Abstract
AbstractThe eggshell of the fruit flyDrosophila melanogasteris a useful model for understanding the synthesis of a complex extracellular matrix. The eggshell is synthesized during mid-to-late oogenesis by the somatic follicle cells that surround the developing oocyte. We previously reported that female flies mutant for the genedrop-dead(drd) are sterile, but the underlying cause of the sterility remained unknown. In this study, we examined the role ofdrdin eggshell synthesis. We show that eggs laid bydrdmutant females are fertilized but arrest early in embryogenesis, and that the innermost layer of the eggshell, the vitelline membrane, is abnormally permeable to dye in these eggs. In addition, the major vitelline membrane proteins fail to become crosslinked by nonreducible bonds, a process that normally occurs during egg activation following ovulation, as evidenced by their solubility and detection by Western blot in laid eggs. In contrast, the Cp36 protein, which is found in the outer chorion layers of the eggshell, becomes crosslinked normally. To link thedrdexpression pattern with these phenotypes, we show thatdrdis expressed in the ovarian follicle cells beginning in mid-oogenesis, and, importantly, that alldrdmutant eggshell phenotypes could be recapitulated by selective knockdown ofdrdexpression in the follicle cells. To determine whetherdrdexpression was required for the crosslinking itself, we performedin vitroactivation and crosslinking experiments. The vitelline membranes of control egg chambers could become crosslinked either by incubation in hyperosmotic medium, which activates the egg chambers, or by exogenous peroxidase and hydrogen peroxide. In contrast, neither treatment resulted in the crosslinking of the vitelline membrane indrdmutant egg chambers. These results indicate thatdrdexpression in the follicle cells is necessary for vitelline membrane proteins to serve as substrates for peroxidase-mediated cross-linking at the end of oogenesis.
Publisher
Cold Spring Harbor Laboratory