cxcr5 is sufficient to increase ventricular cell proliferation after injury in the adult zebrafish telencephalon. (A) Cxcr5 is a seven-span transmembrane protein with an extracellular receptor domain, seven transmembrane domains and a C-terminus intracellular domain. We generated full-length and dominant-negative versions of Cxcr5. Both variants are inserted into a transgenesis cassette containing the enhanced green fluorescent protein (EGFP) reporter and self-cleaving T2A. The whole cassette is expressed under heat-inducible hsp70l promoter. (B) Heat shock scheme. Four heat shocks, three of which are after the lesion or sham operation, were given before sacrifice and analysis. (C) Proliferating cell nuclear antigen (PCNA) and green fluorescent protein (GFP) immunohistochemistry (IHC) in telencephalons of unlesioned (sham-operated) non-transgenic animals. C′ and C′′ are PCNA and GFP. (D) PCNA and GFP IHC in telencephalons of unlesioned (sham-operated) Tg(hsp:egfp-t2a-dncxcr5) animals. D′ and D′′ are PCNA and GFP. (E) PCNA and GFP IHC in telencephalons of unlesioned (sham-operated) Tg(hsp:egfp-t2a-FLcxcr5) animals. E′ and E′′ are PCNA and GFP. (F) Quantification graph for the relative amounts of GFP and PCNA double-positive cells in unlesioned telencephalons, where the transgenic misexpression of cxcr5 does not effect cell proliferation. (G) PCNA and GFP IHC in telencephalons of 3 days post-lesion (dpl) non-transgenic animals. G′ and G′′ are PCNA and GFP. (H) PCNA and GFP IHC in telencephalons of 3 dpl Tg(hsp:egfp-t2a-dncxcr5) animals. H′ and H′′ are PCNA and GFP. (I) PCNA and GFP IHC in telencephalons of 3 dpl Tg(hsp:egfp-t2a-FLcxcr5) animals. I′ and I′′ are PCNA and GFP. (J) Quantification graph for the relative amounts of GFP and PCNA double-positive in 3 dpl telencephalons, where transgenic misexpression of the full-length cxcr5 increases but the dominant negative variant does not affect the ventricular cell proliferation. Scale bars 25 μm; n = 4 telencephalons for each set of analyses.
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