Understanding cellular mechanisms that preserve totipotency or pluripotency in cells are central goals of both stem cell and developmental biology. Primordial germ cells (PGCs), precursors to the gametes, are the ultimate stem cell as they retain full developmental potential while other embryonic cells become progressively restricted in their fates. Our long term goal is to establish the key regulatory pathways that operate during the specification and differentiation of PGCs in the vertebrate model organism Xenopus. Genetic studies in Drosophila and C. elegans and molecular embryological studies in Xenopus have identified gene products in PGCs that appear to protect them from inappropriate somatic differentiation. How the identified gene products are """"""""networked"""""""" and importantly, the real mechanism through which these preserve totipotency, remain largely unknown. The present application will address this gap in our knowledge. Xenopus is the system of choice for these studies because it offers a unique combination of total accessibility at both the biochemical and embryological level. Based on our previous data, we hypothesize that during the time that germ layers are established in the embryo, totipotency is preserved in PGCs by a combination of targeted translational repression of specific maternal mRNAs and global repression of mRNA transcription controlled by Xcat2. To test our central hypothesis we will complete the following specific aims: 1) Determine how maternal VegT function is restricted in PGCs by assessing VegT stability, translation, and nuclear localization in isolated PGCs using a combination of Real Time RT-PCR, injected tagged VegT transcripts and immunolocalization. 2) Determine the role Xcat2 plays as a translational repressor in preserving PGC totipotency by identifying the RNAs that co-purify with Xcat2 using a pull-down, RT-PCR, and cloning approach. Mis-expression of these RNAs in PGCs will be tested for their effects on PGC fate. 3) Determine what mechanism is responsible for the transient repression of transcription in primordial germ cells by selecting for Xcat2 interacting factors that repress transcription in vivo functional assays. Relevance of this research to public health: Stem cells are of high therapeutic value because of their ability to develop into a wide variety of cell types that could be used in the treatment of degenerative diseases including diabetes, Parkinson's and cardiovascular disease. Our studies on PGCs, prime exemplars of totipotency, will explore how different mechanisms cooperate to preserve genetically naive or ground states in cells. Our findings will be relevant to how adult somatic stem cells maintain a pluripotent condition, important issues in stem cell biology. ? ? ?
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