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Ser168 of Hwa is phosphorylated in zebrafish and is responsible for axis-inducing activity. a Immunoblotting of pHwa in zebrafish embryos injected with 200 pg WT or S168A mutant of hwa-HA mRNA at the 1-cell stage. b Quantifications of relative pHwa levels in embryos treated as in (a), N = 6. c Immunoblotting of pHwa in zebrafish injected with 200 pg hwa-HA mRNA at the 1-cell stage, followed by treatment with AZD5438 from 2–4 hpf. (d) Quantifications of relative pHwa levels in embryos treated as in (c), N = 4. e Overexpression of Myc-cdk16 and ccnyl1-HA in wild-type embryos resulted in dorsalized phenotypes (D1, D2), some with double head/axis (DH), N = 2. f Rescue efficiency of hwa-HA mRNA alone or together with Myc-cdk16 & ccnyl1-HA injected at the 1-cell stage in Mhwatsu01sm/tsu01sm embryos, N = 2. g Expression levels of the organizer-specific genes (boz and chd) in embryos rescued by different mRNA combinations of hwa-HA and Myc-cdk16/ccnyl1-HA as in (f), N = 3. h Rescue efficiency of 5 pg hwa-HA mRNA alone or together with Myc-cdk2 injected at the 1-cell stage in Mhwatsu01sm/tsu01sm embryos, N = 3. i Expression levels of boz and chd in embryos rescued by different mRNA combinations of hwa-HA and Myc-cdk2 as in (h), N = 3. j Rescue efficiency of lower dose (1.0 or 0.5 pg) of hwa-HA mRNA alone or together with Flag-mGSK3β and Myc-cdk2 injected at the 1-cell stage in Mhwatsu01sm/tsu01sm embryos, N = 3. phenotypes were grouped as in (h). b–i A two-tailed unpaired t-test was performed; (e–j) A two-tailed Fisher’s exact test was performed (all phenotypes were divided into two groups: Unchanged and Changed). N, number of biological replicates; n, total number of embryos in each treatment; Significant differences are indicated by ns ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Source data are provided as a Source Data file.
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