Molecular mechanisms that regulate target cell sensitivity to Hedgehog morphogens

项目摘要/摘要 Hhedgehog(HH)信号对发育、再生、组织构型和细胞增殖是必不可少的。 和疾病。作为经典的形态因子，HH配体以一种依赖于 信号强度。信号强度的精确调节对于正常的组织发育和 维护，突出的事实是，即使是信号幅度的轻微变化也可能导致人类 先天缺陷。而大量的研究表明，信号强度受浓度的影响 以及HH配体的暴露时间，这一同样重要的调节层仍然没有被认识和研究： 靶细胞对形态因子的敏感性是如何调节的？在我博士后的第一阶段 在培训期间，我使用全基因组CRISPR筛查发现了三个能够减弱HH信号的基因 在目标细胞中：Mosmo、Megf8和Mgrn1(“MMM模块”)。两地的MMM模块均发生故障 成纤维细胞和神经前体细胞(NPC)对HH配体和 改变神经细胞命运的决定，由跨膜转导水平的显著增加驱动 主纤毛光滑(SMO)。这些研究导致了以下假设：MMM模块调节 通过调节HH途径组件的亚细胞定位在靶细胞中的信号强度。为了测试 这一假设，我将(1)确定MMM模块管理SMO贩运的机制，(2) 阐明泛素化在MMM调节的SMO贩运和HH敏感性中的作用，以及(3)确定 MMM模块在胚胎发育中的作用。这些研究将揭开分子基础 以及一种新机制的生理功能，该机制允许靶细胞修改其对 细胞外信号，并因此提出新的策略来调节疾病中的HH信号强度 各州。在研究生院，我接受了老鼠遗传学家和胚胎学家的培训。在我的第一阶段 博士后，我学会了如何使用CRISPR技术进行全基因组功能丧失筛查，并 在复杂的体外分化实验中测试特定基因的功能。来自K99的支持 计划，斯坦福大学可用的资源，以及我的顾问小组的专业知识，将使我能够 在高级显微镜、蛋白质生物化学和质谱学领域开发关键的新技能 了解MMM模块等发育调节因子的生化和生物学功能。这就做 通过我的导师Rajat Rohatgi博士(生物化学和癌症生物学)的培训，我的同事- 导师Tim Stearns博士(纤毛生物学)，以及由具有以下专业知识的成员组成的强大顾问小组 蛋白质运输、计算生物学、发育生物学和质谱学。培训和 我在K99/R00奖项期间得到的指导将为我实现我的 学术目标是建立一个充满活力的独立研究项目，能够回答 使用从小鼠遗传学到机械生物化学等方法的发育信号。

PROJECT SUMMARY/ABSTRACT Hedgehog (Hh) signaling is essential for tissue patterning and cell proliferation in development, regeneration, and disease. As classical morphogens, Hh ligands direct cell fate decisions in a manner dependent on signaling strength. Precise regulation of signaling strength is critical for proper tissue development and maintenance, highlighted by the fact that even modest changes in the signaling amplitude can result in human birth defects. While a large body of work has shown that signaling strength is influenced by the concentration and exposure time of Hh ligands, an equally important layer of regulation remains unrecognized and unstudied: How is the sensitivity of target cells to morphogens regulated? During the first phase of my postdoctoral training, I used genome-wide CRISPR screens to discover three genes that function to attenuate Hh signaling in target cells: Mosmo, Megf8, and Mgrn1 (the “MMM module”). Disruption of the MMM module in both fibroblasts and neural progenitor cells (NPCs) resulted in a ~10-fold increase in sensitivity to Hh ligands and altered neural cell-fate decisions, driven by a marked increase in levels of the transmembrane transducer Smoothened (SMO) at primary cilia. These studies lead to the hypothesis that the MMM module regulates signaling strength in target cells by regulating the sub-cellular localization of Hh pathway components. To test this hypothesis, I will (1) determine the mechanism by which the MMM module regulates SMO trafficking, (2) illuminate the role of ubiquitination in MMM-regulated SMO trafficking and Hh sensitivity, and (3) identify the function of the MMM module during embryonic development. These studies will unravel the molecular basis and physiological function of a novel mechanism that allows target cells to modify their responses to extracellular cues and consequently suggest new strategies to modulate Hh signaling strength in disease states. In graduate school, I trained as a mouse geneticist and embryologist. During the first phase of my postdoc, I learned how to use CRISPR technology to conduct genome-wide loss-of-function screens and to test the function of specific genes in sophisticated in vitro differentiation assays. Support from the K99 program, the resources available at Stanford University, and the expertise of my advisory panel will allow me to develop critical new skills in the areas of advanced microscopy, protein biochemistry, and mass spectrometry to understand the biochemical and biological function of developmental regulators like the MMM module. I will accomplish this with training from my mentor Dr. Rajat Rohatgi (biochemistry and cancer biology), my co- mentor Dr. Tim Stearns (cilia biology), and a strong advisory panel composed of members with expertise in protein trafficking, computational biology, developmental biology, and mass spectrometry. The training and mentorship I receive during my K99/R00 award will provide a critical stepping stone for me to achieve my academic goal of establishing a vibrant independent research program that can answer important questions in developmental signaling using approaches ranging from mouse genetics to mechanistic biochemistry.