Fig. 9

Sema3d overexpression rescues hapln1a knockdown phenotypes.

(A) Experimental design. Three groups of fish were analysed: G1, G2 and G3. Fins from all three groups were amputated at 50% level. On 3 dpa all animals (G1, G2 and G3) were treated for hapln1a knockdown. Four hours post knockdown, G1 and G3 animals were heat shocked for 1hr at 37°C. G2 animals were not treated for heat shock. The following day, 1 dpe, G1 animals were selected for GFP positive fins. For GFP detection, immuno-staining (IS) and in situ hybridization experiments (ISH), fins were harvested 1 dpe and for regenerate and segment length analyses fins were harvested at 4 dpe. (B) Heat shock induces Sema3d-GFP (green) expression in Tg(hsp70:sema3d-gfp). GFP is not detected in the absence of heat shock. The white line indicates the amputation plane. Scale bar represents 50µm. (C) Following hapln1a knockdown the experimental group (G1) that is positive for both transgene and heat shock alone shows rescue for the phenotypes (i.e., shows high percent similarity compared to the un-injected side) whereas the control groups either negative for heat shock (G2) or negative for transgene (G3) fail to show rescue (i.e., shows reduced percent similarity compared to the un-injected side). Students t-test was performed (p<0.05) to determine significance, and the error bars indicate standard error of mean.