Anchoring of maternal RNAs in the oocyte of the clawed frog, Xenopus laevis

grantor: University of TorontoThe body plan of the embryo is established by a polarized source of developmental information in the oocyte. The 'X. laevis' oocyte creates polarity by anchoring mRNAs in the vegetal cortex, including ' Vg1', which may function in body plan specification, and ' Xwnt-11' and 'Xcat-2', which may function in germ cell development. To identify components of the RNA anchoring mechanism, I used manually isolated vegetal cortices (IVC) to assay loss or change in spatial arrangement of RNAs caused by disruption of cortical elements. The role of cytoskeleton in RNA anchoring was tested by treating oocytes with inhibitors that selectively disrupted actin filaments and cytokeratin filaments, and by using the IVC to assay for RNAs. Treatment of oocytes with cytochalasin B caused clumping of 'Vg1' and 'Xwnt-11' as revealed by 'in situ' hybridization of IVCs, but did not cause their release as confirmed by RT-PCR analysis. These RNA clumps did not match the distribution of actin filament clumps, but they appeared to be similarly distributed as the remnant cytokeratin filaments. Treatment of oocytes with anti-cytokeratin antibody C11 released these RNAs from the cortex. C11 altered the texture of the cytokeratin network, but did not affect the actin meshwork. From these results, I concluded that 'Vg1' and 'Xwnt-11 ' are retained by a cytokeratin filament-dependent mechanism, and that organization of the cytokeratin network depended on an intact actin meshwork. Colcemid did not disrupt 'Vg1' and 'Xwnt-11' retention by the IVC, so anchoring of these RNAs are independent of microtubules. Membrane disruption in the IVC by Triton X-100 decreased 'Vg1 ' and 'Xwnt-11'. The disappearance of these RNAs was mainly due to degradation by ribonuclease activity released from membrane components. However, more 'Vg1' and 'Xwnt-11' were recovered in the supernatant upon treatment of IVCs on ice, which diminished ribonuclease activity. This suggested that a fraction of these RNAs required membranes to be retained in the cortex. Cytochalasin B, C11, colcemid, and Triton X-100 on ice neither released nor degraded 'Xcat-2' mRNA, so no cortical element could be implicated in its anchoring. 'Xcat-2' was also more resistant to endogenous ribonuclease activity compared to 'Vg1 ' and ' Xwnt-11'.Ph.D