Translational regulation of limb bud initiation

肢芽起始的翻译调控

• 批准号: 10538940
• 负责人: Can Cenik
• 金额: $ 19.81万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-19 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10538940
• 关键词: Cells; Data; Development; Developmental Process; Embryo; Epithelial; FGF10 gene; Fibroblast Growth Factor; Gene Expression; Genes; Genetic Transcription; Hour; Lateral; Limb Bud; Limb Development; Limb structure; Malignant Neoplasms; Maps; Measurement; Mediating; Mesenchymal; Mesoderm; Messenger RNA; Methods; Microfluidics; Molecular; Mus; Neoplasm Metastasis; Organogenesis; Phase; Play; Process; Proteins; RNA; Regulator Genes; Resolution; Ribosomes; Role; Running; Sampling; System; Techniques; Technology; Testing; Tissues; Translating; Translational Regulation; Translations; Variant; cancer type; cell type; differential expression; gene network; gene regulatory network; high reward; high risk; improved; in vivo; mRNA Expression; novel; novel strategies; ribosome profiling; single-cell RNA sequencing; transcriptome sequencing

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.

大多数常见发育系统的基因调控网络的特征在于， 使用mRNA表达作为主要读数的越来越细的细节。然而，mRNA表达水平是 与蛋白质水平的相关性很差。人们认为，蛋白质水平上无法解释的变化中有很大一部分 翻译效率是指每个mRNA合成的蛋白质数量。平移地 可通过核糖体分析鉴定主动翻译的mRNA来获得调控基因。在 结合传统的基因表达技术，该方法提供了一种计算 mRNA的翻译效率。然而，这种方法需要大量的单元（多于1个 百万），这对于应用于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 开发一种新的微流体方法用于核糖体分析，称为Ribo-ITP， 从超低输入样品（~100个细胞）的覆盖率和高分辨率核糖体占有率测量。 该方法使得即使在动态发育中也能够鉴定出受调控的基因。 细胞材料有限的系统。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。这个过程是由FGF 10介导的，FGF 10对于细胞的生长是必需的。 侧板中胚层的上皮向间充质转化（EMT）以及随后的肢体 萌芽生长我们假设肢芽EMT和FGF 10反应基因调控网络是 严格监管。在具体目标1中，我们将确定翻译调节对肢体的贡献。 芽上皮至间充质转化（EMT）。在具体目标2中，我们将鉴定转录和 由FGF 10介导的翻译网络，其对于介导肢芽起始是必要且充分的 通过不太了解的机制。在完成这些研究后，我们将确定这一过程的特征 的EMT在肢芽启动，并确定了翻译控制的贡献，这一进程。 此外，我们将确定FGF 10介导的基因转录网络，并确定是否 它们受到神经调节，大大促进了我们对肢芽发育的理解。总的来说， 这一建议有可能对我们理解翻译如何产生变革性的影响， 调节影响脊椎动物器官发生。