High Through-put Analysis Reveals Novel Maternal Germline RNAs Critical for PGC Survival, Migration, and Differentiation

Xenopus laevis embryonic development requires the selective localization of maternal RNAs to the animal or vegetal pole. These RNAs include somatic and germline determinants, essential in establishing the embryo’s body plan. Such determinants are involved in primary germ layer formation and organization, dorsal/ventral axis specification, anterior/posterior axis specification, and germline development. In Xenopus laevis, germline development requires maternally inherited germplasm, a vegetally localized material that both specifies and protects primordial germ cells (PGCs) from adopting somatic fates throughout embryogenesis. The germline is unique in its ability to maintain heritable genetic material for many metazoans, including humans. It is, therefore, of great interest to understand what keeps the integrity of this cell population from becoming somatic cells. Previous studies utilizing microarray have confidently identified localized RNAs in the Xenopus laevis oocyte, but these studies were limited to known transcripts only. Therefore, we hypothesized that using high through-put RNA sequencing would identify novel vegetally localized transcripts that are required for PGC specification. In this dissertation, I present substantial computational data derived from our RNA-seq analysis on fully grown Xenopus laevis oocytes, revealing 411 (198 annotated) and 27 (15 annotated) mRNAs as being enriched at the vegetal and animal pole, respectively. Of the vegetally and animally enriched mRNAs, ninety were novel and over 4-fold enriched and six were over 10-fold enriched at the vegetal and animal poles, respectively. In addition to mRNAs, identified microRNAs were found to not be localized within our oocyte samples. We also show that within the vegetally enriched transcripts, we are able to construct a gene regulatory network (GRN) linking 47 genes with 5 defining hubs which identify protein-modifying enzymes, receptors, ligands, RNA-binding proteins, transcription factors and co-factors. To address and validate our vegetally localized transcript set for its inclusion of germ plasm mRNAs, we performed a comparison of our data with that of Butler et al. (2017), who recently identified enriched transcripts in zygotically active PGCs of Xenopus laevis. Over half of the 198 vegetally enriched transcripts were zygotically expressed in the PGC sample. Further similarity of both maternal and zygotic germ plasm containing samples was highlighted during GRN analysis. Each sample set generated GRNs containing the transcription factors sox7 and e2f1. Taken together, these data guided expression and functional studies for the investigation of germ plasm enriched RNAs in both sample types. Outside of computationally generated data, my dissertation work also provides expression and functional data for transcripts identified in maternal and/or zygotic germ plasm containing samples. Transcripts were chosen for Gain-of Function and Loss-of Function experiments by either anticipated biological function in PGC development or presence in our GRN. Whole mount in situ hybridization analysis confirmed 17 and 11 RNAs were localized to the vegetal pole and the zygotic PGC, respectively. Functional studies provided evidence that efnb1, sox7, e2f1, wwtr1, otx1, parn, p300, and rras2 are all involved in promoting the normal development of PGCs. Additional studies indicate novel functions for efnb1 and sox7 in PGCs, where ephrinB1 (efnb1) plays a role in migration and sox7 activity must be tightly regulated to maintain PGCs. Here, I present the initial and required steps for identifying germ plasm components involved in Xenopus laevis germline development. However, the full utility of my RNA-seq data has yet to be realized. The proposed pathways operating in both the vegetal pole and zygotic PGC samples highlight where future investigations will be most fruitful