The exclusion of germ cells from somatic cell fates in early development is an essential process in metazoans that ensures continuation of the species. Primordial germ cells (PGCs) execute at least four activities that are required to both protect them from somatic differentiation and to initiate their own unique gene expression programs: 1) activation of sequestered maternal germline mRNAs; 2) repression of maternal somatic messages; 3) transient genome-wide suppression of transcription to ensure that somatic programs are not activated when zygotic transcription initiates in the rest of the embryo; and 4) transcriptional activation of PGC-specific genes after degradation of maternal somatic mRNAs. All of these activities occur in the absence of transcription and therefore must be regulated primarily at the level of translation. The goal of the proposed project is to define key players in this intricate program, to uncover mechanistic details of their activities, and to construct a viable working model for the network that protects and specifies the germline. The RNA-binding proteins Nanos and Dazl are translational regulators in the germlines of diverse species, including frogs and humans. Both are translationally activated in PGCs by an unknown mechanism. Nanos and Dazl both interact with another (sequence-specific) RNA-binding protein, Pumilio (Pum), to regulate translation, but with opposite effects: Nanos represses translation of target RNAs, while Dazl promotes it. Our work in Xenopus has shown that PGCs lacking Nanos prematurely initiate Pol II transcription, inappropriately express somatic genes, and do not survive. Xenopus PGCs deficient in dazl fail to migrate to the primordial gonads and are lost from the germline. Key questions then are what activates Nanos and dazl, and the identities of their target mRNAs. Preliminary studies support a new role for the RNA-binding protein, Dead-end, as a translational activator of Nanos. Our working model is that preservation of the germline is initiated by Dnd, which activates the translation of Nanos and probably other germline mRNAs including dazl. Nanos then represses translation of maternal mRNAs essential for somatic fates, while Dazl promotes translation of RNAs that activate PGC-specific traits.
The aims of this project are: 1) to define the Dnd/RNA interactions that result in translational activation of Nanos and other potential RNA targets; the definitive test for relevance will be to reconstitute Nanos translation in a defined in vitro system with any required protein partners and Dnd; 2) to identify the maternal RNAs whose repression by Nanos/Pum is required to prevent aberrant expression of somatic RNAs; using a novel assay for translational regulation in PGCs, we will authenticate Nanos/Pum/RNA interactions in vivo; 3) to identify the maternal RNAs whose activation by Dazl is required for PGC identity; candidate mRNAs that co-immunoprecipitate with Dazl will be validated in vivo by assessing their abilities to restore PGC traits in Dazl-depleted embryos. With this information, we expect to be able to construct a relatively detailed working model for the regulation of germ-line fate in the earliest stages of development.
Our studies will contribute to identifying the gene products that control entry into the germline and that preserve the characteristics of totipotency, essentil information to addressing the causes of human infertility as well as the future use of pluripotent stem cells in the treatment of degenerative diseases such as diabetes, Parkinson's and cardiovascular disease.
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