HB-EGF is necessary and sufficient for Müller glia dedifferentiation and retina regeneration

Abstract

Müller glia (MG) dedifferentiation into a cycling population of multipotent progenitors is crucial to zebrafish retina regeneration. The mechanisms underlying MG dedifferentiation are unknown. Here we report that heparin-binding epidermal-like growth factor (HB-EGF) is rapidly induced in MG residing at the injury site and that pro-HB-EGF ectodomain shedding is necessary for retina regeneration. Remarkably, HB-EGF stimulates the formation of multipotent MG-derived progenitors in the uninjured retina. We show that HB-EGF mediates its effects via an EGFR/MAPK signal transduction cascade that regulates the expression of regeneration-associated genes, like ascl1a and pax6(b). We also uncover an HB-EGF/Ascl1a/Notch/hb-egf(a)-signaling loop that helps define the zone of injury-responsive MG. Finally, we show that HB-EGF acts upstream of the Wnt/β-catenin-signaling cascade that controls progenitor proliferation. These data provide a link between extracellular signaling and regeneration-associated gene expression in the injured retina and suggest strategies for stimulating retina regeneration in mammals.

Figure 1. Injury-dependent hb-egf_a induction is necessary for MG dedifferentiation and proliferation

(A) RT-PCR showing the temporal expression pattern of genes encoding EGFR ligands in the uninjured and injured retina. (B) In situ hybridization and BrdU immunofluorescence shows hb-egf_a is induced in proliferating progenitors in the injured retina (* marks the injury site). (C) MO-mediated HB-EGF_a knockdown reduces the number of proliferating MG-derived progenitors in the injured retina at 4 dpi (those cells that do proliferated do not contain the lissamine-labeled MO, arrowheads). (D) Quantification of (C) at different hb-egf_a MO concentrations. *P<0.01. Error bars are standard deviation. Scale bars, 50 µm. Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; MO, antisense morpholino-modified oligonucleotide.

Figure 2. HB-EGF stimulates MG dedifferentiation into multipotent retinal progenitors in the uninjured retina

(A) Diagram of experimental protocol and low magnification photomicrographs showing HB-EGF injection into the eye of 1016 tuba1a:gfp fish stimulates GFP expression and BrdU incorporation throughout the INL of the uninjured retina. Scale bar, 150 µm. (B) High magnification view of (A) showing GFP⁺/BrdU⁺ cells express the MG marker glutamine synthetase (GS). Scale bar, 50 µm. (C) RT-PCR and (D) in situ hybridization assays showing HB-EGF stimulates the expression of genes characteristic of dedifferentiated MG. In (D), the PBS + BSA treated retinas did not show an in situ hybridization signal above background for any of the probes tested – data only shown for the ascl1a probe. Scale bars, 50 µm. (E) The matrix metalloprotease inhibitor, GM6001, suppresses injury-dependent proliferation of MG-derived progenitors and this regulation is rescued by soluble HB-EGF. (F) Quantification of the effects observed in (E). (G) BrdU and retinal cell type-specific immunfluorescence shows HB-EGF generated progenitors in the uninjured retina at 14 days post injection are multipotent and generate all major retinal cell types. Scale bar, 50 µm. (H) Quantification of regenerated cell types observed in (G). *P<0.05. Error bars are standard deviation. Abbreviations: as in Figure 1. See also Figures S1 and S2.

Figure 3. HB-EGF and retinal injury stimulate MG dedifferentiation via an EGFR/MAPK/Ascl1a signaling cascade

(A) EGFR inhibition (PD153035) or MAPK inhibition (PD98059, SL327) reduces the number of proliferating progenitors generated following retinal injury. (B) Quantification of (A). *P<0.01. (C) RT-PCR shows that EGFR or MAPK inhibition blocks induction of genes associated with MG dedifferentiation. (D) EGFR or MAPK inhibition reduces the number of proliferating progenitors generated following HB-EGF treatment of the uninjured retina. (E) HB-EGF_a knockdown suppresses induction of regeneration associated genes in the injured retina. [hb-egf MO] is in mM. C-MO is 0.5 mM. (F) Ascl1a knockdown has little effect on hb-egf_a induction in the injured retina (MOs are 0.25 mM). (G) Real-time PCR quantification of (E and F) normalized to uninjured value. The hb-egf MO is at 0.5 mM and ascl1a MOs are at 0.25 mM. *P<0.01. (H) ascl1a and hb-egf_a in situ hybridization assays along with BrdU immunofluorescence show co-localization in the injured retina. (I) MO-mediated knockdown of Ascl1a in the presence of HB-EGF suppresses HB-EGF-dependent generation of BrdU⁺ progenitors. BrdU⁺ progenitors that do form in the Ascl1a knockdown retina generally lack lissamine labeled MO (arrowheads). Control MO does not block the generation of BrdU⁺ progenitors (arrows). (J) Quantification of BrdU⁺ cells in (I). *P<0.01. Error bars are standard deviation. All scale bars, 50 µm. Abbreviations: as in Figure 1. See also Figure S3.

Figure 4. HB-EGF and Ascl1a regulate Notch signaling component genes in the injured retina

(A) Real-time PCR shows injury-dependent induction of Notch signaling components. (B) RT-PCR shows HB-EGF activates Notch signaling component genes in the uninjured retina. (C) RT-PCR shows MO-mediated HB-EGF_a knockdown suppresses injury-dependent induction of Notch signaling component genes. (D) Real-time PCR quantification of (C). *P<0.01. (E) RT-PCR shows Ascl1a knockdown suppresses injury-dependent induction of Notch signaling component genes. (F) Real-time PCR quantification of (E). *P<0.01. (G) Ascl1a knockdown suppresses HB-EGF-dependent induction of notch1 and her4 mRNAs. Error bars are standard deviation. Scale bar, 50 µm. Abbreviations: as in Figure 1. See also Figures S4 and S5.

Figure 5. Notch inhibition stimulates MG proliferation and expands the zone of dedifferentiated MG in the injured retina via induction of hb-egf_a gene expression

(A) DAPT treatment of 1016 tuba1a:gfp transgenic fish results in an expansion of the zone of dedifferentiated MG (GFP⁺/BrdU⁺) in the injured retina. Scale bar, 50 µm. (B) RT-PCR (gel) and Real-time PCR (4 dpi, graph) showing DAPT induces ascl1a, hb-egf_a and egfr mRNA expression in the injured retina. **P<0.01 for ascl1a and hb-egf_a; *P<0.05 for egfr. (C) In situ hybridization showing DAPT expands ascl1a mRNA expression in the injured retina (* marks the injury site). Scale bar, 50 µm. (D) ascl1a:gfp transgenic fish exhibit injury- and Notch-dependent regulation of transgene expression. Scale bar, 50 µm. (E) DAPT stimulates hb-egf_a mRNA expression in proliferating MG-derived progenitors of the injured retina. Scale bar, 50 µm. (F) MAPK inhibition (PD98059 and SL327) and EGFR inhibition (PD153035) suppress DAPT-dependent expansion of MG proliferation in the injured retina. *P<0.01. (G) BrdU immunofluorescence on retinal sections reveals BrdU incorporation in the injured retinas of WT and hsp70:gal4;uas:nicd-myc transgenic fish over-expressing NICD. Scale bar, 50 µm. *P<0.01. (H) In situ hybridization assays for hb-egf_a and ascl1a mRNA expression in WT and hsp70:gal4;uas:nicd-myc fish over-expressing NICD (* marks the injury site). Scale bar, 50 µm. (I) RT-PCR shows ascl1a, hb-egf_a and pax6_b mRNA expression are inhibited by NICD over-expression. (J) NICD-myc overexpression suppresses HB-EGF-induced cell proliferation in the uninjured retina. BrdU⁺ cells in HB-EGF-treated retinas over-expressing NICD-myc are generally NICD negative (arrows). Scale bar is 150 µm in low magnification images and 50 µm in higher magnification images. Error bars are standard deviation. Abbreviations: as in Figure 1. See also Figure S6.

Figure 6. Notch signaling regulates the differentiation of MG-derived progenitors

(A, C) Diagram of the experimental protocol and representative photomicrographs showing BrdU and retinal cell type-specific immunofluorescence. (B, D) Quantification of double-labeled cell types. *P<0.01. Error bars are standard deviation. Scale bar, 50 µm. Abbreviations: as in Figure 1.

Figure 7. HB-EGF activates a Wnt/β-catenin signaling cascade

(A) β-catenin immunofluorescence shows HB-EGF stimulates β-catenin stabilization in proliferating progenitors of the uninjured retina. Scale bar, 50 µm. (B,C) Pyrvinium (Pyrvin)-mediated inhibition of Wnt/β-catenin signaling suppresses HB-EGF-dependent generation of proliferating progenitors in the uninjured retina. Error bars are standard deviation. *P<0.01. Scale bar, 50 µm. (D) RT-PCR shows pyrvinium suppresses HB-EGF-dependent induction of regeneration associated genes in the uninjured retina. (E) In situ hybridization and BrdU immunofluorescence shows HB-EGF-dependent induction of transgene expression in TOP:dGFP Wnt signaling reporter fish is inhibited by pyrvinium (pyrvin). (F) Ascl1a knockdown suppresses HB-EGF-dependent β-catenin stabilization and BrdU incorporation in the uninjured retina. (G) Summary of signaling cascades and genes regulated by HB-EGF.