Localized maternal mRNAs can provide the molecular basis for regional information that patterns the early embryo. We have identified the vegetal cortex as a region in which localized maternal RNAs are concentrated and have isolated seven of them. Four of these appear to be involved in two critical pathways one specifying the dorsal axis and the other establishing the germ cell lineage. Xcat-2, Xcat-3 and B6 are components of the germ plasm and are inherited exclusively by the primordial germ cells (PGCs). Bei is a putative transcription factor related to brachyury (T-gene) that can induce a secondary dorsal axis. In this proposal experiments are outlined to: (1) determine the spatial and temporal pattern of RNA localization and protein synthesis for Xcat- 2, Xcat-3 and B6 in relation to PGC development, migration, and differentiation; (2) determine the function of Xcat-2, Xcat-3 and B6 as components of the germ plasm in germ cell specification; (3) identify other genes expressed in the germ plasm of PGCs; (4) determine the spatial and temporal pattern of bei expression and synthesis in relation to its nuclear entry, cortical rotation, and dorsalizing activity; and (5) establish whether bei activates the transcription of the early response genes involved in patterning the mesoderm in Xenopus. The general strategy will be to establish the expression pattern at the RNA and protein level for Xcat-2, Xcat-3, B6 and bei using whole mount in situ hybridization, immunohistological analysis, and RNase protection. Loss of function, overexpression, and ectopic expression phenotypes of these genes will be studied to determine their possible functions during development. By studying localized RNAs we hope to understand how they direct axial patterning in the early embryo and establish the future germ line. How pluripotency is maintained in the germline as it differentiates remains unknown and is important to studies of human organ regeneration and cancer. Our studies should also provide insights into how the dorsal axis (neural tube, hind brain, correct alignment of somites) is determined in vertebrates. Such information will allow an understanding of what genes may be involved in birth defects which affect this axis.
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