The orb gene of Drosophila encodes sex-specific genn-line proteins that contain two RRM-type RNA-binding domains. Here we report the distribution of Orb protein in wild-type, tumorous, and orb mutant ovaries. The wild-type distribution of Orb protein during oogenesis resembles that of its RNA, preferentially accumulating in the cytoplasm of the developing oocyte shortly after the formation of the 16-cell cyst. As anticipated from its germ-line expression, mutations in orb lead to female sterility. Analysis of the effect of orb mutants on the distribution of RNAs known to be required for oocyte differentiation and polarity suggests that orb functions in RNA localization at multiple points during oogenesis. In addition, phenotypic characterization of the orb mutants indicates that the gene is required early in oogenesis for formation of the 16-cell cyst. It then functions in the differentiation of the oocyte and is required for the three-dimensional reorganization of the germ cells in the cyst as well as for the establishment of normal germ-line-soma interactions in the egg chamber.

The establishment of polarity axes in the Drosophila egg and embryo depends upon the localization and on-site expression of maternal mRNAs. The critical step in the targeting of posterior determinants is the localization of oskar (osk) mRNA to the pole and its on-site translation. Osk protein then recruits other posterior group gene products involved in the formation of pole plasm and in the localization and regulation of the posterior determinant, nanos. Here we have investigated the role of the Drosophila CPEB homolog, the orb gene, in the osk mRNA localization pathway. We demonstrate that the expression of Osk protein is dependent upon the orb gene. In strong orb mutants, Osk protein expression is undetectable, while in the hypomorphic mutant, orb(mel), little or no on-site expression of Osk protein at the posterior pole is observed. The defects in Osk protein accumulation in orb mutant ovaries are correlated with a reduction in the length of the osk poly(A) tails. We show that osk mRNA is in immunoprecipitable complexes with Orb protein in ovaries and that the osk 3' UTR can be UV cross-linked to Orb protein in ovarian extracts. These data suggest that Orb is required to activate the translation of osk mRNA and at that this may be accomplished by a mechanism similar to that used by the Xenopus CPEB protein to control translation of "masked" mRNAs.

The orb gene encodes an RNA recognition motif (RRM)-type RNA-binding protein that is a member of the cytoplasmic polyadenylation element binding protein (CPEB) family of translational regulators. Early in oogenesis, orb is required for the formation and initial differentiation of the egg chamber, while later in oogenesis it functions in the determination of the dorsoventral (DV) and anteroposterior axes of egg and embryo. In the studies reported here, we have examined the role of theorb gene in the gurken (grk)-Drosophila epidermal growth factor receptor (DER) signaling pathway. During the previtellogenic stages of oogenesis, the grk-DER signaling pathway defines the posterior pole of the oocyte by specifying posterior follicle cell identity. This is accomplished through the localized expression of Grk at the very posterior of the oocyte. Later in oogenesis, thegrk-DER pathway is used to establish the DV axis. Grk protein synthesized at the dorsal anterior corner of the oocyte signals dorsal fate to the overlying follicle cell epithelium. We show that orb functions in both the early and late grk-DER signaling pathways, and in each case is required for the localized expression of Grk protein. We have found thatorb is also required to promote the synthesis of a key component of the DV polarity pathway, K(10). Finally, we present evidence that Orb protein expression during the mid- to late stages of oogenesis is, in turn, negatively regulated by K(10).

The RRM-type RNA binding protein Orb plays a central role in the establishment of polarity in the Drosophila egg and embryo. In addition to its role in the formation and initial differentiation of the egg chamber, orb is required later in oogenesis for the determination of the dorsoventral (DV) and anteroposterior (AP) axes. In DV axis formation, Orb protein is required to localize and translate gurken mRNA at the dorsoanterior part of the oocyte. In AP axis formation, Orb is required for the translation of oskar mRNA. In each case, Orb protein is already localized at the appropriate sites within the oocyte before the arrival of the mRNAs encoding axis determinants. We present evidence that an autoregulatory mechanism is responsible for directing the on site accumulation of Orb protein in the Drosophila oocyte. This orb autoregulatory activity ensures the accumulation of high levels of Orb protein at sites in the oocyte that contain localized orb message.