Epiboly in zebrafish requires reactive oxygen species produced by NADPH oxidases for the regulation of vesicular trafficking

Abstract

Epiboly is the first morphogenetic cell movement that occurs at the onset of gastrulation in zebrafish. During epiboly, the blastoderm thins out and spreads cells over the massive yolk cell. Epiboly progression is controlled by a complex regulatory network that involves diverse molecular effectors. Previously, we reported that reactive oxygen species (ROS) derived from NADPH oxidases (Nox) are required for normal epiboly progression, embryo survival, and early development. We also found that the inhibition of Nox activity during gastrulation downregulates E-cadherin abundance at the enveloping layer (EVL) cell margins. Since the dynamic localization of E-cadherin at the plasma membrane is highly regulated by endocytosis and vesicular trafficking during epiboly, in the present study, we investigated the effects of Nox inhibition and hydrogen peroxide (H2O2) on endocytosis and in the localization of different proteins important for endosomal trafficking in zebrafish embryos. We show that the simultaneous treatment with the Nox inhibitor VAS2870 and the dynamin 2 (Dnm2) inhibitor dynasore rescues the effects of VAS2870 on epiboly delay, embryo mortality and E-cadherin abundance at EVL cell margins. Furthermore, we found that H2O2 impacts the endocytic rate of fluorescent fluid-phase markers at the EVL, as well as the localization and abundance of Rab11, a small GTPase protein involved in recycling endosomes. Our results suggest that Nox-derived ROS participate in the regulation of the initial steps of endocytosis and in the endosomal trafficking required for epiboly progression during early zebrafish development. HIGHLIGHTS- NADPH oxidase (Nox) activity is required for the epiboly and localization of E- cadherin. - Dynamin inhibition rescues the developmental defects produced by the loss of Nox activity. - Nox-derived reactive oxygen species (ROS) participate in the regulation of endosome and E-cadherin trafficking, which is required for epiboly. - Nox inhibition increases the rate of fluorescent fluid-phase markers of endocytosis in EVL cells. - H2O2 decreases fluid-phase internalization in EVL cells. - H2O2 regulates Rab11 localization