Fig. 7

Fig. 7 The endogenous function of the S168A mutation in zebrafish embryos.

a The CRISPR/Cas9-based editing strategy was applied to construct the S168A mutation in zebrafish embryos. The gRNA targeting site and PAM sequence are highlighted in yellow and blue, respectively; Cas9 mRNA, gRNA, and single-strand donor DNA were coinjected at the 1-cell stage. b Sanger sequencing of WT and Mhwa^S168A/S168A females confirmed the successful construction of the hwa^S168A homozygous mutant line. c Phenotypes of Mhwa^S168A/S168A embryos at 24 hpf could be divided into two classes: both lacking an embryonic axis, and Mhwa^S168A/+ embryos were all normal. d Marker genes were quantified by RT-qPCR at 4 hpf and 6 hpf for WT and Mhwa^S168A/S168A embryos, N = 3. e Loss of expression of dorsal/organizer markers (chd and gsc at 6 hpf) was detected by WISH in Mhwa^S168A/S168A embryos. f The working model of the Hwa receptor. In wild-type embryos, Hwa protein is phosphorylated at Ser168 by multiple kinases and activated, enhancing Tnks1/2-mediated degradation of the Axin protein, increasing stability of β-catenin in the cytosol. β-catenin then translocates into the nucleus and turns on downstream target genes. However, in Mhwa^S168A/S186A mutant embryos or Mhwa^{tsu01sm/tsu01sm} embryos supplied with Hwa(S168A) mutant protein, the point mutation switches off the activity of the Hwa receptor. d A two-tailed unpaired t-test was performed and data were presented as mean ± SD. N, number of biological replicates; Significant differences are indicated by ns ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, with individual p-values illustrated. Source data are provided as a Source Data file.