High-resolution Analysis of Oocyte RNAs Reveals Gene Pathways Specific for the Vegetal Pole

TRISTÁN AGÜERO; DAWN OWENS; DEREK VAN BOOVEN; KAREN NEWMAN; MARY LOU KING

Pacific Grove

Conferencia; 15th International Xenopus Conference; 2014

Genetics Society of America

During Xenopus 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. To date, identification of localized maternal mRNAs has relied on microarray analysis, an approach limited toknown mRNAs.  Here we present our results using RNA-seq to identify, in an unbiased way, RNAs highly enriched at the vegetal pole. In situhybridization and qPCR were used to validate localized RNAs. Complete details of the RNA-seq procedures and parameters will be provided at themeeting. Vegetal and animal pole tips were cut from stage VI oocytes.  Samples from three different frogs were pooled for one set; a total of 3 setswere made from a total of nine frogs.  Data were aligned to the Xl. (v6.0) and Xt. (v7.1) annotated genomes, in addition to de novo sequencing. Overthree thousand transcripts were found differentially expressed between the animal and vegetal poles.  Reads aligning to a ribosomal specific referenceor mitochondria sequences represented <5% and 1.28% of the total respectively. The top 355 RNAs enriched >900 to 5-fold over the animal pole samplewere selected for GeneGo analysis. Protein-modifying enzymes, receptors, ligands, RNA binding proteins and 4 key transcription factors defining hubswere selected for functional analyses. 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.  Interestingly, known genes in neurogenicpathways were well represented.  miRNA analysis identified only 8 and all uniformly distributed. 16 localized mRNAs were identified containing at leastone recognition sequence conserved between Xt. and humans for these miRNAs. Early embryonic patterning appears not regulated by localizedmaternal miRNAs, but rather localized mRNAs.  Interestingly, 5 miRNAs are found densely clustered on the human X chromosome and are dysregulatedin cancers. (NIH HD072340 to MLK)