Translational regulation of limb bud initiation

大多数常见发育系统背后的基因调控网络已被描述为 使用信使核糖核酸表达作为主要读数的越来越细粒度的细节。然而，mRNA的表达水平是 与蛋白质水平的相关性很差。蛋白质水平的不可解释的变化有很大一部分被认为 要归因于翻译效率，每个信使核糖核酸合成的蛋白质的数量。翻译过来就是 通过核糖体图谱鉴定主动翻译的mRNAs可以获得受调控的基因。在……里面 与传统的基因表达技术相结合，该方法提供了一种计算 MRNAs的翻译效率。但是，这种方法需要大量的单元格(多于1个 百万)，这对大多数体内发育系统是不可行的。我们中的一个人最近开创了 一种新的微流控核糖体分析方法Ribo-ITP的开发 来自超低输入样本(~100个细胞)的覆盖率和高分辨率核糖体占有率测量。 这种方法使得即使在动态发育过程中也能够识别翻译调控的基因 具有有限细胞材料的系统。我们建议应用这种新的方法来确定机制 调节肢体生长的初始阶段。这一过程是由FGF10介导的，FGF10对 侧板中胚层及其后肢的上皮向间质转化(EMT) 芽长出来了。我们假设肢芽EMT和FGF10反应的基因调控网络是 翻译上受管制。在具体目标1中，我们将确定翻译调控对肢体的贡献 芽上皮向间充质转化(EMT)。在具体目标2中，我们将确定转录和 FGF10介导的翻译网络是调控肢芽发生的必要条件和充分条件 通过鲜为人知的机制。在这些研究完成后，我们将确定这一过程的特征 并确定了翻译控制在这一过程中的作用。 此外，我们将确定FGF10介导的基因转录网络，并确定 它们受翻译调控，极大地促进了我们对肢芽启动的理解。总而言之， 这一提议有可能对我们对翻译如何 调节影响脊椎动物的器官发生。

Gene regulatory networks underlying most common developmental systems have been characterized in increasingly granular detail using mRNA expression as a primary readout. Yet, mRNA expression levels are poorly correlated with protein levels. A large component of the unexplained variation in protein levels is thought to be attributable to translation efficiency, the number of proteins synthesized per mRNA. Translationally regulated genes can be obtained by identifying actively translated mRNAs by ribosome profiling. In combination with conventional gene expression techniques, this approach provides a method for calculating the translational efficiency of mRNAs. However, this approach requires large numbers of cells (more than 1 million) that are unfeasible for application to most in vivo developmental systems. One of us recently pioneered the development of a novel microfluidic approach for ribosome profiling termed Ribo-ITP that delivers high coverage and high-resolution ribosome occupancy measurements from ultra-low input samples (~100 cells). This method enables the identification of translationally regulated genes even in dynamic developmental systems with limited cellular material. We propose to apply this new approach to determine the mechanisms regulating the initial phase of limb outgrowth. This process is mediated by FGF10, which is essential for the epithelial to mesenchymal transformation (EMT) of the lateral plate mesoderm as well as for subsequent limb bud outgrowth. We hypothesize that limb bud EMT and FGF10-responsive gene regulatory networks are translationally regulated. In specific aim 1, we will determine the contribution of translational regulation to limb bud epithelial to mesenchymal transformation (EMT). In specific aim 2, we will identify transcriptional and translational networks mediated by FGF10, which is necessary and sufficient for mediating limb bud initiation via poorly understood mechanisms. At the completion of these studies, we will have characterized the process of EMT in limb bud initiation and determined the contribution of translational control to this process. Furthermore, we will have identified the FGF10-mediated transcriptional network of genes, and determined if they are translationally regulated, substantially advancing our understanding of limb bud initiation. Collectively, this proposal has the potential to have a transformative impact on our understanding of how translational regulation influences vertebrate organogenesis.