Fig. S4
Larval phenotypes upon cxcr5 misexpression; and transgene activity 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. Dominant negative variant lacks the last three transmembrane domains and the intracellular domain. Both variants are inserted into a transgenesis cassette that contains the coding sequence for enhanced green fluorescent protein (EGFP) and self-cleaving T2A peptide. The whole cassette is expressed under heat-inducible hsp70l promoter. (B) Non-transgenic sibling at 3 days post-fertilization (dpf). (B1) High-magnification image of the yolk-sac in B. (B2) High-magnification image of the yolk-tube extension in B. (B3) High-magnification image of the ventral fin fold in B. (C)Tg(hsp:egfp-T2A-dncxcr5) transgenic animals at 3 dpf after two heat shocks in gastrula. (C1) High-magnification image of the yolk-sac in C. (C2) High-magnification image of the yolk-tube extension in C. (C3) High-magnification image of the ventral fin fold in C. (D)Tg(hsp:egfp-T2A-FLcxcr5) transgenic animals at 3 dpf after two heat shocks in gastrula. (D1) High-magnification image of the yolk-sac in D. (D2) High-magnification image of the yolk-tube extension in D. (D3) High-magnification image of the ventral fin fold in D. (E) Quantification graph for the number of pigment cells in different regions of the non-transgenic, Tg(hsp:egfp-T2A-dncxcr5) and Tg(hsp:egfp-T2A-FLcxcr5) larvae. Note that dominant negative variant of cxcr5 significantly reduces while full-length cxcr5 significantly increases the number of pigment cells in comparison to the non-transgenic siblings. (F) Quantitative real-time PCR analyses at 3 dpf after the misexpression of cxcr5 with two heat shocks at gastrula. (G) Heat-shock scheme for adult zebrafish telencephalon expression. (H) GFP and DAPI staining in heat-shocked non-transgenic animals. (I) GFP and DAPI staining in heat-shocked Tg(hsp:egfp-T2A-dncxcr5) animals. (J) GFP and DAPI staining in heat-shocked Tg(hsp:egfp-T2A-FLcxcr5) animals. (K-M) GFP IHC on Tg(hsp:egfp).(K) Olfactory bulb at 1 day after heat-shock. (L) Olfactory bulb at 2 days after heat-shock. (M) Telencephalon at 1 day after heat-shock. (N) Telencephalon at 2 days after heat-shock. GFP protein as a result of heat shock paradigm reduces significantly after 24 hours after heat shock. |