Regulatory Mechanisms Governing Vertebral Segmentation

描述（由申请人提供）：基因表达振荡在生物系统中广泛存在。分割时钟就是这样一个控制脊柱分割的振荡器。它的破坏导致人类先天性椎体缺陷。我们建立了一个基于转录反馈回路的数学起搏器模型。我们的模型预测，适度增加Her蛋白或mrna的稳定性应延长周期，而进一步增加将消除振荡。为了建立Her蛋白和mRNA转换的机制，并阐明将这种周期性信息传递给下一个形成片段的细胞的机制，我们将：1。发现快速循环振荡蛋白的翻译后机制：我们将对调节Her蛋白快速降解的候选蛋白进行功能丧失实验。我们将评估是否有任何这些基因调节her家族蛋白的降解。最后，为了验证我们模型的预测，我们将通过减少触发其降解的蛋白质的表达来稳定Her蛋白到不同的水平，并测量分割过程中的相应变化。2. 发现快速循环振荡rna的转录后机制：我们将确定her- rna结合蛋白稳定其mrna并导致椎体分割缺陷的功能丧失。我们将通过减少rna结合蛋白的表达来增加她的mrna的半衰期，并确定分割过程是如何受到影响的，从而测试我们模型的预测。3. 发现从分割时钟到分割机器的信息传递机制：我们将对mesp基因进行时间分辨过表达和功能丧失实验，以确定其对分割过程的影响。我们将确定从片段时钟开始的调控级联，继续通过动态表达的mesp转录因子，并以片段边界的形成结束。Hes蛋白也在神经祖细胞、卵巢细胞和胚胎干细胞中振荡，这些振荡似乎控制着从增殖到分化的时间转换。Hes蛋白的功能获得与癌症有关，其抑制可恢复分化。阐明椎体分割过程中he /Her振荡的动力学不仅对理解和潜在地预防椎体畸形具有重要意义，而且对开发各种组织中受控干细胞增殖和分化以及治疗癌症进展的方法也具有重要意义。因此，该应用程序与国家卫生研究所的使命有很强的相关性。

DESCRIPTION (provided by applicant): Gene expression oscillations are widespread in biological systems. The segmentation clock is one such oscillator controlling segmentation of the vertebral column. Its disruption results in congenital vertebral defects in humans. We have built a mathematical pacemaker model that is based on a transcriptional-feedback loop. Our model predicts that a moderate increase in the stabilities of Her proteins or mRNAs should lengthen the period, while a further increase would abolish the oscillations. To establish the mechanisms of Her protein and mRNA turnover, and to elucidate the mechanism that transfers this periodic information to cells of the next- forming segments, we will: 1. Discover the post-translational mechanism that rapidly recycles oscillating proteins: We will perform loss-of- function experiments for proteins that are candidates to regulate rapid degradation of Her proteins. We will assess whether any of these genes regulate the degradation of the Her-family proteins. Finally, to test the prediction of our model, we will stabilize Her proteins to varying levels by reducing expression of proteins that trigger its degradation and measure corresponding changes in the segmentation process. 2. Discover the post-transcriptional mechanism that rapidly recycles oscillating RNAs: We will determine loss- of-function of which her-RNA-binding proteins stabilize her mRNAs and result in vertebral segmentation defects. We will test the prediction of our model by increasing the halflives of her mRNAs through reduced expression of RNA-binding proteins and determining how the segmentation process is affected. 3. Discover the information transfer mechanism from the segmentation clock to the segmentation machinery: We will perform time-resolved overexpression and loss-of-function experiments for the mesp gene to determine its impact on the segmentation process. We will determine the regulatory cascade starting with the segmentation clock, continuing through the dynamically expressed mesp transcription factor and ending with the formation of segment boundaries. Hes proteins also oscillate in neural progenitor cells, ovary cells and embryonic stem cells, where the oscillations appear to control the temporal switch from proliferation to differentiation. Gain-of-function of Hes proteins is correlated with cancer and their inhibition restores differentiation. Elucidating the dynamics of the Hes/Her oscillations during vertebral segmentation is significant not only for understanding and potentially preventing vertebral malformations, but also for developing approaches for controlled stem cell proliferation and differentiation in various tissues and therapies against cancer progression. Therefore, this application has strong relevance to the mission of the National Institute of Health.