Molecular mechanisms that regulate target cell sensitivity to Hedgehog morphogens

调节靶细胞对刺猬形态发生素敏感性的分子机制

• 批准号: 10732871
• 负责人: Jennifer Kong
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10732871
• 关键词: Affect; Animals; Area; Attenuated; Award; Biochemical; Biochemistry; Biological Assay; Biological Process; Biology; Brain; CRISPR screen; CRISPR/Cas technology; Cancer Biology; Cell Differentiation process; Cell Proliferation; Cell surface; Cells; Cilia; Complex; Computational Biology; Congenital Abnormality; Cues; Defect; Degenerative Disorder; Development; Developmental Biology; Disease; Embryo; Embryonic Development; Erinaceidae; Event; Fibroblasts; Future; Genes; Goals; Heart; Human; Impairment; In Vitro; Integral Membrane Protein; Lead; Learning; Ligands; Limb structure; Lung; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Membrane; Mentors; Mentorship; Microscopy; Microtubules; Minor; Molecular; Mus; Mutation; Natural regeneration; Neurons; Outcome; Pathway interactions; Pattern; Phase; Physiological; Play; Postdoctoral Fellow; Proliferating; Protein Biochemistry; Proteins; Regulation; Research; Resources; Role; Set protein; Signal Transduction; Spinal Cord; Testing; Time; Tissues; Training; Transducers; Translating; Ubiquitination; Universities; Work; bone; congenital anomaly; extracellular; gene function; genome-wide; graduate school; loss of function; member; morphogens; mouse genetics; mutant; neglect; nerve stem cell; novel; post-doctoral training; programs; protein transport; response; skills; targeted treatment; tissue regeneration; trafficking; ubiquitin-protein ligase

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.

项目总结/摘要 Hedgehog（Hh）信号传导对于发育、再生中的组织图案化和细胞增殖是必不可少的， 和疾病作为经典的形态发生剂，Hh配体以依赖于细胞生长的方式指导细胞命运决定。 信号强度信号强度的精确调节对于适当的组织发育和组织分化是至关重要的。 维持，突出的事实是，即使是信号幅度的适度变化也会导致人类 出生缺陷虽然大量的工作表明，信号强度受到浓度的影响， 和Hh配体的暴露时间，同样重要的调节层仍然未被认识和研究： 靶细胞对形态发生素的敏感性是如何调节的？在我博士后的第一阶段， 在培训中，我使用全基因组CRISPR筛选发现了三个能够减弱Hh信号传导的基因 在靶细胞中：Mosmo、Megf 8和Mgrn 1（“MMM模块”）。MMM模块的中断， 成纤维细胞和神经祖细胞（NPC）导致对Hh配体的敏感性增加约10倍， 改变神经细胞命运的决定，由跨膜转导子水平的显著增加驱动 初级纤毛平滑（SMO）。这些研究导致了MMM模块调节 通过调节Hh途径组分的亚细胞定位来增强靶细胞中的信号强度。测试 这个假设，我将（1）确定MMM模块调节SMO贩运的机制，（2） 阐明泛素化在MMM调节的SMO运输和Hh敏感性中的作用，以及（3）确定 MMM模块在胚胎发育过程中的功能。这些研究将揭开 和生理功能的一种新的机制，允许靶细胞修改他们的反应， 细胞外的线索，从而提出了新的策略，以调节Hh信号强度的疾病 states.在研究生院，我是一名老鼠遗传学家和胚胎学家。在我的第一阶段 在博士后期间，我学会了如何使用CRISPR技术进行全基因组功能缺失筛查， 在复杂的体外分化试验中测试特定基因的功能。支持K99 计划，斯坦福大学的可用资源，以及我的顾问小组的专业知识将使我能够 在先进的显微镜，蛋白质生物化学和质谱领域发展关键的新技能 了解发育调节因子（如MMM模块）的生化和生物功能。我会 在我的导师Rajat Rohatgi博士（生物化学和癌症生物学）的培训下，我完成了这一目标， 导师蒂姆·斯特恩斯博士（纤毛生物学），以及一个强大的顾问小组，由具有以下专业知识的成员组成： 蛋白质运输、计算生物学、发育生物学和质谱分析。培训和 我在K99/R 00奖励期间获得的指导将为我实现我的目标提供重要的垫脚石。 建立一个充满活力的独立研究计划，可以回答重要的问题， 使用从小鼠遗传学到机械生物化学的方法进行发育信号传导。