 group with neurites shorter than 10 µm and another group with neurites longer than 30 µm; there was a significant difference in neurite length between cells expressing caß-catenin-EGFP or caLEF-EGFP (Fig. 1D). As a possible reason for the less convincing results of caLEF-EGFP obtained in dissociated culture, we surmised that the endogenous Wnt signals are less effectively worked in the dissociated culture, which has far less density of cells in compare with that of the explant culture.



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Fig. 1. Morphology of neural retina cells transfected with retrovirus containing EGFP, dnLEF-EGFP, caß-catenin-EGFP or caLEF-EGFP. (A) Retinal explants were prepared from E17.5 mouse embryos and infected with retrovirus encoding EGFP, dnLEF-IRES-EGFP, caß-catenin-IRES-EGFP or caLEF-IRES-EGFP. After 2 weeks in culture, the explants were fixed and frozen-sectioned. Immunohistochemistry used anti-GFP antibody, and DAPI was used to visualize nuclei. Bars, 10 µm (left panels); 50 µm (right panels). (B) Subretinal localization of EGFP-positive virus-infected cells was examined in several sections. More than 100 cells of each sample were counted and each set of experiments was conducted in triplicate. The percentage of the total EGFP-positive cell number at each location is shown. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (C) Neurite length distribution of the retinal cells expressing EGFP, dnLEF-EGFP, caß-catenin-EGFP or caLEF-EGFP. Retinal explants were prepared from E17.5 mouse embryos and infected with retrovirus encoding EGFP, dnLEF-IRES-EGFP, caß-catenin-IRES-EGFP or caLEF-IRES-EGFP. After 3 days of culture, cells were disaggregated, replated into chamber slides and cultured for a further 11 days. Cells were then stained with anti-GS and anti-GFP antibodies and the neurite length of more than 45 cells of GS-negative/GFP-positive neural cells of each samples was measured. (D) Average neurite length from Fig. 1C is shown in the left panel. Populations of cells with neurite length less than 10 µm (middle panel) and more than 30 µm (right panel) in Fig. 1C are compared. More than 50 cells of each sample were measured and each set of experiments was conducted in triplicate. Error bars represent s.e.m. *P<0.05; **P<0.01.

 
Activation or inhibition of the ß-catenin-Lef-1 pathway does not affect proliferation of E17.5 retinal explant cells
Since Wnt-ß-catenin-Lef-1 signaling is known to play important roles in the proliferation of various cells (Novak and Dedhar, 1999Go), we next examined the proliferation activity of retinal cells expressing EGFP, caß-catenin-EGFP, caLEF-EGFP or dnLEF-EGFP in terms of BrdU incorporation. Retinal explants were transfected with these genes by using retroviruses and then incubated with 5 µM BrdU for 24 hours starting 2, 6 or 13 days after the retrovirus infection. When the control EGFP virus was used, nearly 20% of the EGFP-expressing retinal cells were BrdU positive at the third day; and the positive population was reduced to less than 3% at the seventh day (Fig. 2A,B). At the fourteenth day, BrdU-positive cells expressing EGFP were hardly detected (Fig. 2A,B). Since the number of immature progenitor population may be reduced during retinal development, the observed reduction in the number of BrdU-positive cells was expected. When the caß-catenin-EGFP or caLEF-EGFP viruses were used for the infection, BrdU-positive cells were rather slightly decreased in number at the third day compared with those in the EGFP-infected control (Fig. 2A,B). At the seventh or fourteenth day, their percentage was nearly the same as that for the control (Fig. 2A,B). When we infected cultures with dnLEF-EGFP retrovirus, the percentage of BrdU-positive cells was almost the same as that for the control and slightly higher than that for the other group of cells. These data suggest that activation of the ß-catenin pathway either did not enhance proliferation of the retinal cells, or affected the proliferation somewhat negatively.



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Fig. 2. Proliferation and differentiation of retinal cells perturbed by the ß-catenin signaling pathway. (A,B) BrdU incorporation into retinal cells expressing EGFP, caß-catenin-EGFP, caLEF-EGFP or dnLEF-EGFP was examined. Retinal explants were infected with retrovirus and incubated with 5 µM BrdU at 2, 6 or 13 days after retrovirus infection and harvested 24 hours later. (A) Patterns of frozen sections immunostained with anti-BrdU and anti-GFP antibodies were visualized with secondary antibodies conjugated to Alexa 546 and Alexa 488 (Molecular Probes), respectively. (B) Percentage of BrdU-incorporated retinal cells in virus-infected cells. (C,D) Expression of rod photoreceptor and Muller glia markers was examined by immunohistochemistry. Retinal explants were infected with retrovirus and cultured for 2 weeks. Frozen sections were then made (C), or the explants were disaggregated with trypsin and replated into chamber slides (D). In D, the number of Rho4D2-positive cells were counted. More than 100 cells were measured for each sample and s.d. was calculated from three independent experiments. Both samples were subjected to immunohistochemistry for rod photoreceptor cells by using anti-Rho4D2 antibody and for Muller glia cells by using anti-GS antibody. These first antibodies were visualized by using secondary antibody conjugated to Alexa 546. Nuclei were stained with DAPI and infected cells were detected by anti-GFP antibody. Bars, 50 µm (A,C).

 

Sub-retinal localization of cells in the early period of culture showed that the cells expressing caß-catenin-EGFP tended to be localized in the INL as well as in the outer edge of the ONL, whereas the EGFP-expressing cells were distributed throughout the retinal area by the third day (Fig. 2A,B). At the seventh day, most of the former were localized in the INL and process extension had not occurred (Fig. 2A,B). However, the cells expressing caLEF-EGFP became localized normally in the early period of the culture. In the control EGFP-expressing cells, process extension was first observed at 6-7 days; and the extensions became longer by 13-14 days (Fig. 2A,B). The morphology of retinal cells expressing dnLEF-EGFP looked normal until 7 days; but on day 13 their processes extended farther than those of control EGFP-expressing cells, indicating that the initiation cue of process budding may not be perturbed by dnLEF but that the regulation of process length may be affected by it.

Retinal cells expressing caß-catenin, caLEF or dnLEF differentiate into appropriate retinal cells
We next examined the expression of retinal differentiation markers of the retrovirus-infected cells by immunohistochemistry. Retinal cultures were infected with retrovirus as in previous experiments, and frozen sections were made after 2 weeks of culture and used for immunohistochemistry. Approximately 80% of the EGFP-positive retinal cells in retinal cultures infected with the control EGFP virus expressed a rod photoreceptor marker (Rho4D2), and the other 20% reacted with antibody against GS (Fig. 2C). Although the cells expressing caß-catenin or caLEF did not extend their processes, rod photoreceptor and glia cell markers were detected in these cells. Also, both markers were observed in the subsets of retinal cells expressing dnLEF-EGFP (Fig. 2C). We also examined markers of amacrine cells (syntaxin), horizontal cells (Neurofilament160), and amacrine and ganglion cells (Hu and ßIII tubulin) by immunohistochemistry and detected these markers in a total 2 or 3% of all EGFP-positive cells. There was no significant difference in marker frequency between the control-EGFP-infected cells and Wnt-ß-catenin signaling-modulated cells (data not shown). To examine differentiation of cells expressing ß-catenin-related genes more convincingly, we disaggregated retinal explant culture as described, and replated these cells onto chamber slides and immunostained with either Rho4D2 or anti-GS antibody. As shown in Fig. 2D, about 75% of cells expressed antigen for Rho4D2 and no significant difference between cells expressing different genes was observed. Similarly, about 20% of cells expressed GS and no significant difference was observed between samples. In addition, immunostaining of anti-musashi and anti-nestin antibodies showed that none of the transfected cells expressed these immature neuronal markers (data not shown), supporting the idea that transfected cells were really differentiated. Taken together, these findings suggest that perturbation of the Wnt-Lef-1 signaling pathway did not affect the differentiation of retinal progenitor cells.



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Fig. 3. Effects of perturbation of the Wnt signaling pathway on neurite extension by PC12 cells. (A) Morphology of PC12 cells infected with EGFP, caß-catenin-EGFP, caLEF-EGFP or dnLEF-EGFP retroviruses. PC12 cells were infected with retrovirus encoding EGFP, caß-catnin-EGFP, caLEF-EGFP or dnLEF-EGFP; the cells were then incubated with 50 ng/ml NGF for 5 days to cause differentiation, and subsequently fixed. Cells were immunostained with anti-ßIII-tubulin and anti-GFP antibodies and visualized with Alexa 546 and Alexa 488, respectively. DAPI was used for staining nuclei. Bar, 100 µm. (B-E) Neurite length distribution of the PC12 cells expressing EGFP, dnLEF-EGFP, caß-catenin-EGFP or caLEF-EGFP. Neurite lengths of more than 75 cells of EGFP-positive and -negative cells were measured. Each experiment was conducted in triplicate. Error bars represent s.e.m. of three independent results. (F) Normalized neurite length of PC12 cells expressing EGFP, caß-catenin-EGFP, caLEF-EGFP or dnLEF-EGFP. The average neurite length of EGFP-positive transfected cells (n>75) were normalized by the average neurite length of EGFP-negative non-transfected cells (n>75). Each experiment was conducted in triplicate. Error bars represent s.e.m. *P<0.05; ***P<0.005 by the Student's t-test. (G) Effect of the ß-catenin-Lef-1 signaling pathway on MAPK-dependent transcription activity. PC12 cells were co-transfected with the c-fos promoter-luciferase plasmid (0.1 µg) together with caMEK or caLEF plasmids (0.1 µg), as described in Materials and Methods. After 10 hours of culture, cells were harvested and the luciferase activities were measured.

 
Activation of the ß-catenin-Lef-1 signal pathway inhibits NGF-induced neurite outgrowth in PC12 cells
The PC12 cell (Greene and Tischler, 1976Go) is a well-studied model for investigating the regulation of neurite extension. So, for further study of the role of the ß-catenin-Lef-1 signaling pathway in neurite extension, we used PC12 cells. We first examined whether activation of ß-catenin-Lef-1 signaling would also inhibit neurite outgrowth of PC12 cells or not. We expressed EGFP, caß-catenin-EGFP, caLEF-EGFP or dnLEF-EGFP in PC12 cells by the retrovirus method and then stimulated these cells with NGF for 5 days.

When the control EGFP virus was used to infect the PC12 cells, the length of neurites of EGFP-positive infected cells and EGFP-negative noninfected cells was indistinguishable (Fig. 3A). Histogram analysis revealed that the main population of both EGFP-positive and EGFP-negative cells showed a neurite length of approx. 10-40 µm (Fig. 3B). The average neurite length of control EGFP-expressing cells was 20.6 µm. The neurite outgrowth of PC12 cells expressing either the caß-catenin-EGFP or caLEF-EGFP was strongly inhibited (Fig. 3A,C,D). Although the neurite length of the EGFP-negative (non-infected) cells and the control EGFP virus-infected cells varied between 10 and 40 µm, the neurite length of the caß-catenin-EGFP, or caLEF-EGFP-expressing cells was mostly distributed in the 0-10 µm range (Fig. 3C,D). The average neurite length of cells expressing caß-catenin-EGFP or caLEF-EGFP was 5.08 µm and 5.92 µm, respectively. As expected, the neurite length of dnLEF-EGFP-expressing cells was longer than that of EGFP-expressing cells, and their average neurite length was 29.7 µm. Statistical analysis also showed that the inhibitory effects of caß-catnin and caLEF, and enhancement by dnLEF expression were significant (Fig. 3F). Therefore we concluded that the ß-catenin-Lef-1 pathway also suppresses neurite extension in PC12 cells. Proliferation of these cells, which was examined by BrdU incorporation for 24 hours at the fourth day after retrovirus infection, was not modulated by the expression of EGFP, caß-catenin-EGFP, caLEF-EGFP or dnLEF-EGFP (data not shown).

Inhibition of neurite extension by activation of the ß-catenin signal does not occur through modulation of the MAPK signal pathway
Neurite outgrowth of PC12 cells is known to be positively regulated by MAPK/ERK signaling activity (Noda et al., 1985Go). We confirmed this phenomenon in our experimental system by expressing dominant-negative Ras (dnRas) or constitutively active Mek-1 (caMEK) in PC12 cells (data not shown). We then examined the possibility that the ß-catenin exerts its effect by inhibiting MAPK signaling. To examine this possibility, we evaluated the expression of the c-fos gene, which is known as a target gene of the MAPK pathway (Marais et al., 1993Go). In addition, a recent report showed that c-fos-activated phospholipid synthesis is required for neurite elongation in PC12 cells (Gil et al., 2004Go). Transient transfection analysis was carried out using a plasmid containing a c-fos promoter fused to the coding region of luciferase (Fig. 3G) (Watanabe et al., 1993Go). Expression of caLEF slightly reduced c-fos transcriptional activity, but this inhibition was not statistically significant. As expected, constitutively activated Mek-1 enhanced c-fos promoter activity dramatically. Co-transfection with both caLEF and caMEK did not suppress the caMEK-induced c-fos promoter transcriptional activity, suggesting that the inhibition of neurite extension by the ß-catenin-Lef-1 signal pathway was not due to negative regulation of MAPK signaling. Similar results were obtained by using a mammalian 1 hybrid system of elk-1 (PathDetect, Elk1 trans-reporting system, Stratagene), thus confirming a MAPK-independent mechanism of suppression of neurite extension by ß-catenin-Lef-1 in PC12 cells.

The MAPK pathway does not play a role in process extension during retinal development in explant cultures
We examined whether or not the MAPK pathway plays a role in neurite extension in the neural retina by using explant cultures. We first examined whether the MAPK pathway was activated or not in retinal explant cultures by immunohistochemistry of frozen-sectioned explant cultures with anti-phospho-MAPK-specific antibody (Fig. 4A). At day 6 of culture, phosphorylated MAPK was observed in the INL and in most of the outer edge of the ONL (Fig. 4A, right panel). In 14-day cultures, weak phospho signals were observed in all areas except the INL, where the signals were relatively strong. To confirm the activation of MAPK, we next performed western blotting of whole-cell lysates of retinal explants (Fig. 4B). The explants were cultured in the presence or absence of PD98059, a Mek-1 inhibitor, and western blotting was carried out by using either anti-MAPK antibody or anti-phospho-MAPK antibody. As a positive control, whole-cell lysates from cells of the hematopoietic cell line Ba/F3 expressing human GM-CSF receptor (Watanabe et al., 1993Go) and stimulated with human GM-CSF (10 ng/ml) were prepared. The addition of PD98059 to Ba/F3 cultures blocked phosphorylation of hGM-CSF-induced MAPK, as expected (Fig. 4B). In the lysate from retinal explant cultures, a band of phosphorylated MAPK was observed both at day 1 and day 3 (Fig. 4B). The addition of either 10 or 50 µM PD98059 significantly suppressed the phosphorylation of MAPK in the explant cultures. We then analyzed the effects of PD98059 on neurite extension of retinal cells. To examine neurite extension more easily, we first introduced EGFP by retrovirus into retinal explants at the beginning of the culture, and then added PD98059 at the desired times during the culture period. Frozen-sectioned samples were then prepared and examined for neurite extension. The presence of 10 µM PD98059 during the whole period of culture (Fig. 4C, 0-14 days) or during the last half (Fig. 4C, 7-14 days) had no significant effect on neurite extension, which was similar to that in the control sample (Fig. 4C, control). We also examined neurite extension of retinal cells in the presence of 50 µM PD98059 and found that the inhibition of Mek1 signals at this higher concentration also did not significantly affect neurite extension of the retina in the explant culture (data not shown). We then analyzed the effects of the expression of dnRas or dominant-negative MEK1 (dnMEK) on neurite extension of the retina. Retroviruses containing either dnRas or dnMEK cDNA followed by IRES-EGFP were introduced into retinal explant cultures at the beginning of the culture; 2 weeks later, neurite extension was examined by inspecting frozen-sectioned samples. As shown in Fig. 4D, neurite extension was indistinguishable among these three samples. We also examined the subretinal localization of retrovirus-infected cells and found that the cells expressing EGFP, dnRas or dnMEK were distributed similarly in the retina (Fig. 4E). To examine neurite extension more quantitatively, we measured neurite length by dissociation culture and found that, in cells expressing control EGFP, dnMEK and dnRas, the distribution of populations of the same neurite length was almost the same (Fig. 4F). In addition, there was no significant difference in average neurite length between cells expressing these molecules in this culturing system (Fig. 4G). These data thus suggest that Ras-MAPK signals may not play a significant role in neurite extension in the neural retina.



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Fig. 4. Activation of the MAPK pathway and its inhibition during neural retina development. (A,B) Phosphorylation of MAPK in neural retina. (A) Retinal explant cultures were frozen-sectioned at day 6 and 14, and immunostained using anti-phospho p44/42 MAPK antibody ({alpha}-p-MAPK) or control IgG; the first antibody was visualized by using a horseradish peroxidase-labeled secondary antibody. (B) Retinal explant cultures in the presence or absence of PD98059 were harvested at day 1 or 3, and whole-cell lysates were subjected to western blotting using anti-MAPK antibody ({alpha}-MAPK) or anti-phospho p44/42 MAP antibody ({alpha}-pMAPK). Ba/F3 cells expressing human GM-CSF receptor were stimulated or not for 10 minutes in the presence or absence of PD98059 and used as a positive control. (C) Effects of PD98059 on neurite extension in retinal explant cultures. Retrovirus encoding EGFP was introduced into E17.5 retinal explant cultures at day 1, and the cultures were continued in the presence of 10 µM PD98059 from day 1-14 or day 7-14. The cultures were harvested and frozen-sectioned to examine EGFP-positive cells by immunostaining with anti-GFP antibody. (D) Retinal explants were prepared from E17.5 mouse embryos and infected with retrovirus encoding dnMEK-IRES-EGFP, dnRas-IRES-EGFP or EGFP. After 2 weeks in culture, the explants were fixed and frozen-sectioned. Immunohistochemistry used anti-GFP antibody, and DAPI was used to visualize nuclei. (E) Subretinal localization of EGFP-positive virus-infected cells was examined in several sections. More than 100 cells of each sample were counted and each set of experiments was conducted in triplicate. ONL, outer nuclear layer, INL, inner nuclear layer, GCL, ganglion cell layer. Bars, 50 µm (A,C,D). (F,G) Neurite length was measured in cells from dissociated retinal culture expressing EGFP, dnMEK-EGFP or dnRas-EGFP, as described in the legend to Fig. 1C. Neurite length distribution of the retinal cells (F) and the average neurite length±s.e.m. (G) are shown. More than 70 cells of each sample were measured and each set of experiments was conducted in triplicate.

 

    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
For development of a functional visual system, proper neurite extension and wiring between optic nerve and neural retina are important as a final stage of morphogenesis of the eye, but their molecular mechanisms in the early stage of neurite extension are not yet well understood. Here, we showed that forced activation of the Wnt signaling pathway suppressed the neurite extension of neural retina cells by a Lef-1 transcription factor-dependent mechanism. Although the neurite extension was completely suppressed by the expression of ß-catenin, differentiation markers such as those for photoreceptor cells and Muller glia cells were expressed, which suggests that the early differentiation occurred in the neural retina but that the final morphological differentiation was suppressed by ß-catenin-Lef-1 signals. We previously reported that early differentiation and final morphological differentiation could be separated in terms of their regulation in zebrafish (Kurita et al., 2003Go). In that study, we found that the lens plays a critical role in plexiform layer formation, but has no role in the expression of several marker proteins that occur during the early differentiation of retinal cells during eye development in zebrafish embryos (Kurita et al., 2003Go). These findings mean that process extension is regulated by mechanisms different from those regulating early differentiation. Here, we showed that the Wnt pathway is one such signal specifically regulating neurite extension.

The activation of ß-catenin in the retinal explants resulted in strong inhibition of neurite outgrowth but did not act to promote proliferation. Many reports suggesting a role for the Wnt-ß-catenin signaling pathway in cell proliferation have accumulated, and aberrant Wnt signaling has been associated with tumorigenesis (Polakis, 2000Go). Wnts exert their activities by stabilizing ß-catenin, which consequently leads to transcriptional activation of specific target genes (Novak and Dedhar, 1999Go). Mutations in ß-catenin or other Wnt pathway components, which result in ß-catenin accumulation, are found in a wide range of human cancers (Polakis, 2000Go). Interestingly, involvement of the Wnt signaling pathway in tumorigenesis in the nervous system has mainly been reported for embryonic tumors of the cerebellum, such as medulloblastoma, which are most likely derived from neuronal stem cells (Koesters and von Knebel Doeberitz, 2003Go). Thus, it should be considered that although a substantial population of the retinal cells did not respond to ß-catenin in terms of proliferation, a subset of cells such as stem cells proliferated by a ß-catenin-dependent mechanism. In fact, cells localized in the ciliary marginal zone (CMZ) proliferate in response to Wnt stimulation in the chicken system (Kubo et al., 2003Go). This phenomenon is thought to be due to the local expression of Wnt-2b. However, whether some cell-intrinsic feature or other permissive signals also determine the specific response is not yet known. The expression of various Wnt genes, such as Wnt-1, Wnt-3, Wnt-5a, Wnt-5b, Wnt-7b and Wnt-2b, are known to be expressed in the mouse neural retina at various developmental stages (Liu et al., 2003Go). As in the chicken system, Wnt-2b is expressed in the cells at the tip in the mouse CMZ (Fig. 5), and the expression pattern is dynamically changed along with retinal development (Liu et al., 2003Go). We found that the strong expression of TCF-dependent-EGFP was observed in E17-derived neural retina and the expression disappeared after 1 week of culture by explant. However, TCF-dependent transcription in the eye examined by using TCF-LacZ transgenic mice (Liu et al., 2003Go) revealed that TCF-Lef-1 activity was localized to the outer neuroblast layer of the neural retina, and the level of ß-gal activity in the neural retina was substantially reduced from E13.5 to E14.5 (Liu et al., 2003Go). This observation was in contrast to our finding of strong activity in E17-mice-derived retina, and we surmised that the difference may be caused by the different sensitivity of the reporter gene.



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Fig. 5. The expression and activation of the relevant Wnts in the mouse retinas. The expression of Wnts in mammalian retina is schematically summarized from a previous report (Liu et al., 2003Go). Tcf-binding-site-dependent EGFP expression was examined in the explanted retina from E17 by using TCF-binding-site-EGFP plasmid as described in the Results section.

 

Several studies showed that the Wnt regulates neurite extension in the cerebellum and other systems (Lyuksyutova et al., 2003Go), but the role of Lef-1 in regulating neurite extension has not been well understood. We showed that the inhibition of Lef-1 transcription by using dnLef-1 resulted in enhanced neurite extension in the explant culture, suggesting the importance of its transcriptional activation to suppress neurite outgrowth. This notion was supported by our finding that constitutively active Lef-1 suppressed neurite extension. ß-Catenin is known to act as a component of the cadherin-catenin complex in addition to functioning as a transcription activator in the Wnt signal pathway (Nelson and Nusse, 2004Go). Axonal remodeling and synaptic differentiation in the commissural axons were shown to be regulated by the Wnt-7a-GSK-3ß signaling pathway (Hall et al., 2000Go). Phosphorylation targets of GSK-3ß were not only ß-catenin but also cytoskeleton proteins such as MAP-1B and Tau, which were thought to be involved in the Wnt-GSK-3ß signal pathway. The caLEF used in the present study was composed of the C-terminal transcriptional activation domain of ß-catenin fused to the N-terminus of the C-terminal half of mouse Lef-1. Since the C-terminal transcriptional activation domain of human ß-catenin does not bind to signal components such as cadherin, APC, GSK-3ß and Axin (Bienz and Clevers, 2000Go), the possibility that the effect of ß-catenin observed in this work was mediated by interaction with those proteins could be excluded.

We found that the activation of the Wnt-ß-catenin-Lef-1 signal pathway in PC12 cells resulted in the inhibition of NGF-induced neurite outgrowth but had no effect on proliferation. These results are consistent with a previous report that PC12 cells expressing Wnt-1 failed in NGF-dependent neurite extension (Shackleford et al., 1993Go). The MAPK pathway is known as a possible positive regulator of neurite extension in PC12 cells (Noda et al., 1985Go), but collaborational regulation of these two signaling pathways in neurite extension has not been reported. Crosstalk between the TAK1-NLK pathway, which is a member of the MAPK family, and the ß-catenin signal was reported (Maneghini et al., 1999Go). So we hypothesized that crosstalk between Wnt and the MAPK pathway may occur in the regulation of neurite extension, and therefore examined modulation of the MAPK signal by Wnt and vice versa. However, we found that the activation of the ß-catenin-Lef-1 signal pathway did not affect MAPK signal pathway activation in PC12 cells. In addition, MAPK did not affect ß-catenin-induced Lef-1 transcriptional activation (data not shown). Thus the effects of ß-catenin on neurite extension occurred not through modulation of the MAPK pathway but through transcriptional activation of Lef-1, which causes transcription of its target. This notion raised the question as to whether MAPK plays a role in neurite extension in the neural retina. MAPK is expressed throughout the mouse retina at least at P0 and P5 (Rhee and Yang, 2003Go) and, in fact, we clearly observed phosphorylation of MAPK during retinal development in the explant cultures. However, we could not obtain evidence of the involvement of the MAPK pathway in neurite extension in the explant cultures. An earlier study suggested a role for the MAPK signaling pathway in the regulation of process extension in the chicken retina (Dimitropoulou and Bixby, 2000Go). In that case, neurite extension was examined for neurons that were on a basal adhesive substrate. Netrin-1 is neurite extension factor and reported to mediate axon guidance of retinal ganglion cells (Deiner et al., 1997Go) and, in this case, neurite extension was examined by in vitro collagen gel assay. Netrin-1 activates the MAPK signaling pathway and involvement of MAPK activation in commissural neurons was reported (Forcet et al., 2002Go). This difference in observations regarding the role of MAPK for neurite extension may be partly explained by the difference in experimental systems or species. However, Wnt is also recognized as an axon guidance molecule (Schnorrer and Dickson, 2004Go). Thus, we propose that the different roles of molecules as guidance cues for growing axons or as signals for growing neurites should be carefully considered for further examination.

Activation of ß-catenin in retinal cells resulted in defects in the proper sub-retinal localization of retinal cells. Interestingly, Lef-1-dependent transcription did not affect the localization of retinal cells, suggesting that this ß-catenin-induced abnormality of retinal localization was not dependent on Lef-1 transcriptional activity. Since caß-catenin is known to act as a component of the cadherin/catenin system (Ben-Ze'ev and Geiger, 1998Go), there is a possibility that perturbation of cadherin-dependent rearrangement of cell membrane and actin cytoskeleton resulted in the abnormal and rounded morphology of the caß-catenin-expressing cells. In fact, mutation of N-cadherin resulted in severe impairment of proper formation of the retina in zebrafish (Malicki et al., 2003Go; Masai et al., 2003Go). However, our preliminary experiments of retrovirus-mediated expression of dominant negative N-cadherin did not show apparent morphological and sub-retinal localization disorders (data not shown).

Dvl (Dishevelled)-Rho-JNK signaling pathway is reported to be involved in planar cell polarity (PCP) (Weston and Davis, 2002Go). Although we could not find previous literature suggesting direct crosstalk between the JNK pathway and ß-catenin, we can hypothesize the effects of caß-catenin on the JNK signaling pathway through modification of localization of Dvl, which is upstream of Rho/JNK in the PCP signaling pathway. Similarly, modification of stoichiometry of binding partners such as APC, GSK-3ß and Axin, which are known to regulate cell morphogenesis, may be candidates for involvement in the mechanism of ß-catenin-dependent abnormal cell localization. However, the role of LEF-dependent transcription cannot be excluded from the possible mechanisms. Smad is reported to bind to LEF, and LEF might bind to other proteins with various target sequences. There is a possibility that our caLEF mimicked only some of the activities of the wild-type LEF because of the lack of binding domains with unknown partners.

In view of our findings, we propose a model in which the Wnt signal plays a role in promoting proliferation of progenitor cells in the marginal zone in the early developmental stage of the retina, and then, after differentiation takes place, functions to suppress neurite extension in order to execute the developmentally staged program properly. For the final step of differentiation, cells would have to be released from suppression of neurite extension. As a candidate for the downregulation of Wnt expression and its signaling pathway, we propose SFRPs, which are a family of extracellular antagonists of Wnt (Liu et al., 2003Go) expressed in the neural retina. Such molecules may switch off the Wnt signaling pathway to promote the final differentiation of neural retina cells.


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 Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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