CONICET | Buscador de Institutos y Recursos Humanos

The Xenopus oocyte is a highly polarized cell along the animal/vegetal axis. During oogenesis, specific maternal RNAs are localized to the vegetal pole that will determine germ layer identity, dorsal/ventral patterning, and the germ cell lineage in the embryo. Using RNA-seq, we have identified RNAs highly enriched at the vegetal pole. In situ hybridizationand qPCR served to validate localized RNAs. In this study, vegetal and animal pole tips were cut from stage VI oocytes and frozen immediately. Samples from 3 different frogs were pooled and comprised one sample set; a total of 3 sets were made from a total of 9 frogs. Data were aligned to the Xenopus laevis (v7.1) and Xenopus tropicalis (v7.1) annotated genomes. Using the 0.05 q-value cutoff determined by cuffdiff, 5,720 total transcripts were found differentially expressedbetween the animal and vegetal pools. Reads aligning to a ribosomal specific reference or mitochondria sequences represented <5% and 1.28% respectively. The top 197 RNAs enriched >4-fold over the animal pole sample were selected for GeneGo analysis. Of these, protein-modifying enzymes, receptors, ligands, and 4 key transcription factors defining hubs will be functionally tested in future experiments. The maternal transcription factor Creb1 was found in diverse gene pathways involving localized mRNA, suggesting its function as an embryonic determinant is based on its regulating expression of localized RNAs. Known genes in neurogenic pathways were well represented. miRNA analysis identified eight uniformly distributed in the oocyte. Interestingly, all are up-regulated in cancers and found clustered on the human X chromosome., Sixteen vegetally localized mRNAs contained at least one recognition sequence conserved between Xt. and humans for these miRNAs. Our data suggest that early embryonic patterning is not regulated by localized maternal miRNAs, but rather localized mRNAs. Funding from NIH GM102397; HD072340.

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