Translational regulation of limb bud initiation

肢芽起始的翻译调控

• 批准号: 10686265
• 负责人: Can Cenik
• 金额: $ 23.78万
• 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/10686265
• 关键词: Cells; Data; Development; Developmental Process; Embryo; Epithelium; 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; Protein Biosynthesis; 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.

大多数常见发展系统的基因调节网络已在 使用mRNA表达作为主要读数的日益颗粒状细节。但是，mRNA表达水平是 与蛋白质水平的相关性不佳。人们认为，蛋白质水平无法解释的很大一部分被认为是 归因于翻译效率，每个mRNA合成的蛋白质数量。翻译 可以通过通过核糖体分析鉴定主动翻译的mRNA来获得调节的基因。在 该方法与常规基因表达技术结合使用，提供了一种计算方法 mRNA的翻译效率。但是，这种方法需要大量细胞（超过1个 百万）对于大多数体内发育系统的应用是不可行的。我们中的一个最近开创了 开发一种新型的微流体方法，用于核糖体分析称为Ribo-ITP，可提供高度 超低输入样品（〜100个细胞）的覆盖范围和高分辨率核糖体占用度测量值。 该方法即使在动态发展中也可以鉴定翻译调节的基因 细胞材料有限的系统。我们建议采用这种新方法来确定机制 调节肢体生长的初始阶段。此过程由FGF10介导，这对于 侧板中胚层的上皮到间充质转化（EMT）以及随后的肢体 芽的生长。我们假设肢体芽EMT和FGF10响应基因调节网络是 翻译监管。在特定目标1中，我们将确定翻译调节对肢体的贡献 芽上皮到间充质转化（EMT）。在特定目标2中，我们将确定转录和 FGF10介导的翻译网络，这是介导四肢芽启动的必要和足够的 通过知之甚少的机制。这些研究完成时，我们将表征该过程 EMT在肢体芽的启动中，并确定了翻译控制对这一过程的贡献。 此外，我们将确定FGF10介导的基因转录网络，并确定是否是否 它们受翻译的监管，实质上推进了我们对肢体芽启动的理解。共同 该提议有可能对我们对翻译的理解产生变革性的影响 调节会影响脊椎动物器官发生。

项目成果

Single-cell quantification of ribosome occupancy in early mouse development.

• DOI: 10.1038/s41586-023-06228-9
• 发表时间: 2023-06
• 期刊: NATURE
• 影响因子: 64.8
• 作者: Ozadam, Hakan;Tonn, Tori;Han, Crystal M.;Segura, Alia;Hoskins, Ian;Rao, Shilpa;Ghatpande, Vighnesh;Duc Tran;Catoe, David;Salit, Marc;Cenik, Can
• 通讯作者: Cenik, Can