During the early embryogenesis of C. elegans wnt and src pathways are redundantly involved in the determination of the spindle orientation in EMS and ABar blastomeres and endoderm induction. We performed an RNAi/4D microscopy screening to identify the genes in the wnt and src pathways by observing ABar spindle orientation. The candidate genes were examined for the endoderm induction by checking gut granules and were determined for the pathway in which the candidate genes are involved using simultaneous RNAi with wnt pathway genes
dsh-2 and
mig-5, or src pathway gene
src-1. We identified
hmp-2, which encodes a beta-catenin, as a src pathway component. We focused on the function of
hmp-2 in endoderm induction. 32% of
hmp-2;
dsh-2:
mig-5(RNAi) embryos lacked endoderm, whereas neither
hmp-2(RNAi) nor
hmp-2;
src-1(RNAi) embryos showed endoderm induction defect. Furthermore, POP-1 was symmetrically localized to nuclei of MS and E blastomere in 36% of
hmp-2;
dsh-2;
mig-5(RNAi) embryos. These results indicate that HMP-2 contributes to endoderm induction in src pathway by regulating the asymmetric localization of POP-1. We examined the localization of HMP-2 by immunostainning and found that anti-HMP-2 antibody stains nuclei of all blastomeres as well as cell-cell boundaries at 4-cell stage. This staining was diminished in
hmp-2(RNAi) embryos. We examined the position of HMP-2 in src pathway. The nuclear localization of HMP-2 was diminished in
src-1(RNAi) embryos indicating HMP-2 is downstream of SRC-1. SRC-1 is tyrosine kinase and anti-phospho-tyrosine antibody pY99 stains all cell-cell boundaries at 4-cell stage with strong signal on P2/EMS boundary. We found that pY99 also stains all nuclei at 4-cell stage SRC-1 and HMP-2 dependently, indicating HMP-2 is required for kinase activity of SRC-1 or a target of SRC-1. We also found that the protein detected by anti-HMP-2 antibody is also detected by pY99
src-1dependently on western blotting. These data indicate HMP-2 is a target of SRC-1. HMP-2 is anchored at cortex by binding to HMR-1 (cadherin). Phosphorylation of mammalian beta-catenin by Src disturbs interaction between beta-catenin and cadherin. We assumed phosphorylation of HMP-2 by SRC-1 causes dissociation of HMP-2 from HMR-1 and allows translocation of HMP-2 to nuclei. Indeed the addition of
hmr-1(RNAi) to
src-1(RNAi) caused the restoration of the nuclear HMP-2. We also found that endoderm induction defect in
dsh-2;
mig-5;
src-1(RNAi) was suppressed by
hmr-1(RNAi)
hmp-2 dependently. These data indicate phosphorylation of HMP-2 by SRC-1 leads to dissociation of HMP-2 from cadherin and nuclear translocation, and contributes to endoderm induction.