fgf8a Gene Regulation during Retina Regeneration

(A) RT-PCR analysis of fgf8a, fgf8b, and Fgf-responsive genes in uninjured and needle-poke-injured retinas.

(B) In situ hybridization assays and BrdU immunofluorescence for fgf8a expression and MG proliferation, respectively, before and after injury to central retina in 6-month-old fish. Arrowheads point to fgf8a RNA in uninjured retina; arrows point to fgf8a RNA enriched in proliferating MG-derived progenitors at 4 dpi. Asterisk indicates injury site. Scale bar, 50 μm.

(C) RT-PCR analysis of ascl1, fgf8a, and gapdh RNAs in GFP+ MGs and GFP− non-MGs (retinal neurons) in uninjured and injured retinas that were FACS purified from gfap:GFP transgenic fish retinas.

See also Figure S1.

Sustained Fgf8a Expression Inhibits MG Proliferation

(A) BrdU immunofluorescence was used to visualize and quantify MG proliferation in injured (Inj) retinas following 2 days of sustained HS in WT and hsp70:fgf8a fish. Asterisk indicates injury site (central retina of 6-month-old fish). Scale bar, 100 μm. Graph shows quantification of BrdU+ cells; n = 3 individual experiments. Error bars indicate SD. ∗∗p < 0.01.

(B) BrdU immunofluorescence was used to visualize and quantify MG proliferation in uninjured retinas following intravitreal injection of HB-EGF/insulin (once a day for 3 days) and HS (for 4 days) in WT and hsp70:fgf8a fish. Scale bar, 150 μm. Graph shows quantification of BrdU+ cells; n = 3 individual experiments. Error bars indicate SD. ∗∗∗p < 0.001.

(C) qPCR analysis of gene expression in uninjured and injured (2 dpi) WT and hsp70:fgf8a fish retinas with HS; n = 3 individual experiments. Error bars indicate SD. p < 0.05.

See also Figure S2.

Cessation of Forced Fgf8a Expression Stimulates MG Proliferation in Injured or Growth-Factor-Treated Retina

(A) BrdU immunofluorescence in injured (Inj) retinas from WT and hsp70:fgf8a fish that received HS for 2 days and were assayed for MG proliferation 2 days later; Asterisk indicates injury site (central retina, 6-month-old fish). Scale bar, 100 μm. Graph shows quantification of BrdU+ cells; n = 3 individual experiments. Error bars indicate SD. ∗∗∗p < 0.001.

(B) Graph quantifying BrdU+ and EdU+/BrdU+ double-labeled cells in injured retinas from WT and hsp70:fgf8a (Fgf8a) fish that received HS for 2 days and then received an i.p. injection of BrdU and EdU at the indicated times; n = 3 individual experiments. Error bars indicate SD. ∗∗p < 0.01.

(C) Quantification of BrdU immunofluorescence in injured retinas of WT and hsp70:fgf8a fish that received HS for 2 days and were then sacrificed 3 hr after an i.p. injection of BrdU at the indicated times post-HS; n = 3 individual experiments. Error bars indicate SD.

(D) BrdU immunofluorescence in injured retinas at 2 dpi from WT and hsp70:fgf8a fish that received a 1-hr HS at the time of injury and were immersed in fish water with or without SU5402 for the indicated time. Asterisk indicates injury site (central retina, 6-month-old fish). Scale bar, 100 μm. Graph shows quantification of BrdU+ cells; n = 3 individual experiments. Error bars indicate SD. ∗∗p < 0.01.

(E) qPCR analysis of insm1a and ccnd1 gene expression at different times post-injury in WT and hsp70:fgf8a fish that received a 1-hr HS at the time of injury; n = 3 individual experiments. Error bars indicate SD. p < 0.05.

(F and G) BrdU immunofluorescence in uninjured (F) and injured (G) retinas electroporated with control and fgf8a-targeting MO at the indicated times. Asterisk indicates injury site (central retina, 6-month-old fish). Scale bar, 100 μm. Graph in (G) is quantification of proliferating MGs in injured retina treated with control (Ctrl) and fgf8a-targeting MO; n = 3 individual experiments. Error bars indicated SD. ∗∗p < 0.01.

(H and I) BrdU immunofluorescence in uninjured retinas from WT and hsp70:fgf8a fish that received a 1-hr HS and intravitreal injection of indicated growth factor (HB-EGF, 50 ng/μL; FGF2, 200 ng/μL; insulin growth factor 1 [IGF1], 200 ng/μL; insulin, 500 ng/μL) (H) or PBS/BSA (I) at the indicated times. Scale bar, 150 μm.

See also Figure S3.

Fgf8a Stimulates Notch Signaling and MG Quiescence

(A) mCherry and glutaminesynthetase (GS) immunofluorescence in tp1:mCherry transgenic fish treated with or without the Notch signaling inhibitors DAPT or RO 4929097. Scale bar, 100 μm.

(B) mCherry and BrdU immunofluorescence in tp1:mCherry transgenic fish at various times post-retinal injury. Arrowheads point to areas of reduced mCherry expression in the INL. Asterisk indicates injury site (central retina, 6-month-old fish). Scale bar, 100 μm.

(C) BrdU immunofluorescence in WT fish retina 4 days after intravitreal injection of DMSO or DAPT. Scale bar, 100 μm.

(D) mCherry and BrdU immunofluorescence in injured (Inj) retinas from tp1:mCherry and hsp70:fgf8a;tp1:mCherry transgenic fish that received HS from 1–2 dpi. Asterisk indicates injury site (central retina, 6-month-old fish); arrows point to BrdU+/mCherry− cells. Scale bar, 100 μm.

(E) BrdU immunofluorescence at 4 dpi in WT and hsp70:fgf8a fish that were immersed in fish water with or without DAPT and received HS over 4 days. Asterisk indicates injury site (central retina, 6-month-old fish). Scale bar, 100 μm. Graph shows quantification of BrdU+ cells; n = 3 individual experiments. Error bars indicate SD. ∗∗p < 0.01.

(F) RT-PCR analysis of indicated RNAs in uninjured (Uninj) and injured (6 hpi) retinas from WT fish (1, 2, and 3 are triplicate samples).

(G) qPCR quantification of dll4 and hey1 gene expression in WT and hsp70:fgf8a fish that received a 1-hr HS at the time of injury and were sacrificed 5 hr later; n = 3 individual experiments. Error bars indicate SD. p < 0.05.

(H) pPCR as in (G), but HS was for 2 days, and gene expression was assayed 2 days later; n = 3 individual experiments. Error bars indicate SD. p < 0.05.

See also Figure S4.

Age-Dependent Switch in Fgf8a Signaling

(A and B) BrdU immunofluorescence in uninjured retinas from 2- (B) and 6-month-old (A) WT and hsp70:fgf8a fish that received HS for 4 days before sacrifice. Scale bar, 100 μm.

(C) Quantification of spontaneous MG proliferation in the central (2/3) and remaining periphery of 2- and 6-month-old fish retina isolated from WT fish immersed in BrdU-containing water for 9 days; n = 3 individual experiments. Error bars indicate SD. ∗∗∗p < 0.001.

(D) BrdU and Edu immunofluorescence in the injured (Inj) central retina of WT and hsp70:fgf8a fish that received i.p. injections of BrdU and EdU at 4 and 14 dpi, respectively, and received HS from 10 to 14 dpi. Graph is quantification of the percentage of BrdU+ cells that co-label with EdU in the INL. Scale bar, 100 μm; n = 3 individual experiments. Error bars indicate SD. p < 0.05.

(E) Quantification of the number of BrdU+ cells per injury site in retinas from WT and hsp70:fgf8a fish of different ages that received HS for 4 days; n = 3 individual experiments. Error bars indicate SD. ∗∗p < 0.01; ∗∗∗p < 0.001. Inj, injured.

(F) BrdU immunofluorescence in central and peripheral regions of injured retinas from WT and hsp70:fgf8a fish that received HS for 4 days. Asterisk indicates injury site. Scale bar, 100 μm. Graph is quantification of BrdU+ cells per injury site; n = 3 individual experiments. Error bars indicate SD. ∗∗p < 0.01.

(G) Quantification of BrdU+ cells per injury site in peripheral retinas from WT and hsp70:fgf8a fish that received HS for 2 dpi and then BrdU at 4 dpi. Values are the difference between injured fish and uninjured fish; n = 3 individual experiments. Error bars indicate SD. ∗∗p < 0.01.

(H) Quantification of BrdU immunofluorescence in injured 2-month-old WT and hsp70:fgf8a fish retinas that received HS for 2 days and were then sacrificed 3 hr after an i.p. injection of BrdU at the indicated times post-HS; n = 3 individual experiments. Error bars indicate SD.

(I and J) BrdU immunofluorescence in uninjured (I) and injured (J) 2-month-old WT fish retina electroporated with control and fgf8a-targeting MO at the indicated times. Asterisk in (J) indicates injury site. Scale bars, 100 μm. Graph in (J) is quantification of proliferating MGs in injured retina treated with control and fgf8a-targeting MO; n = 3 individual experiments. Error bars indicate SD. ∗∗p < 0.01.

See also Figure S5.

Signaling Pathways Contributing to Fgf8a-Dependent MG Proliferation in the Uninjured Retina

(A and B) mCherry and BrdU immunofluorescence in uninjured retinas from 2-month-old (A) and 6-month-old (B) hsp:70:fgf8a;tp1:mCherry fish that received HS for 4 days (4d). Arrows point to BrdU+/mCherry− cells in the INL of the central retina (A) and retinal periphery (B). Scale bar, 100 μm.

(C) qPCR analysis of dll4 mRNA expression in central retina (2/3) and remaining retinal periphery in WT and hsp70:fgf8a (Fgf8a) fish that received HS for 4 days; n = 3 individual experiments. Error bars indicate SD. p < 0.05. Uninj, uninjured.

(D and E) BrdU immunofluorescence in uninjured retinas from 2-month-old (D) and 6-month-old (E) hsp70:fgf8a fish treated with DMSO, MAPK inhibitor (UO126), PI3K inhibitor (LY294002), or Jak/Stat3 inhibitor (JSI-124). Shown is the whole retina for the 2-month-old fish and the peripheral retina for the 6-month-old fish. Scale bars, 150 μm in (D) and 100 μm in (E). Quantification of BrdU+ cells per retina is shown below the images; n = 3 individual experiments. Error bars indicate SD. ∗∗∗p < 0.001.

(F) qPCR analysis of indicated RNAs isolated from whole retina, central retina (2/3), and remaining peripheral retina of a 6-month-old uninjured WT or hsp70:fgf8a (Fgf8a) fish with and without HS for 4 days before sacrifice; n = 3 individual experiments. Error bars indicate SD. p < 0.05.

See also Figure S6.

Related to Figure 2. Fgf8a inhibits MG proliferation.

(A) qPCR analysis of fgf8a expression at various times after a 1 hr HS in hsp70:fgf8a fish.

(B) BrdU immunofluorescence in injured retinas from Wt and hsp70:fgf8a fish that received HS for 4 days; asterisk indicates injury site (central retina, 6 mo old fish); scale bar is 100 µm. Graph is quantification of BrdU+ cells/injury. Asterisk indicates injury site; n=3 individual experiments, error bars are s. d. **P<0.01.

(C, D) BrdU immunofluorescence in injured retinas from Wt fish that received daily intravitreal injections of PBS/FGF2 2 days (C) or 4 days (D); Asterisk indicates injury site (central retina, 6 mo old fish); scale bar is 100 µm; Graphs show quantification of BrdU+ cells/injury; n=3 individual experiments, error bars are s. d. (E) RT-PCR analysis of indicated genes in uninjured and injured retinas from Wt and hsp70:fgf8a (Fgf) fish that received HS for the indicated times.

Related to Figure 3. Transient Fgf8a expression stimulates MG proliferation.

(A) Quantification of BrdU+ cells in injured retina from Wt and hsp70:fgf8a fish that received a 1 hr HS at the time of injury; n=3 individual experiments, error bars are s. d. **P<0.01.

(B) BrdU immunofluorescence in injured retinas from Wt and hsp70:fgf8a fish that received a 1 hr heat shock just before retinal injury. Asterisk indicates injury site (central retina, 6 mo old fish); scale bar is 100 µm. Graph shows quantification of BrdU+ cells/injury; n=3 individual experiments, error bars are s. d. ***P<0.001.

(C) As in (B) but BrdU incorporation quantified at 2 dpi; n=3 individual experiments, error bars are s. d. ***P<0.001.

(D) BrdU immunofluorescence in injured retinas from Wt and hsp70:fgf8a fish that received a 1 hr heat shock at 1 dpi. Asterisk indicates injury site (central retina, 6 mo old fish); scale bar is 100 µm. Graph shows quantification of BrdU+ cells/injury; n=3 individual experiments, error bars are s. d. **P<0.01.

(E, F) TUNEL assay was used to quantify cell death at various times in injured retinas from Wt and hsp70:fgf8a fish that received HS for 2 days (E) or for 1 hr (F); n=3 individual experiments, error bars are s. d.

(G) Quantification of BrdU+ cells at 2 dpi in Wt and hsp70:fgf8a fish that received a 1 hr HS 3 hrs before sacrifice; n=3 individual experiments, error bars are s. d. ***P<0.001.

Related to Figure 4. Fgf8a stimulates Notch signaling in the injured retina.

(A) mCherry fluorescence in injured retina from Tp1:mCherry and hsp70:fgf8a;tp1:mCherry fish that received a 1h HS and then sacrificed 5h later when Fgf8a levels are still high. Asterisk and arrows mark the injury site (central retina, 6 mo old fish); scale bar is 100 µm.

(B) mCherry and BrdU immunofluorescence in injured retinas from Tp1:mCherry and hsp70:fgf8a;tp1:mCherry fish that received HS from 3-4 dpi and sacrificed at 4 dpi. Asterisks and arrows mark the injury site (central retina, 6 mo old fish) and arrows point to BrdU+ cells; scale bar is 100 µm.

(C) mCherry fluorescence and BrdU immunofluorescence in injured retina from Tp1:mCherry and hsp70:fgf8a;tp1:mCherry fish that received a 2 day HS and then sacrificed 2 days later. Asterisk and arrows mark the injury site (central retina, 6 mo old fish); scale bar is 100 µm.

(D) BrdU immunofluorescence in injured retinas from Wt and hsp70:gal4:uas:NICD fish that received HS for 1h at 1 dpi and sacrificed at 4 dpi. Asterisk indicates injury site (central retina, 6 mo old fish); scale bar is 100 µm. Graph is quantification of BrdU+ cells/injury; n=3 individual experiments, error bars are s. d. ***P<0.001.

(E) RT-PCR analysis of hey1 and dll4 gene expression in uninjured and injured retina.

(F) In situ hybridization reveals spatial changes in dll4 expression in the injured retina. Asterisk indicates injury site (central retina, 6 mo old fish). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Related to Figure 5. Age-dependent change in Fgf8a signaling.

(A) BrdU immunofluorescence in uninjured retinas from Wt and hsp70:fgf8a fish at different ages that received HS for 2 or 4 days. Arrows point to BrdU+ MG; scale bar is 150 µm.

(B) BrdU immunofluorescence in injured retinas from Wt and hsp70:fgf8a fish at different ages that received HS for 4 days. Asterisk indicates injury site; scale bar is 100 µm.

(C) Glutamine synthetase immunofluorescence was used to quantify MG in the central and peripheral regions of 2 mo and 6 mo old fish retinas; n=3 individual experiments, error bars are s. d. *P<0.05.

(D) BrdU-based lineage tracing strategy was used to follow the fate of MG-derived progenitors in injured retinas from Wt fish and uninjured and injured retinas from hsp70:fgf8a fish that received a 2 day HS treatment at the time of injury; n=3 individual experiments, error bars are s. d. Antibodies used are: anti-Zpr1, cones; anti-GS, Muller glia; anti-HuC/D, amacrine cells in INL (inner nuclear layer) and ganglion cells in GCL (ganglion cell layer); anti-PKC, bipolar cells.

Related to Figure 6. Fgfr RNA levels.

(A, B) mCherry and BrdU immunofluorescence in 2 mo (A) and 6 mo (B) old hsp70:fgf8a;Tp1:mCherry transgenic fish retina. Arrow in (B) identifies proliferating retinal progenitors in the retinal ciliary marginal zone.

(C) qPCR analysis of delta-like (dl) gene expression in central and peripheral regions of uninjured retinas from 6 mo old hsp70:fgf8a fish that received HS for 4 days.; n=3 individual experiments, error bars are s. d.

(D) qPCR analysis of fgfr gene expression in MG that were FACS purified from uninjured gfap:GFP fish retinas at 2 and 6 mo of age; n=3 individual experiments, error bars are s. d. **P<0.01.

(E) qPCR analysis of fgfr gene expression in MG that were FACS purified from central and peripheral regions of uninjured gfap:GFP fish retinas at 2 and 6 mo of age; n=2 individual experiments, error bars are s. d.

(F) qPCR analysis of fgfr gene expression in central and peripheral regions of uninjured retinas from hsp70:fgf8a fish at 6 mo of age and that received HS for 4 days; n=3 individual experiments, error bars are s. d. Data was normalized to gapdh levels.

Acknowledgments
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