Expanded progenitor populations, vitreo-retinal abnormalities, and Müller glial reactivity in the zebrafish leprechaun/patched2 retina
© Bibliowicz and Gross; licensee BioMed Central Ltd. 2009
Received: 1 May 2009
Accepted: 19 October 2009
Published: 19 October 2009
The roles of the Hedgehog (Hh) pathway in controlling vertebrate retinal development have been studied extensively; however, species- and context-dependent findings have provided differing conclusions. Hh signaling has been shown to control both population size and cell cycle kinetics of proliferating retinal progenitors, and to modulate differentiation within the retina by regulating the timing of cell cycle exit. While cell cycle exit has in turn been shown to control cell fate decisions within the retina, a direct role for the Hh pathway in retinal cell fate decisions has yet to be established in vivo.
To gain further insight into Hh pathway function in the retina, we have analyzed retinal development in leprechaun/patched2 mutant zebrafish. While lep/ptc2 mutants possessed more cells in their retinas, all cell types, except for Müller glia, were present at identical ratios as those observed in wild-type siblings. lep/ptc2 mutants possessed a localized upregulation of GFAP, a marker for 'reactive' glia, as well as morphological abnormalities at the vitreo-retinal interface, where Müller glial endfeet terminate. In addition, analysis of the over-proliferation phenotype at the ciliary marginal zone (CMZ) revealed that the number of proliferating progenitors, but not the rate of proliferation, was increased in lep/ptc2 mutants.
Our results indicate that Patched2-dependent Hh signaling does not likely play an integral role in neuronal cell fate decisions in the zebrafish retina. ptc2 deficiency in zebrafish results in defects at the vitreo-retinal interface and Müller glial reactivity. These phenotypes are similar to the ocular abnormalities observed in human patients suffering from Basal Cell Naevus Syndrome (BCNS), a disorder that has been linked to mutations in the human PTCH gene (the orthologue of the zebrafish ptc2), and point to the utility of the lep/ptc2 mutant line as a model for the study of BCNS-related ocular pathologies. Our findings regarding CMZ progenitor proliferation suggest that, in the zebrafish retina, Hh pathway activity may not affect cell cycle kinetics; rather, it likely regulates the size of the retinal progenitor pool in the CMZ.
During retinal development, proliferation and differentiation must be tightly coordinated in order to produce a tissue of the proper size and containing the correct cell types . The Hh pathway has been shown to play a critical role in controlling these two seemingly opposite processes . Early in retinal development, the optic vesicle is composed of a population of proliferating neuroepithelial cells that will ultimately give rise to the mature retina . Differentiation of the retinal neuroepithelium occurs in a succession of temporally overlapping waves . In the zebrafish, the first cells to exit the cell cycle become retinal ganglion cells (RGCs), which differentiate in a Sonic Hh (Shh)-dependent wave . A second Hh-dependent wave of differentiation in the inner nuclear layer (INL) occurs almost simultaneously with the first wave, and is responsible for the differentiation of the multiple cell types of the INL (horizontal, amacrine, and bipolar cells, and Müller glia) and the rod and cone photoreceptors of the outer nuclear layer (ONL) . In addition, extra-retinal Hh signaling originating in the retinal pigment epithelium (RPE) has been suggested to direct photoreceptor differentiation . While the role of the Hh pathway in cell cycle exit and differentiation of retinal progenitors is well described, comparatively less is known about its possible influence on cell fate decisions. In Xenopus, over-expression of p27Xic1, which promotes cell cycle exit, results in increased numbers of early-born cell types (RGCs), while the over-expression of cyclin E1, which inhibits cell cycle exit, biases cell fate towards late-born cell types (e.g. Müller glia) . Similarly, Shh has been shown to promote early cell cycle exit in the Xenopus retina ; however, a direct role of the Hh pathway in dictating retinal cell fate has yet to be established in vivo.
While the cells of the central retina of the zebrafish exit the cell cycle by 60 hours post fertilization (hpf) , a population of retinal progenitors at the CMZ is maintained and continues to proliferate throughout the animal's lifetime [11, 12]. The spatial pattern of cells within the CMZ, with retinal stem cells at the most peripheral region followed by proliferative retinal progenitors and finally differentiating progenitors more centrally, is thought to recapitulate the temporal sequence of early retinal development . Some of the factors that control early retinal development, such as notch, rx1, pax6a, and vsx2/chx10, are expressed in the zebrafish CMZ . In Xenopus, expression of gli2, gli3, and smoothened, is found at the retinal margin, suggesting a role for the Hh pathway in the stem cell/progenitor population in the CMZ . Shh over-expression studies in chick support a role for the Hh pathway as a mitogen in the CMZ , consistent with its described mitogenic effects on retinal progenitors in early zebrafish and mouse retinal development [16, 17].
Invaluable knowledge regarding Hh function in the developing retina has been gained from the analysis of Hh pathway mutants in zebrafish. For example, the zebrafish mutants syu (shh) and smu (smoothened) have helped elucidate the complex mechanisms of Hh-dependent neural differentiation and proliferation [7, 16]. However, retinal differentiation is severely defective or altogether absent in these mutants due to defective Hh signaling, making it difficult to arrive at definitive conclusions about the possible role of the Hh pathway in cell fate decisions. To address this issue, we analyzed retinal development in the zebrafish lep/ptc2 mutant, in which the Hh pathway is 'over-active'. lep/ptc2 mutants possess a non-sense mutation in the exon encoding the sixth trans-membrane domain of Patched2 , which is predicted to abolish its function [18, 19]. Loss of Patched function results in an 'over-active' Hh pathway due de-repression of Smoothened . Normally, in the absence of Hh ligand, the Patched protein inhibits the activity of Smoothened. Binding of the Hh ligand to its Patched receptor relieves this inhibition, ultimately resulting in increased transcription of Hh target genes [21, 22]. A non-functional Patched would therefore result in loss of inhibition on, and constitutive activity of, Smoothened in ptc2 deficient cells. lep/ptc2 mutants possess increased proliferation in multiple tissues including the retinal CMZ . Utilizing this mutant as a tool, we sought to gain further insight into Hh function with respect to retinal cell fate decisions and to determine how Hh activity influences progenitor proliferation at the CMZ. Our results revealed no significant change in neuronal composition within the retina, but we did note a reduction in differentiated Müller glia that was accompanied by localized Müller glial reactivity and abnormalities at the vitreo-retinal interface. In addition, the results of cell cycle analyses suggest that the over-proliferation at the CMZ likely results from an expansion of the progenitor cell population, and not from direct effects of the mutation on cell cycle kinetics within progenitor cells.
lep/ptc2 mutants possess enlarged retinas and overgrown irises, while lens and RPE morphology are largely normal
Neuronal composition of the lep/ptc2 retina is normal, while Müller glia are under-represented
Hh signaling has been shown to be required for the differentiation of multiple cell types in the vertebrate retina [6, 7, 29] and is thought to promote cell fate decisions by modulating the timing of cell cycle exit . Shh gain-of-function experiments in Xenopus link a faster cell cycle with early cell cycle exit , which in turn results in an over-production of early-born retinal cell types at the expense of late-born cell types . We therefore reasoned that in lep/ptc2 retinas, where the Hh pathway is over-active, early-born cell types would be over-represented while late-born cell types would be reduced in number. Cell counts from histological sections of lep/ptc2 mutants revealed a statistically significant increase in cell number in all three retinal nuclear layers (GCL, INL, and ONL), as well as in total retinal cells, when compared to those from phenotypically wild-type siblings (Figure 2E). However, no significant change in the proportion of each layer was observed when the number of cells per layer was calculated as a percentage of total retinal cells (Figure 2F). This suggests that all three retinal cell layers are proportionally increased in lep/ptc2 retinas.
lep/ptc2 mutants possess abnormalities at the vitreo-retinal boundary and localized Müller glial reactivity
Immunohistochemical analysis of GFAP revealed that its levels in Müller glia were indeed higher in lep/ptc2 mutant retinas as compared to their phenotypically wild-type siblings (Figure 5C, C', D, D'). Higher levels of GFAP were often localized to Müller glial endfeet in the central retina, adjacent to the optic nerve. However, BrdU incorporation assays in lep/ptc2 mutants at 5dpf did not reveal ectopic proliferation in the central retina (see Additional file 1), suggesting that the upregulation of GFAP is not coupled with Müller glial proliferation in this context. In addition, the localized nature of Müller glial reactivity in not due to abnormal cell death in the lep/ptc2 retina and/or optic nerve since no apoptotic nuclei were detected by TUNEL assays (see Additional file 1).
patched2 (ptc2) is expressed in the progenitor/stem cell populations of the CMZ
The number of proliferating progenitors, but not the rate of progenitor proliferation, is increased in the lep/ptc2 CMZ
Retinal patterning is normal in lep/ptc2 mutants
Characterization of lep/ptc2 retinas with respect to cell type composition revealed that over-activity of the Hh pathway did not affect retinal patterning. Cell count analysis of all major retinal neuronal cell types revealed no change in the proportion of each cell type. This finding was surprising in light of the well established role of the Hh pathway in patterning and cell fate decisions throughout the CNS in a number of model systems [20, 38, 39], as well as studies in Xenopus which show that the over-expression of Shh results in early cell cycle exit , and that can, in turn, influence cell fate decisions . While mouse Ptc1+/- (the orthologue of the zebrafish ptc2 ) mutants were found to possess no defects in retinal cell fate specification, Ptc1-/- mice could not be examined since they die in utero . Our results indicate that Ptc2-dependent Hh signaling does not likely play an integral role in neuronal cell fate decisions in the zebrafish retina. These findings raise the possibility that the second zebrafish Patched protein (Patched1) might compensate for the lack of a functional Patched2. While patched1 (blowout) mutant retinas contain all major retinal cell types, the lack of a retinal phenotype might be due to the hypomorphic nature of the mutation [40, 41]. However, this seems unlikely since patched1 transcripts are not expressed at detectable levels in retinal tissue throughout development (data not shown and ). The reduced number of differentiated Müller glia, a late born cell type , could feasibly be due to a bias towards early born cell types in lep/ptc2 mutants; however, this potential bias does not affect neuronal composition of the retina at 5dpf. It is possible that a transient bias towards early neuronal cell types could occur early in retinal development, but any differences could masked by neurons added from the CMZ later in development. Conversely, any bias could also be present in neuronal populations that arise at the CMZ, resulting in an unnoticeable change in neuronal composition of the 5dpf lep/ptc2 retina. With this in mind, it will be interesting to examine the proportion of retinal cell types in juvenile lep/ptc2 mutants, as some homozygous fish reach two months of age . Finally, the Hh pathway could play a role in neuronal cell fate decisions in the retina through Hh ligand interactions with other Hh receptors. The Ihog Hh receptors have recently been suggested to compete with Patched for Hh binding  and act at levels at, or upstream of, Patched . Indeed, the vertebrate Ihog homologue Cdo is expressed in the developing mouse retina . In the future, it would be interesting to test whether certain aspects of retinal development, such as cell fate decisions, might be Ihog-dependent.
lep/ptc2 mutants as a possible model for the study of BCNS-related ocular pathologies
In BCNS patients, abnormalities at the interface between the retina and the vitreous, known as epiretinal membranes (ERMs), are associated with disruptions of the ILM and are thought to result from the overproliferation and ectopic presence of multiple cell types, including glia . BCNS ocular pathologies have been linked to mutations in the human PTCH gene [34, 46, 47], and have been shown to be associated with reactive Müller glia . Müller glial reactivity is often marked by upregulation of GFAP, and lep/ptc2 mutants possessed regions of elevated GFAP levels. Elevated GFAP levels were consistently localized in the inner retina, where Müller glial endfeet terminate and contribute to the ILM. Staining with GS, which labels the Müller glial endfeet, revealed disruptions in ILM integrity in ~40% of mutant retinas assayed. In these cases, Müller glial endfeet did not terminate properly in the ILM, and the ILM was discontinuous. In some human patients, ERMs are found adjacent to retinal arteries ; interestingly, we consistently detected the presence of reactive glial endfeet in localized retinal regions adjacent to the optic nerve and embryonic retinal vasculature. Importantly, no increases in cell death were detected in the retina or optic nerve of lep/ptc2 mutants, or in Müller glia themselves (see Additional file 1), ruling out reactivity due to apoptosis. Finally, in other contexts, Müller glia reactivity is often coupled with ectopic proliferation of these cells during 'reactive gliosis' . BrdU analysis did not detect ectopic proliferation in the central retinas of lep/ptc2 mutants (Figure 7 and Additional file 1), indicating that while a subset of these cells was reactive, they did not undergo a proliferative response. While reactive Müller glia have been shown to be associated with ERMs, it is unclear whether reactive Müller glia are the cause of these pathologies or, conversely, whether Müller glia become reactive in response to ERM formation. Further studies will be required to answer this question and to shed light on the cellular and molecular causes of ERM formation.
The retinal progenitor population of the CMZ is expanded in lep/ptc2 mutants
Exact roles for Hh pathway activity in proliferating retinal progenitor cells remain unclear. Over-expression studies in Xenopus suggest a direct role for the Hh pathway in regulating proliferation by influencing the length of the cell cycle in CMZ progenitors . In Ptc1+/- mice, more retinal progenitors are allocated to the CMZ throughout development and into adulthood . BrdU pulse-chase analysis of the progenitor population at the lep/ptc2 CMZ suggests that Patched-dependent Hh signaling controls the number of retinal progenitors in the zebrafish CMZ, and does not directly affect the length of the cell cycle in these cells. Importantly, cell counts at an earlier time-point, before the formation of the CMZ, revealed no increase in cell number in lep/ptc2 retinas as compared to their wild-type siblings (Figure 2E), suggesting that the increase in proliferating progenitors at the CMZ is not simply due to an earlier proliferative event. Shh has been shown to control stem cell maintenance in multiple organs, including the adult brain (reviewed in ). Indeed, the Hh pathway genes smo, gli2, and gli3 are expressed in the putative stem cell region of the Xenopus CMZ . In light of our findings, it is possible that the increase in retinal progenitors in the lep/ptc2 CMZ is an indirect result of mis-regulation of the stem cell population rather than a direct effect on progenitor proliferation.
The possible role for the Hh pathway in retinal cell fate decisions has yet to be established in vivo. Our results indicate that Patched2-dependent Hh signaling is not likely to play an integral role in dictating neuronal cell fate decisions in the zebrafish retina. In addition, ocular phenotypes in lep/ptc2 mutants that are similar to those found in human BCNS patients point to the utility of the lep/ptc2 mutant line as a model for the study of BCNS ocular pathologies. With regards to progenitor proliferation, our data support a role for Patched2-dependent Hh signaling in the control of the size of progenitor populations at the retinal CMZ, and not in regulating their rate of proliferation, during zebrafish eye development.
Zebrafish maintenance and strains
Zebrafish (Danio rerio) were maintained at 28.5°C on a 14 h light/10 h dark cycle. Embryos were obtained from the natural spawning of heterozygous carriers setup in pairwise crosses. Embryos were collected and raised at 28.5°C  and were staged according to . ptc2 tj222 outcrosses were obtained from the Zebrafish International Resource Center and were propagated by repeated outcrosses to AB fish. All animals were treated in accordance with provisions established at the University of Texas at Austin governing animal use and care.
Histology was performed as described in . Briefly, mutant and wild-type sibling embryos were collected and fixed overnight at 4°C in a solution of 1% (w/v) paraformaldehyde (PFA), 2.5% glytaraldehyde and 3% sucrose in phosphate in a 2% OsO4 solution, washed 3 × 5 min in PBS at room temperature and further dehydrated 2 × 10 min in propylene oxide and infiltrated 1-2 h in a 50% propylene oxide/50% Epon/Araldite mixture (Polysciences, Inc.). Embryos were then incubated overnight at RT in 100% Epon/Araldite resin with caps open to allow for propylene oxide evaporation and resin infiltration, embedded and baked at 60°C for 2-3 days. Sections 1-1.25 μm were cut, mounted on glass slides and stained in a 1% methylene blue/1% borax solution. Sections were mounted in DPX (Electron Microscopy Sciences) and photographed on a Leica DMRB microscope mounted with a DFC320 digital camera. For 5dpf cell counts, the number nuclei in each retinal layer were counted in ten eye sections obtained from different embryos. Horizontal cells were identified by location and morphology. Averages were analyzed and compared using a Student's t-test.
Immunohistochemistry was performed as described in . Briefly, embryos were collected and fixed overnight at 4°C in a 4% PFA solution in PBS. Embryos were then washed 3 × 10 min in PBS and incubated in a 25% sucrose/PBS solution for 1 h followed by 35% sucrose/PBS for 1 h. Embryos were then mounted in Tissue Freezing Medium (Triangle Biomedical Sciences, Inc.) and immediately transferred to -80°C freezer for storage. Frozen blocks were sectioned at 12 μm, mounted on gelatin coated slides, and let dry for 2 h. Slides were then re-hydrated in PBTD [0.1% Tween, 1% DMSO in 1× PBS] and then blocked using 5%NGS/PBTD for 2 h. Slides were incubated in primary antibody diluted in block in a humid chamber over-night at 4°C. When necessary, nuclear staining was obtained by applying Sytox-Green (Molecular Probes) diluted 1:10,000 in block immediately after removal of primary antibody. Slides were washed 3 × 10 min with PBTD and then applied with secondary antibody in block for 1 h. After 3 × 10 min washes in PBTD, slides were mounted with Vectashield (Vector Laboratories, Inc.) and coverslipped. Samples were stored at 4°C for up to one week and imaged on a Zeiss LSM5 Pascal laser scanning confocal microscope. The following primary antibodies were used: bipolar cells (PKC, 1:200, Santa Cruz Biotechnology), Müller glia (GFAP-zrf1, 1:100, ZIRC and GS, 1:500, Millipore), rods (zpr3, 1:100, ZIRC), green/red cones (zpr1, 1:100, ZIRC), PH3 (1:200, Upstate). Blue opsin (1:500) and UV opsin (1:1,000) antibodies were provided by David Hyde (Univerity of Notre Dame). Amacrine cell antibody (5E11, 1:100) was the gift of Jim Fadool. For cell counts, positively stained cells and nuclei were counted in four eye sections obtained from different embryos. For total cell counts at 33hpf, Sytox-Green staining was used. Averages were analyzed and compared using a Student's t-test.
BrdU incorporation assays
Embryos were dechorionated and incubated in fish water with 10 μM 5-Bromo-2-deoxyuridine (BrdU, Sigma) and 15% DMSO at 4°C for mentioned time periods and either immediately sacrificed or washed three times in fish water and grown at 28.5°C prior to sacrifice after . Embryos were processed for immunohistochemistry as in . Mouse anti-BrdU was used at a 1:50 dilution and Cy3 anti-mouse secondary at a 1:200 dilution. Nuclei were counterstained with Sytox:Green (1:10,000, Molecular Probes). For cell counts, Brdu-positive cells and nuclei were counted in four eye sections obtained from different embryos. Averages were analyzed and compared using a Student's t-test.
Hybridizations were performed as described in  using digoxigenin labeled antisense RNA probes. ptc2 probe synthesis construct was provided by Brian Perkins (Texas A&M University).
This work was funded by NIH RO1-EY18005 to J.M.G. We are grateful to David Hyde, Jim Fadool and Brian Perkins for providing reagents, to Ian Mackaye for technical assistance and to members of the Gross lab for helpful suggestions on this work, and comments on this manuscript. cDNA constructs and antisera were obtained from the Zebrafish International Resource Center, supported by NIH-NCRR grant P40 RR012546.
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