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 MhwaS168A/S168A females confirmed the successful construction of the hwaS168A homozygous mutant line. c Phenotypes of MhwaS168A/S168A embryos at 24 hpf could be divided into two classes: both lacking an embryonic axis, and MhwaS168A/+ embryos were all normal. d Marker genes were quantified by RT-qPCR at 4 hpf and 6 hpf for WT and MhwaS168A/S168A embryos, N = 3. e Loss of expression of dorsal/organizer markers (chd and gsc at 6 hpf) was detected by WISH in MhwaS168A/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 MhwaS168A/S186A mutant embryos or Mhwatsu01sm/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.