Fig. 2

Inhibition of Fgf signaling, using a heat shock-inducible dominant negative Fgf receptor, prevents CS formation.

(A) The cldn:GFP∗hsp70l:dnfgfr1-EGFP transgenic zebrafish line was used to induce dominant negative fgfr1 expression. Heat shock was induced at 24 hpf and 38 ​°C for 45 ​min and the CS development was evaluated 24 ​h later by confocal microscopy or whole mount in situ hybridization for stc1. (B) An example of normally developed CS in a CTR – control embryo that has not been heat-shocked. (C) An example of abnormally developed CS in a cldn:GFP∗hsp70l:dnfgfr1-EGFP embryo that has been heat-shocked. (D) Heat shock of cldn:GFP∗hsp70l:dnfgfr1-EGFP embryos increased the number of defective CS (left and middle bars; ctr – not heat-shocked embryos of the cldn:GFP∗hsp70l:dnfgfr1-EGFP line; HS - heat-shocked embryos of the cldn:GFP line). However, heat shock-induced expression of dnfgfr1 resulted in reduction of CS formation that was significantly different (p ​= ​0.007, Cochran-Mantel-Haenszel test) from the effect caused by heat shock alone (HS dnfgfr1 - heat-shocked embryos of the cldn:GFP∗hsp70l:dnfgfr1-EGFP line). The numbers of scored embryos are shown within each bar. (E) Representative images of embryos with normal (top), strongly reduced (middle) or absent (bottom) stc1 expression after heat shock-induced expression of dnfgfr1. (F) An example of control (ctr) embryo of the cldn:GFP∗hsp70l:dnfgfr1-EGFP line that has not been heat-shocked showing normal expression of stc1. (G) Defective CS formation correlated with reduced stc1 expression after heat shock-induced expression of dnfgfr1 (p ​= ​0.016, Cochran-Mantel-Haenszel test). The numbers of scored embryos are shown within each bar. ctr – not heat-shocked embryos of the cldn:GFP∗hsp70l:dnfgfr1-EGFP line; HS - heat-shocked embryos of the cldn:GFP line; HS dnfgfr1 - heat-shocked embryos of the cldn:GFP∗hsp70l:dnfgfr1-EGFP line.