1. Loss of Grk2 impairs Hh signal transduction in zebrafish
Loss of Grk2 impairs Hh signal transduction in zebrafish Western blot analysis of the different forms of Gli2a in MZgrk2 embryos compared to wild‐type (AB), shh mRNA‐ and dnPKA mRNA‐injected wild‐type and cyclopamine (Cyc(A))‐treated wild‐type 20hpf embryos. Gli2a‐FL levels are low relative to Gli2aR levels in wild‐type embryos, but are elevated in response to pathway activation (shh and dnPKA mRNA injected). Gli2aR levels are increased while Gli2a‐FL is undetectable in CycA‐treated and MZgrk2 mutant embryos. Probing the same blot with rabbit anti‐GRK3 (which recognises the zebrafish Grk2 protein) reveals a complete loss of full‐length Grk2 in MZgrk2 embryos. Probing with anti‐γ‐tubulin was performed as a loading control. Three biological replicates of this analysis were performed.Phenotype of wild‐type (WT), MZgrk2 and grk2‐GFP mRNA‐injected MZgrk2 embryos at 24hpf (n = 20 for each sample). The red lines indicate the shape of the somites (middle panels) and the separation of the eyes (right hand panels). Scale bar, 200 μm.In situ hybridisation of ptch2, nkx2.2a and olig2 transcripts in wild‐type, MZgrk2‐and grk2‐GFP‐injected MZgrk2 24hpf embryos. Each panel shows a full view of the embryo on the left and a cross‐sectional view of a somite on the right (n = 30 for each sample). Scale bars, 200 μm (whole mounts), 50 μm (sections).Expression of Prox1a and Engrailed (Eng) proteins in somites of wild‐type, MZgrk2‐ and grk2‐GFP mRNA‐injected MZgrk2 embryos at 30hpf. Each panel shows Prox1a in green, Eng in red and the merged images with DAPI staining in blue (n = 10 for each sample). Scale bar, 50 μm.
Zhonghua Zhao et al. EMBO Rep. 2016;17:
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