RNA anchoring in the vegetal cortex of the Xenopus oocyte.

The body plan of the embryo is established by a polarized source of developmental information in the oocyte. The Xenopus laevis oocyte creates polarity by anchoring mRNAs in the vegetal cortex, including Vg1 and Xwnt-11, which might function in body plan specification, and Xcat-2, which might function in germ cell development. To identify components of the RNA anchoring mechanism, we used the manually isolated vegetal cortex (IVC) to assay loss or change in spatial arrangement of mRNAs caused by disruption of cortical elements. The role of cytoskeleton in mRNA anchoring was tested by treating oocytes with inhibitors that selectively disrupted actin microfilaments and cytokeratin filaments. Treatment of oocytes with cytochalasin B caused clumping of Vg1 and Xwnt-11 as revealed by in situ hybridization of the IVC, but did not cause their release, as confirmed by RT-PCR analysis. These mRNA clumps did not match the distribution of actin microfilament clumps, but were distributed similarly to the remnant cytokeratin filaments. Treatment of oocytes with monoclonal anti-cytokeratin antibody C11 released these mRNAs from the cortex. C11 altered the texture of the cytokeratin network, but did not affect the actin meshwork. These results show that Vg1 and Xwnt-11 are retained by a cytokeratin filament-dependent mechanism, and that organization of the cytokeratin network depend on an intact actin meshwork. Colcemid did not disrupt Vg1 and Xwnt-11 retention in the IVC, so anchoring of these mRNAs are independent of microtubules. Membrane disruption in the IVC by Triton X-100 decreased Vg1 and Xwnt-11. Loss of these mRNAs was due mainly to ribonuclease activity released from membrane components. However, when ribonuclease activity was suppressed under cold temperature, a higher amount of Vg1 and Xwnt-11 was recovered in the supernatant. This result suggested that a fraction of these mRNAs required membranes to be retained in the cortex. By contrast, Xcat-2 mRNA was neither released nor degraded following treatments with cytochalasin B, C11, colcemid and Triton X-100 under cold temperature, so no cortical element could be implicated in its anchoring.

Cytokeratins in the liver of the sea lamprey (Petromyzon marinus) before and after metamorphosis.

Cytokeratins of the liver were examined during the lamprey life cycle. This organ transforms, at metamorphosis, from a ductal larval structure to an aductal adult organ lacking the biliary passageways. Specifically, the relationship between liver morphology and cytokeratins 7, 18, and 19, and several unidentified cytokeratins was compared in larvae, transformers, and adults. Immunofluorescence staining showed that spatiotemporal cytokeratin distributions are cell-type-specific among hepatocytes, bile-duct cells, and gallbladder cells. In the larva, cytokeratins 7 and 19, and other unknown cytokeratins occur diffusely in the hepatocyte cytoplasm and prominently in the apices of hepatocytes near a bile canaliculus, so that collectively these cytokeratins form a pericanalicular ring. At mid-metamorphosis, the ring disappears, and cytokeratins are located predominantly in the periphery of bile-duct cells. In the adult, cytokeratin 19 has a sexually dimorphic distribution. Electrophoretic separations of cytoskeletal extracts of larval and adult livers have revealed larval cytokeratins 46, 49, 58, and 63.5, and adult male cytokeratins 46, 48, 49, 52.5, 58, 63.5, 64.5, 65, and 66. Immunoblotting has shown that certain cytokeratins are each related to at least two mammalian cytokeratins. We suggest that lamprey-specific liver cytokeratins exist, and that the metamorphosing liver can be used as a model for studying the relationship between cytokeratin organization and cell function.

Identification of new localized RNAs in the Xenopus oocyte by differential display PCR.

We have identified localized transcripts in full-grown Xenopus oocytes by differential display PCR. One clone, An4a, has two transcripts, which localize to the animal half of the stage VI oocyte. The transcripts are expressed throughout early development, with embryonic expression primarily in anterior neural tissues. An4a has a high degree of sequence identity to a human cDNA clone of unknown function. Another clone, the previously identified beta-transducin repeat containing protein (beta-TrCP), has three transcripts with a unique pattern of localization, one localized to the animal half and two localized primarily to the vegetal cortex. This cDNA has previously been shown to rescue a yeast cell division cycle mutant, raising the possibility that the different Xenopus transcripts are involved in animal and vegetal cell cycles. Embryonic expression is primarily in the cement gland. These new localized transcripts contribute to the general observation that the vegetal cortex, but not the animal cortex, is a specific site for RNA localization.