Regulatory Mechanisms Governing Vertebral Segmentation

描述(申请人提供)：基因表达振荡在生物系统中广泛存在。分割时钟是控制脊柱分割的一个这样的振荡器。它的破坏会导致人类先天性脊椎缺陷。我们已经建立了一个基于转录反馈回路的起搏器数学模型。我们的模型预测，适度增加HER蛋白质或mRNAs的稳定性应该会延长这一周期，而进一步增加将消除这种振荡。为了建立HER蛋白和mRNA周转的机制，并阐明这种周期性信息传递到下一个形成片段的细胞的机制，我们将：1.发现快速回收振荡蛋白的翻译后机制：我们将对可能调节HER蛋白快速降解的蛋白质进行功能丧失实验。我们将评估这些基因中是否有任何基因调节HER家族蛋白的降解。最后，为了测试我们模型的预测，我们将通过减少引发其降解的蛋白质的表达来将她的蛋白质稳定到不同的水平，并测量分割过程中的相应变化。2.发现快速回收振荡RNA的转录后机制：我们将确定HER-RNA结合蛋白稳定HER mRNAs并导致脊椎节段缺陷的功能丧失。我们将通过减少RNA结合蛋白的表达来增加Her mRNAs的半衰期，并确定分割过程是如何受到影响的，以测试我们模型的预测。3.发现从分割时钟到分割机制的信息传递机制：我们将对MESP基因进行时间分辨的过表达和功能丧失实验，以确定其对分割过程的影响。我们将从片段时钟开始，通过动态表达的MESP转录因子持续到片段边界的形成来确定调控级联。HES蛋白也在神经前体细胞、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蛋白的功能增强与癌症相关，抑制HES蛋白的功能可以恢复分化。阐明脊椎分割过程中HES/HER振荡的动态变化不仅对于理解和潜在地预防脊椎畸形具有重要意义，而且对于开发各种组织中控制干细胞增殖和分化的方法以及抗癌进展的治疗都具有重要意义。因此，这项申请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.