Examining post-transcriptional regulation of the segmentation clock by microRNAs

描述（申请人提供）：脊椎动物的胚胎轴由连续重复的元件组成，其中最明显的是体节。 体节发生过程中体节的正确产生对中轴骨架的形成至关重要。 体节发生的过程部分受到遗传时钟的调节。 Notch、Wnt和Fgf途径中的关键基因表现出周期性表达，其周期与体节形成速率相匹配。 扰乱这些基因的正常摆动的突变可导致中轴骨骼的先天性缺陷，包括脊椎肋骨发育不全。 因此，了解控制分段时钟功能的调节机制对我们理解骨骼发育至关重要。 几个小组已经研究了与分割时钟相关的振荡基因表达的转录控制，但对调节时钟功能的转录后机制知之甚少。 研究人员假设microRNA可能在与时钟相关的基因的转录后控制中发挥重要作用。 miRNA是小的保守RNA，其结合靶转录物并促进mRNA周转和/或抑制mRNA翻译。 她提出，这些miRNAs的功能可能有助于延迟负反馈调节环，而负反馈调节环是振荡RNA表达的基础。 她已经鉴定出富含与分段时钟中的潜在功能一致的模式的miRNAs，并发现这些miRNAs中的许多被预测靶向振荡基因的3 'UTR。 识别在分割时钟中起作用的miRNA将为理解分割时钟的转录后调控提供新的范例。 为了实现这一想法，将这些候选miRNAs与生物钟中的潜在功能联系起来的进一步初步数据至关重要。 因此，研究者提出在体节发生过程中通过验证这些候选miRNAs的富集来检查这些miRNAs，检查它们在整个胚胎发生过程中的表达模式，并功能性地测试它们靶向和调节振荡基因表达的能力。 这里提出的实验直接属于R 03计划的范围，因为它们代表了一个小型的，独立的项目，将为未来的研究提供关键的初步数据。 此外，这项研究福尔斯直接落入NICHD的使命，研究胚胎发生过程中基因表达的空间和时间控制的机制，以调节体节发生。 据预测，这里提出的研究将提供miRNA调控和分割时钟功能之间的关键联系，并将提供一个基础的初步数据，将支持进一步研究的实际功能的miRNA在体节发生以及分割时钟在未来的R 01应用。 相关性：产生脊柱和肋骨的胚胎发育过程由遗传时钟控制。 当这个时钟被扰乱时，结果可能是先天性缺陷。 这项研究检查了控制与这个时钟相关的基因表达的不同机制。 通过了解生物钟中基因的表达是如何控制的，我们将能够更好地针对生物钟失调引起的缺陷和疾病进行治疗。

DESCRIPTION (Provided by Applicant): The vertebrate embryonic axis consists of serially repeated lements, the most evident of which are the somites. The proper production of somites during somitogenesis is critical to the formation of the axial skeleton. The process of somitogenesis is regulated in part by a genetic clock. Key genes in the Notch, Wnt, and Fgf pathways exhibit cyclic expression with a period that matches the rate of somite formation. Mutations that perturb the proper oscillation of these genes can result in congenital defects of the axial skeleton, including spondylocostal dysostoses. Thus, understanding the regulatory mechanisms that control segmentation clock function is critical to our understanding of skeletal development. Several groups have examined the transcriptional control of oscillatory gene expression linked to the segmentation clock, but comparatively little is known about the post-transcriptional mechanisms that regulate clock function. The investigator hypothesizes that microRNAs may play important roles in the post-transcriptional control of genes linked to the clock. miRNAs are small conserved RNAs that bind to target transcripts and promote mRNA turnover and/or inhibit mRNA translation. She proposes that the function of these miRNAs may contribute to the delays in the negative feedback regulatory loops that underlie oscillatory RNA expression. She has identified miRNAs that are enriched in a pattern that is consistent with potential functions in the segmentation clock, and find that many of these miRNAs are predicted to target the 3'UTRs of oscillatory genes. Identifying miRNAs that function in the segmentation clock would provide a new paradigm to understand the posttranscriptional regulation of the segmentation clock. To pursue this idea it will be critical to generate further preliminary data connecting these candidate miRNAs to potential functions in the clock. The investigator therefore proposes to examine these miRNAs during somitogenesis by validating the enrichment of these candidate miRNAs, examining their expression patterns throughout embryogenesis, and functionally testing their ability to target and regulate expression of oscillatory genes. The experiments proposed here fall directly in the scope of the R03 program, as they represent a small, self-contained project that will produce critical preliminary data for future research. Further, this research falls directly into the mission of the NICHD, examining the mechanisms by which the spatial and temporal control of gene expression during embryogenesis functions to regulate somitogenesis. It is predicted that the research proposed here will provide critical links between miRNA regulation and segmentation clock function, and will provide a base of preliminary data that will support further research into the actual functions of miRNAs during somitogenesis as well as the segmentation clock in a future R01 application. RELEVANCE: The processes of embryonic development that produce the spine and ribs are controlled by a genetic clock. When this clock is perturbed, the results can be congenital defects. This research examines 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 misregulation of the clock.