Journal: Cell reports

Article Title: Leptin and IL-6 Family Cytokines Synergize to Stimulate Müller Glia Reprogramming and Retina Regeneration

doi: 10.1016/j.celrep.2014.08.047

Figure Lengend Snippet: (A) Immunofluorescence on retinal sections shows activated p-Stat3 expression in BrdU+ MG-derived progenitors that are localized to the injury site at 4 dpi. (B) Schematic of gfap:stat3-gfp transgene construct shows the fusion gene, stat3-gfp, under control of the gfap promoter regulatory elements. (C) In gfap:stat3-gfp transgenic fish, Stat3-GFP fusion protein expression is undetectable in MG of the uninjured eye, while restricted to MG-derived progenitors at the injury site at 4 dpi. (D) Confocal images showing co-localization of Stat3-GFP with glutamine synthetase (GS)+/p-Stat3+/BrdU+ MG-derived progenitors at 4 dpi. In (A and C) the asterisk marks the injury site (needle poke) and arrows point to MG-derived progenitors. In (C) white dots indicate autofluorescence unique to the green channel (see Figure S2G). Error bars, s.d. Scale bars, 50 µm (A, C) and 20 µm (D). INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; dpi, days post injury. See also Figures S1and S2.

Article Snippet: Immunofluorescence and Western Blots Anti-GFP and 4C4 immunofluorescence were as previously described ( Craig et al., 2010 ; Fausett and Goldman, 2006 ). p-Stat3 immunoflourescence was performed using mouse anti-phospho-zebrafish Stat3 (Tyr708) antibody (MBL) at 1:100 dilution.

Techniques: Immunofluorescence, Expressing, Derivative Assay, Construct, Control, Transgenic Assay

Journal: Cell reports

Article Title: Leptin and IL-6 Family Cytokines Synergize to Stimulate Müller Glia Reprogramming and Retina Regeneration

doi: 10.1016/j.celrep.2014.08.047

Figure Lengend Snippet: (A) GFP immunofluorescence in 6-ascl1a:gfp fish shows that Jak inhibitors, P6 and JSI-124, applied at the time of retinal injury, inhibit injury-dependent transgene induction. (B) Diagram of ascl1a promoter constructs used to generate transgenic lines. (C) GFP immunofluorescence shows that a distal 1.5 kb fragment of the ascl1a promoter is required for injury-dependent transgene expression and that both consensus Stat3 sites located in this promoter fragment are necessary for this expression. BrdU+ cells indicate the injury site and a normal regenerative response. The asterisk marks the injury site (needle poke). Scale bar, 50 µm. INL, inner nuclear layer; dpi, days post injury. See also Figure S4.

Article Snippet: Immunofluorescence and Western Blots Anti-GFP and 4C4 immunofluorescence were as previously described ( Craig et al., 2010 ; Fausett and Goldman, 2006 ). p-Stat3 immunoflourescence was performed using mouse anti-phospho-zebrafish Stat3 (Tyr708) antibody (MBL) at 1:100 dilution.

Techniques: Immunofluorescence, Construct, Transgenic Assay, Expressing

Journal: Cell reports

Article Title: Leptin and IL-6 Family Cytokines Synergize to Stimulate Müller Glia Reprogramming and Retina Regeneration

doi: 10.1016/j.celrep.2014.08.047

Figure Lengend Snippet: (A) Intravitreal injection of Leptin/CNTF, or Leptin/IL-11 into the eye of gfap:stat3-gfp fish stimulates Stat3-GFP expression and BrdU incorporation throughout the uninjured retina’s INL, while Leptin, CNTF or IL-11 alone had little effect. Note that GFP reports activated p-Stat3 expression (Figure 1; Figure S2). White dots indicate autofluorescence unique to the green channel (see Figure S7K). The arrows point to MG-derived progenitors in the top 4 panels. (B) Quantification of Stat3-GFP+ cells in (A); ***P<0.001 (combination vs individual), n=4. (C) Quantification of BrdU+ cells in (A); ***P<0.001 (combination vs individual), n=4. (D) Western blot shows that retinal injury or intravitreal injection of cytokines into uninjured eye increases p-Stat3 expression. GS serves as loading control. (E) A model showing Leptin and IL-6 family cytokines synergize to stimulate MG reprogramming and retina regeneration via a Jak/Stat3 signaling pathway, which is essential for activating reprogramming genes like hbegf and ascl1a. Error bars, s.d. Scale bars, 50 µm. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Article Snippet: Immunofluorescence and Western Blots Anti-GFP and 4C4 immunofluorescence were as previously described ( Craig et al., 2010 ; Fausett and Goldman, 2006 ). p-Stat3 immunoflourescence was performed using mouse anti-phospho-zebrafish Stat3 (Tyr708) antibody (MBL) at 1:100 dilution.

Techniques: Injection, Expressing, BrdU Incorporation Assay, Derivative Assay, Western Blot, Control