All about orb: A role in polarity establishment and autoregulatory loop driven oocyte specification

Abstract

orb, a translational regulator of the cytoplasmic polyadenylation element binding protein (CPEB) family, is required for multiple steps of Drosophila oogenesis. Here we investigated orb function at two distinct junctures. First, we investigated the role of orb in polarity establishment during midstages of oogenesis. Second, we found that oocyte specification depends on the orb 3’UTR.

orb is needed for translational activation of the dorsal determinant gurken mRNA and the posterior/germline determinant oskar mRNA. Localized translation of these determinants leads to the specification of the future body axes of the fly. We report that orb functions upstream of the translation of the axis determinants and functions in the polarization of the microtubule network. Microtubule polarity within the oocyte depends on Par proteins, which establish anterior and posterior domains within the oocyte through antagonistic cross-regulatory interactions. We find that orb is needed for microtubule polarity and for the localization of Par proteins. Defects in Par protein localization may be due to orb’s role in organization of the cortical actin cytoskeleton.

The second function of orb investigated is its role in oocyte specification. We show that the orb autoregulatory loop, in which Orb protein translationally activates its own mRNA through interactions with the orb 3’UTR, is important for the establishment of oocyte identity. When the orb 3’UTR is deleted orb mRNA and protein fail to localize to within the germline cyst and ultimately all cells differentiate as nurse cells. Other markers of oocyte identity can be localized within the cyst to a subset of cells, but fail to become enriched within a single cell. We identified a fragment of the orb 3’UTR which is partially functional. In wild type, the oocyte is specified in region 2B of the germarium. The partially functional 3’UTR fragment delays oocyte specification until stage 2 of oogenesis. Prior to this point, orb mRNA and protein are not localized to one cell. At stage 2, about 50% of egg chambers successfully localize orb mRNA and protein within the oocyte, suggesting that the autoregulatory loop is activated. Finally, we show that maintenance of oocyte identity also requires orb activity.