Investigating the role of prdm14 in the avian germ cell lineage using a novel inducible dna transposon system.
Glover, James David
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Primordial germ cells (PGCs) are the precursors of the germ cell lineage that eventually differentiate into mature spermatozoa and oocytes. Although present throughout the animal kingdom, the specification and migration of PGCs differs widely between species. In vertebrates, avians are evolutionary divergent from mammals and therefore allow a comparative system in which to study germ cell development in higher organisms. Unlike mouse, PGCs can be isolated from the chicken embryo, expanded and cultured long term in vitro. Analysis of these cells showed that cultured chicken PGCs maintain the characteristics of their in vivo counterparts, including the expression of key germ cell specific markers and cell surface adhesion proteins, and thus, are an ideal system to study germ cell biology. Further characterisation revealed that an avian homologue of the zinc finger transcription factor PRDM14, essential for the specification of the mammalian germ cell lineage, was expressed in chicken PGCs. cPRDM14 was found to be expressed in PGCs in vitro and in vivo from early developmental stages until expression is lost by embryonic day 10 and subsequently re-expressed in the adult testis. The expression of cPRDM14 suggested that this gene may play a conserved role in the avian germ cell lineage. To investigate the function of cPRDM14, a novel single piggyBac transposon vector containing a reverse tetracycline activator protein and a tetracycline response element-regulated promoter was developed. Testing of the integrated transposon revealed that expression was tightly regulated and it was possible to conditionally express one gene product whilst simultaneously reducing the expression of a second gene, both in vitro and in vivo. This vector system was fully functional in PGCs, and was used to create transgenic founder chickens capable of having gene expression manipulated in germ cells at various developmental stages. Transgenic offspring were produced and the transgene was inducible at early developmental stages in the G1 animals. The un-induced transgene proved to be toxic to early embryos so a transgenic line of birds could not be produced. The inducible transposon was used to knockdown cPRDM14 expression in chicken PGCs. Knockdown of this gene led to reduced PGC numbers and increased cell death, both in vitro and in ovo. Expression of the pluripotency factor cNANOG was also significantly reduced which may explain the increased cell death. The knockdown of cPRDM14 also led to an increased susceptibility of PGCs to spontaneously de-differentiate to pluripotent embryonic germ cells (EGCs). cPRDM14 knockdown PGCs exhibited elevated levels of phosphorylated ERK, a target of the FGF signalling pathway. It was possible to prevent de-differentiation of the knockdown PGCs by removing ectopic FGF from the media. Furthermore, a sustained high level of FGF signalling in the media was sufficient to drive the de-differentiation of control PGCs to EGCs, suggesting that increased FGF signalling was key to the de-differentiation process. Extensive epigenetic remodelling of mouse PGCs occurs during embryonic development and PRDM14 was shown to be involved in this process. Chicken PGCs in vitro, contain several key histone modifications (H3K4me3, H3K9me2 and H3K27me3) and are 5-methyl cytosine (5-mC) positive. Immunohistochemical analysis of these markers in PGCs, at various stages during early embryonic development, suggests that these cells do not undergo the extensive epigenetic remodelling found in their mammalian counterparts. In contrast to the mouse germ cell lineage, knockdown of cPRDM14 in cultured PGCs had no noticeable effect on the epigenetic status of chicken PGCs. Together these results demonstrate that cPRDM14 is essential for the survival and maintenance of germ cell identity in chicken PGCs, but may not be critical for maintaining the epigenetic status of these cells.