Transcriptome analysis of Primordial Germ Cells of birds

Abstract

Primordial germ cells (PGCs) are germline competent cells which form the functional gametes of the animal. The potential usage of avian PGCs in producing genetically modified birds has driven research in the derivation, culturing, and genetic manipulation of PGCs. In chicken blastoderm, approximately 50 PGCs are present which proliferate in both male and female embryos until stage HH34 (day 8) and subsequently follow different differentiation pathways in male and female gonads. I investigated the hypothesis that chicken migratory stage PGCs are not initially determined to an oocyte or spermatogonial fate. To understand the differences in genetic mechanisms between male and female chicken PGCs, I studied the RNA transcriptome of PGCs from chicken. Analysis of RNA-Seq data of chicken PGCs reveals transcriptome divergence between the male and female cells and identified 150 differentially expressed genes (DEGs). The cultured female PGCs showed higher expression of cell adhesion genes like NCAM2 and PCDH9, and SMAD7B than male PGCs and also showed that dosage compensation is not maintained throughout the Z sex chromosome.

To identify novel germ cell and stem cell factors in avian PGCs, I compared the transcriptome of chicken PGCs with immortalized chicken cell lines. As a result, a set of genes were identified which are specific to germ cells including DAZL, DDX4, DDX43, PNLDC1, DMRT1, DMRTB1, and FKBP6.

This analysis also helped to identify a suite of pluripotency genes expressed in PGCs: NANOG, OCT4, LIN28, SOX3, GNOT1, TGIF2, PRDM14 and many others.

Furthermore, a cross-species transcriptome comparison between in vitro cultured chicken and goose PGC transcriptomes revealed that the expression of these sets of germ cell-specific genes and pluripotent genes expression is conserved in PGCs from these two avian species. This study also revealed the contrasting gene regulatory networks involved in the selfrenewal are active in chicken and goose PGCs. Chicken PGCs exhibit expression of both Activin and BMP signalling pathway genes whereas BMP signalling pathway genes are active in goose PGCs.

PRDM14 belongs to the family of the transcription factors containing a conserved N-terminal SET regulatory domain. In mouse, Prdm14 gene expression is limited to the pluripotent cells and essential for the development of the germ cell lineage. In chicken, the PRDM14 knockout embryos do not form a primitive streak. I characterized germ cell development in PRDM14 null chicken embryos and found that PRDM14 has a crucial role in the survival and maintenance of germ cells. Extending my transcriptome analysis to wild-type and PRDM14 null embryos identified DEGs and regulatory pathways possibly responsible for the gastrulation phenotype in the null embryos.