Ephb4b/Efnb2b signaling regulates DFC clustering through the RhoA pathway. (A) Ephb4b physically associated with Efnb2b. Myc-tagged zebrafish ephb4b and GST-tagged zebrafish efnb2b were transfected into HEK293T cells. (B) Ephb4b forward signaling activated RhoA signaling. Ephb4b-Flag and GST-rhotekin-RBD were transfected into HEK293T cells. The relative ratio of active RhoA is indicated. (C) Increase of p-MLC level by Ephb4b-Efnb2b interaction. Ephb4b-HA-expressing and Efnb2b-GST-expressing HEK293T cells were mixed and co-cultured, followed by immunoprecipitation and western blotting. The relative ratio of p-MLC is indicated. (D,E) Inhibition of the RhoA signaling pathway or actomyosin activity disaggregated DFCs. Embryos at the 512-cell stage were injected with rhoaN19 or dnrock2a mRNA or treated from 50% to 65% ES with blebbistatin, followed by probing sox17 expression at 65% ES. Representative DFC patterns are shown in dorsal view (D) with quantification in E. (F) Rescue of ephb4b-MO2-induced DFC dispersal by constitutively active RhoA (RhoaV14). One-cell-stage embryos were injected with ephb4b-MO2 alone, or re-injected at the 512-cell stage with different doses of rhoaV14 mRNA, and examined for sox17 expression at 75% ES, The ratios different classes (as shown in D) were calculated. (G) Model of Ephb4b/Efnb2b signaling regulation of DFC aggregation. EVL, envelope layer; MPs, mesodermal progenitors; YSL, yolk syncytial layer. See the Discussion for explanation.
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