The vertebrate segmental body plan is most evident in the organization of the axial skeleton into the repeated structures of the vertebrae and ribs. The embryonic precursors of these structures are the somites, which are transient structures that bud sequentially from an embryonic structure called the presomitic mesoderm (PSM). In the last decade, several genes and signaling pathways have been identified that function within a genetic clock that times the process of somitogenesis (the segmentation clock). Genes in this clock exhibit cyclic transcription, and their gene products oscillate with a period that is equal to the period of somite formation. Dysregulation of clock-linked genes lead to human congenital defects affecting the axial skeleton. In order for oscillatory genes to play a functional role in the clock, their periodic transcription must be coupled to mechanisms that direct rapid turnover of the gene transcript during the off stage of the clock. Hes7 is a critical clock component, and has been suggested to influence the clock period. Models suggest that the half life of the Hes7 transcript influences the clock period, and the work proposed here focuses on understanding this level of post-transcriptional regulation. The mechanisms that regulate transcript turnover in the clock are not well understood. Identifying and clarifying these mechanisms will provide critical data allowing better understanding of the function and regulation of the clock. We find that the Hes7 transcript undergoes alternative polyadenylation in the mouse PSM, and propose two aims to test the functional relevance of this alternative polyadenylation. We will define the utilization of different pA sites in the PSM and examine the effects of different 3'UTR isoforms on transcript stability and translational efficiency. To address potential mechanisms that could provide differential regulation of specific Hes7 isoforms, we will examine functional roles for miRNAs in transcript regulation and will use a zebrafish system to address the conservation of post-transcriptional regulatory mechanisms across vertebrates. Completion of these aims will provide a more complete understanding of the post-transcriptional regulation that controls the stability and turnover of clock-linked transcripts. This will provide important data about regulated RNA stability mechanisms, and provide information relevant to future scientific and mathematical models of the segmentation clock. Finally, we will lay the groundwork for future research into how specific polyadenylation decisions are made, and to what extent alternative UTRs contribute to normal development.
The production of the spine and ribs is controlled by a genetic clock that acts during embryonic development. When the functions of genes in this clock are perturbed, the results can be congenital defects including skeletal deformities such as scoliosis. The research proposed here will examine different mechanisms that control the expression of genes that have been linked to this clock. By understanding how the expression of genes in the clock is controlled, we will be better able to target treatments for defects and diseases that arise from dysregulation of the clock.
