Borday C et al. (2012), Antagonistic cross-regulation between Wnt and H...

XB-ART-45801

Development 2012 Oct 01;13919:3499-509. doi: 10.1242/dev.079582.

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Antagonistic cross-regulation between Wnt and Hedgehog signalling pathways controls post-embryonic retinal proliferation.

Borday C , Cabochette P , Parain K , Mazurier N , Janssens S , Tran HT , Sekkali B , Bronchain O , Vleminckx K , Locker M , Perron M .

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Continuous neurogenesis in the adult nervous system requires a delicate balance between proliferation and differentiation. Although Wnt/β-catenin and Hedgehog signalling pathways are thought to share a mitogenic function in adult neural stem/progenitor cells, it remains unclear how they interact in this process. Adult amphibians produce retinal neurons from a pool of neural stem cells localised in the ciliary marginal zone (CMZ). Surprisingly, we found that perturbations of the Wnt and Hedgehog pathways result in opposite proliferative outcomes of neural stem/progenitor cells in the CMZ. Additionally, our study revealed that Wnt and Hedgehog morphogens are produced in mutually exclusive territories of the post-embryonic retina. Using genetic and pharmacological tools, we found that the Wnt and Hedgehog pathways exhibit reciprocal inhibition. Our data suggest that Sfrp-1 and Gli3 contribute to this negative cross-regulation. Altogether, our results reveal an unexpected antagonistic interplay of Wnt and Hedgehog signals that may tightly regulate the extent of neural stem/progenitor cell proliferation in the Xenopus retina.

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Species referenced: Xenopus laevis
Genes referenced: ccnd1 dhh gli1 gli3 ihh ptch1 rlbp1 rpe sfrp1 shh tcf7l1 wnt16 wnt2b wnt3a wnt7b wnt8b wnt9a wnt9b
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	Fig. 1. Interfering with Hedgehog and Wnt pathways leads to opposite effects on precursor cell destiny during retinogenesis. (A,B)Stage 41 Xenopus retinal sections following in vivo lipofection. (A)The respective morphologies of GFPpositive Mler versus neuroepithelial cells. Arrowheads indicate the cell represented in the adjacent drawing. (B)Anti-CRALBP immunostaining (Mler cell marker; arrowheads) following Ihh-CD2 overexpression. (C-I)Percentage of Mler or neuroepithelial cells observed in stage 41 retinas following in vivo lipofection with the indicated constructs. Cyclopamine treatment was performed from stage 18 onwards on embryos lipofected with GFP. The total number of counted cells per condition is indicated in each bar. P<0.05, P<0.01, **P<0.001 (Student t-test). Mean s.e.m. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars: 10m.
	Fig. 2. Hedgehog and Wnt pathways have opposite impacts on cell cycle exit. Birthdating experiments (from stage 32 to stage 41) following in vivo lipofection with the indicated constructs. Cyclopamine treatment was performed from stage 18 onwards on embryos lipofected with GFP. Transfected cells that have exited the cell cycle before EdU exposure (stage 32) are EdUat stage 41, whereas cells that have exited the cell cycle at any time during the EdU incorporation period are EdU+. (A)Typical stage 41 retinal sections stained for GFP and EdU. The arrow and arrowhead point to a GFP+ EdU+ and to a GFP+ EdUcell, respectively. (B)Percentage of EdU+ nuclei among transfected cells. The total number of analysed retinas per condition is indicated in each bar. P<0.05, *P<0.01 (Student t-test). Mean s.e.m. L, lens. Scale bar: 40m.
	Fig. 3. BIO, IWR-1, purmorphamine and cyclopamine act as efficient Wnt and Hedgehog pathway activators or inhibitors in the Xenopus tadpole retina. (A)In situ hybridisation or immunofluorescence analyses of eGFP expression on stage 40 retinal sections from Wnt-responsive transgenic animals. Enlargements of the ciliary marginal zone (CMZ) region (delineated with dotted line) show that GFP expression is strongest in the peripheral half of the CMZ, including the stem cell zone. Note that Wnt activity is also detected in the peripheral retinal pigment epithelium (RPE). (B,C)In situ hybridisation against eGFP on stage 40 transgenic tadpoles 24 hours following BIO or IWR-1 treatment. (B)Representative images of staining in whole mount (lateral view of the head) and on retinal sections (dorsal side up). (C)Quantification of eGFP staining area per CMZ. (D-G)In situ hybridisation analyses of Ptc1 (D) and Gli1 (E) expression on stage 40 retinal sections 24 hours following purmorphamine or cyclopamine treatment. Note that Ptc1 and Gli1 are detected in the CMZ (arrows) and in the periocular mesenchyme (arrowheads). (F,G)Quantification of Ptc1 and Gli1 staining area per CMZ. The total number of analysed sections per condition is indicated in each bar. ***P<0.001 (Student t-test). Mean s.e.m. L, lens. Scale bar: 40m, except 400m in whole mounts.
	Fig. 4. Interfering with Hedgehog and Wnt pathways leads to opposite proliferative outcomes in the post-embryonic retina. BrdU incorporation assays (3-hour pulse) at stage 41, 24 hours following treatment with the indicated drugs. (A)Control retinal section illustrating how the CMZ, in which BrdU+ cells are counted, is delineated (dotted lines) using Hoechst staining. Images on the right show higher magnifications of the CMZ. (B)Quantification of BrdU+ cells in the CMZ. The total number of analysed retinas per condition is indicated in each bar. P<0.05, P<0.01, **P<0.001 (Student ttest). Mean s.e.m. L, lens. Scale bar: 40m.
	Fig. 5. Wnt and Hedgehog morphogens are expressed in mutually exclusive territories within the post-embryonic retina. (A,B)Retinal sections of stage 39/40 Xenopus tadpoles following in situ hybridisation with the indicated probes. (A)Shh, Ihh and Dhh are detected in the central RPE (arrows). Additionally, Shh labels the ganglion cell layer (arrowhead). (B)In contrast to Hedgehog genes, those encoding Wnt ligands are all expressed in the peripheral retina. Shown beneath each retinal section is a higher magnification of the CMZ region (boxed). Wnt2b, Wnt8b, Wnt9a, Wnt9b and Wnt16 are detected in the peripheral RPE surrounding the CMZ (Wnt8b and Wnt9a exhibit a more intense staining dorsally than ventrally). Wnt8b is additionally expressed in the most peripheral stem cell-containing region of the CMZ, together with Wnt2b, which also labels the peripheral part of the lens. Wnt3a transcripts are present in the presumptive cornea and Wnt7b is expressed in the lens (not shown). (C)Summary of Hedgehog (Hh) and Wnt ligand expression in the retina, and of domains exhibiting Wnt (in the CMZ) and Hedgehog (in the CMZ and periocular mesenchyme) activity. L, lens. Scale bar: 40m.
	Fig. 6. Impact of Wnt pathway perturbations on Hedgehog signalling activity. (A-F)In situ hybridisation analyses of CyclinD1, Gli1 or Ptc1 expression on stage 38 retinal sections 24 hours following treatment with BIO (A,B), induction of TCF3-VP16GR activity in injected embryos (C,D), or treatment with IWR-1 (E,F). Arrows indicate CMZ labelling. (B,D,F) Quantifications of staining area for each transcript. The total number of analysed sections per condition is indicated in each bar. (G,H)qPCR analysis of retinal Ptc1 expression following Wnt signalling activation or inhibition as indicated. P<0.01, *P<0.001 (Student t-test). Mean s.e.m. L, lens. Scale bars: 40m.
	Fig. 7. Hedgehog signalling inhibits Wnt activity. (A-D)Immunofluorescence (A,B) or in situ hybridisation (C,D) analyses of eGFP expression in stage 40 retinas from Wnt-responsive transgenic animals following a 24-hour treatment with either cyclopamine or purmorphamine. (B,D)Quantification of eGFP staining area within the CMZ (delineated by dotted lines). Hoechst staining (for immunolabelling) and Nomarski (for in situ hybridisation) were used to delineate the CMZ in order to exclude RPE eGFP labelling from the quantification. The total number of analysed sections per condition is indicated in each bar. P<0.05, **P<0.001 (Student t-test). Mean s.e.m. L, lens. Scale bars: 40m.
	Fig. 8. The Hedgehog pathway negatively regulates Wnt activity through the transcriptional activation of Sfrp-1. (A)In situ hybridisation analysis of Sfrp-1 expression on stage 41 retinal sections following a 24-hour treatment with purmorphamine (purm.) or cyclopamine (cyclo.). (B)qPCR analysis of Ptc1 (as a control for purmorphamine activity) and Sfrp-1 expression in the head of stage 38 sibling tadpoles following 8 hours of purmorphamine exposure. (C-J)In situ hybridisation analysis of eGFP expression in stage 40 retinas from Wnt-responsive transgenic animals exposed to SRFP-1 protein for 24 hours (C,D,G) or injected with Sfrp-1 Mo (E,F,H). (G-J)Tadpoles were additionally treated for 24 hours with cyclopamine (G,I) or purmorphamine (H,J). (D,F,I,J) Quantification of eGFP staining area per CMZ in each condition. The total number of analysed sections per condition is indicated in each bar. P<0.05, **P<0.001 (Student t-test). Mean s.e.m. L, lens. Scale bar: 40m.
	Fig. 9. The Wnt pathway negatively regulates Hedgehog activity through the transcriptional activation of Gli3. (A,B)In situ hybridisation analysis of Gli3 expression (A) on stage 38 retinal sections 24 hours following BIO or IWR-1 treatment. (B)Quantification of Gli3 staining area per CMZ. The total number of analysed sections per condition is indicated in each bar. (C)qPCR analysis of retinal Gli3 expression following 24-hour BIO or IWR-1 treatment. (D)qPCR analysis of retinal Ptc1 expression following 24-hour BIO treatment on Gli3 Mo-injected tadpoles. P<0.05, **P<0.001 (Student t-test). Mean s.e.m. L, lens. Scale bar: 40m.
	Fig. 10. Model of Wnt and Hedgehog interplay in the postembryonic retina. (A)Schematic highlighting the mutually exclusive expression domains of Wnt and Hedgehog ligands along the central to peripheral axis of the post-embryonic retina. (B-D)Illustration of the activities of Wnt and Hedgehog pathways in the retinal neurogenic niche and of the proposed crosstalk underlying their mutual negative regulation. Shown are the hypothetical physiological situation (B), and the synopsis of our Hedgehog (C) or Wnt (D) activation experiments. Their opposed impacts on stem/progenitor cell proliferation are represented by changes in the CMZ proliferative cell population (grey) in the drawings beneath.

	Fig. 1. Interfering with Hedgehog and Wnt pathways leads to opposite effects on precursor cell destiny during retinogenesis. (A,B)Stage 41 Xenopus retinal sections following in vivo lipofection. (A)The respective morphologies of GFPpositive Mler versus neuroepithelial cells. Arrowheads indicate the cell represented in the adjacent drawing. (B)Anti-CRALBP immunostaining (Mler cell marker; arrowheads) following Ihh-CD2 overexpression. (C-I)Percentage of Mler or neuroepithelial cells observed in stage 41 retinas following in vivo lipofection with the indicated constructs. Cyclopamine treatment was performed from stage 18 onwards on embryos lipofected with GFP. The total number of counted cells per condition is indicated in each bar. P<0.05, P<0.01, **P<0.001 (Student t-test). Mean s.e.m. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bars: 10m.
	Fig. 2. Hedgehog and Wnt pathways have opposite impacts on cell cycle exit. Birthdating experiments (from stage 32 to stage 41) following in vivo lipofection with the indicated constructs. Cyclopamine treatment was performed from stage 18 onwards on embryos lipofected with GFP. Transfected cells that have exited the cell cycle before EdU exposure (stage 32) are EdUat stage 41, whereas cells that have exited the cell cycle at any time during the EdU incorporation period are EdU+. (A)Typical stage 41 retinal sections stained for GFP and EdU. The arrow and arrowhead point to a GFP+ EdU+ and to a GFP+ EdUcell, respectively. (B)Percentage of EdU+ nuclei among transfected cells. The total number of analysed retinas per condition is indicated in each bar. P<0.05, *P<0.01 (Student t-test). Mean s.e.m. L, lens. Scale bar: 40m.
	Fig. 3. BIO, IWR-1, purmorphamine and cyclopamine act as efficient Wnt and Hedgehog pathway activators or inhibitors in the Xenopus tadpole retina. (A)In situ hybridisation or immunofluorescence analyses of eGFP expression on stage 40 retinal sections from Wnt-responsive transgenic animals. Enlargements of the ciliary marginal zone (CMZ) region (delineated with dotted line) show that GFP expression is strongest in the peripheral half of the CMZ, including the stem cell zone. Note that Wnt activity is also detected in the peripheral retinal pigment epithelium (RPE). (B,C)In situ hybridisation against eGFP on stage 40 transgenic tadpoles 24 hours following BIO or IWR-1 treatment. (B)Representative images of staining in whole mount (lateral view of the head) and on retinal sections (dorsal side up). (C)Quantification of eGFP staining area per CMZ. (D-G)In situ hybridisation analyses of Ptc1 (D) and Gli1 (E) expression on stage 40 retinal sections 24 hours following purmorphamine or cyclopamine treatment. Note that Ptc1 and Gli1 are detected in the CMZ (arrows) and in the periocular mesenchyme (arrowheads). (F,G)Quantification of Ptc1 and Gli1 staining area per CMZ. The total number of analysed sections per condition is indicated in each bar. ***P<0.001 (Student t-test). Mean s.e.m. L, lens. Scale bar: 40m, except 400m in whole mounts.
	Fig. 4. Interfering with Hedgehog and Wnt pathways leads to opposite proliferative outcomes in the post-embryonic retina. BrdU incorporation assays (3-hour pulse) at stage 41, 24 hours following treatment with the indicated drugs. (A)Control retinal section illustrating how the CMZ, in which BrdU+ cells are counted, is delineated (dotted lines) using Hoechst staining. Images on the right show higher magnifications of the CMZ. (B)Quantification of BrdU+ cells in the CMZ. The total number of analysed retinas per condition is indicated in each bar. P<0.05, P<0.01, **P<0.001 (Student ttest). Mean s.e.m. L, lens. Scale bar: 40m.
	Fig. 5. Wnt and Hedgehog morphogens are expressed in mutually exclusive territories within the post-embryonic retina. (A,B)Retinal sections of stage 39/40 Xenopus tadpoles following in situ hybridisation with the indicated probes. (A)Shh, Ihh and Dhh are detected in the central RPE (arrows). Additionally, Shh labels the ganglion cell layer (arrowhead). (B)In contrast to Hedgehog genes, those encoding Wnt ligands are all expressed in the peripheral retina. Shown beneath each retinal section is a higher magnification of the CMZ region (boxed). Wnt2b, Wnt8b, Wnt9a, Wnt9b and Wnt16 are detected in the peripheral RPE surrounding the CMZ (Wnt8b and Wnt9a exhibit a more intense staining dorsally than ventrally). Wnt8b is additionally expressed in the most peripheral stem cell-containing region of the CMZ, together with Wnt2b, which also labels the peripheral part of the lens. Wnt3a transcripts are present in the presumptive cornea and Wnt7b is expressed in the lens (not shown). (C)Summary of Hedgehog (Hh) and Wnt ligand expression in the retina, and of domains exhibiting Wnt (in the CMZ) and Hedgehog (in the CMZ and periocular mesenchyme) activity. L, lens. Scale bar: 40m.
	Fig. 6. Impact of Wnt pathway perturbations on Hedgehog signalling activity. (A-F)In situ hybridisation analyses of CyclinD1, Gli1 or Ptc1 expression on stage 38 retinal sections 24 hours following treatment with BIO (A,B), induction of TCF3-VP16GR activity in injected embryos (C,D), or treatment with IWR-1 (E,F). Arrows indicate CMZ labelling. (B,D,F) Quantifications of staining area for each transcript. The total number of analysed sections per condition is indicated in each bar. (G,H)qPCR analysis of retinal Ptc1 expression following Wnt signalling activation or inhibition as indicated. P<0.01, *P<0.001 (Student t-test). Mean s.e.m. L, lens. Scale bars: 40m.
	Fig. 7. Hedgehog signalling inhibits Wnt activity. (A-D)Immunofluorescence (A,B) or in situ hybridisation (C,D) analyses of eGFP expression in stage 40 retinas from Wnt-responsive transgenic animals following a 24-hour treatment with either cyclopamine or purmorphamine. (B,D)Quantification of eGFP staining area within the CMZ (delineated by dotted lines). Hoechst staining (for immunolabelling) and Nomarski (for in situ hybridisation) were used to delineate the CMZ in order to exclude RPE eGFP labelling from the quantification. The total number of analysed sections per condition is indicated in each bar. P<0.05, **P<0.001 (Student t-test). Mean s.e.m. L, lens. Scale bars: 40m.
	Fig. 8. The Hedgehog pathway negatively regulates Wnt activity through the transcriptional activation of Sfrp-1. (A)In situ hybridisation analysis of Sfrp-1 expression on stage 41 retinal sections following a 24-hour treatment with purmorphamine (purm.) or cyclopamine (cyclo.). (B)qPCR analysis of Ptc1 (as a control for purmorphamine activity) and Sfrp-1 expression in the head of stage 38 sibling tadpoles following 8 hours of purmorphamine exposure. (C-J)In situ hybridisation analysis of eGFP expression in stage 40 retinas from Wnt-responsive transgenic animals exposed to SRFP-1 protein for 24 hours (C,D,G) or injected with Sfrp-1 Mo (E,F,H). (G-J)Tadpoles were additionally treated for 24 hours with cyclopamine (G,I) or purmorphamine (H,J). (D,F,I,J) Quantification of eGFP staining area per CMZ in each condition. The total number of analysed sections per condition is indicated in each bar. P<0.05, **P<0.001 (Student t-test). Mean s.e.m. L, lens. Scale bar: 40m.
	Fig. 9. The Wnt pathway negatively regulates Hedgehog activity through the transcriptional activation of Gli3. (A,B)In situ hybridisation analysis of Gli3 expression (A) on stage 38 retinal sections 24 hours following BIO or IWR-1 treatment. (B)Quantification of Gli3 staining area per CMZ. The total number of analysed sections per condition is indicated in each bar. (C)qPCR analysis of retinal Gli3 expression following 24-hour BIO or IWR-1 treatment. (D)qPCR analysis of retinal Ptc1 expression following 24-hour BIO treatment on Gli3 Mo-injected tadpoles. P<0.05, **P<0.001 (Student t-test). Mean s.e.m. L, lens. Scale bar: 40m.
	Fig. 10. Model of Wnt and Hedgehog interplay in the postembryonic retina. (A)Schematic highlighting the mutually exclusive expression domains of Wnt and Hedgehog ligands along the central to peripheral axis of the post-embryonic retina. (B-D)Illustration of the activities of Wnt and Hedgehog pathways in the retinal neurogenic niche and of the proposed crosstalk underlying their mutual negative regulation. Shown are the hypothetical physiological situation (B), and the synopsis of our Hedgehog (C) or Wnt (D) activation experiments. Their opposed impacts on stem/progenitor cell proliferation are represented by changes in the CMZ proliferative cell population (grey) in the drawings beneath.