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Stat3 is required for rod photoreceptor regeneration. (a) Experimental design depicting CRISPR/Cas9-based mutation (MUT) of stat3 in NTR-rod embryos. Control (+Mtz) and crispant NTR-rod larvae (+Mtz, stat3 MUT) were treated with 10mM Mtz from 5–6 dpf and rod fluorescence was assessed via plate reader assay at 7 dpf (to examine the extend of rod cell loss) and 9 dpf (to examine the extent of rod cell regeneration). Created with Biorender.com. (b) Relative stat3 mRNA expression (qRT-PCR assay) in non-injected controls compared to stat3 crispant fish. (c) Quantification of NTR-YFP expression in rod cells by plate reader assay at 7 dpf in non-ablated (-Mtz) wildtype, and ablated control (+Mtz) and stat3 MUT larvae (+Mtz, stat3 MUT). (d-e) Representative in vivo confocal images of regenerated rod cells at 9 dpf in control (+Mtz) and stat3 MUT larvae (+Mtz, stat3 MUT). (f) Quantification of rod cell regeneration by plate reader assay at 9 dpf in control (+Mtz) and stat3 MUT larvae (+Mtz, stat3 MUT). (g-h) Representative immunohistological staining for PCNA at 7 dpf in control (+Mtz) and stat3 MUT larvae (+Mtz, stat3 MUT) fish. (i) Quantification of INL, ONL, and total PCNA+ cells at 7 dpf in control (+Mtz) and stat3 MUT larvae (+Mtz, stat3 MUT). For statistical comparisons, Student’s t test was used to assess the indicated paired conditions. Asterisks indicate the following p-value ranges: * = p<0.05, ** = p<0.01, *** = p<0.001, and **** = p<0.0001, “ns” indicates p>0.05.
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