Molecular mechanisms that regulate target cell sensitivity to Hedgehog morphogens

项目总结/摘要 Hedgehog（Hh）信号对于许多组织和器官的发育是必不可少的。Hh配体是 形态发生素，从而以依赖于信号强度的方式指导细胞命运决定。精确 这种信号强度的调节对胚胎发育至关重要，因为即使是轻微的干扰， 信号幅度可导致严重的出生缺陷。许多研究表明，信号强度 受形态发生剂浓度和形态发生剂暴露持续时间的影响。然而，同样 一个重要的调节层仍然没有被认识和研究不足：靶细胞如何调节其 对形态发生素的敏感性我博士后研究的中心焦点是研究 调节细胞对细胞外形态发生素的敏感性。最初，我使用全基因组CRISPR筛选， 发现三种新型Hh信号衰减剂：Mosmo，Megf 8和Mgrn 1。通过K99的支持，我 显示这三种蛋白质形成膜系泛素连接酶复合物（MMM复合物）， 通过清除细胞中的Hh效应物Smoothened（SMO）抑制细胞对Hh形态发生剂的敏感性 表面和初级纤毛。使用基因敲除小鼠模型，我还证明了MMM复合物是必不可少的， 以保证正确的左-右模式和胚胎心脏发育。总的来说，我的工作已经开始了（1） 揭示了一种新的机制，通过这种机制，靶细胞通过改变细胞外信号来改变它们对细胞外信号的反应， 蛋白质在细胞表面的景观和（2）阐明了复杂的机制 异位和先天性心脏病的遗传学。COVID-19相关研究限制和大学 关闭严重耽误了我的职业规划和个人发展。虽然我的研究进展反映了 成功完成了我最初建议中的目标2和3，COVID-19研究受到严重限制 Aim 1的进展延迟，该项目试图确定MMM复合体 调节蛋白质运输事件。一笔资金的延期将使我能够大规模地发展关键的新技能， 光谱和先进的显微镜，以了解如何MMM复合物调节贩运 蛋白质的细胞表面和初级纤毛。我将在我的导师Rajat博士的培训下完成这一目标 Rohatgi（生物化学），我的共同导师Tim Stearns博士（纤毛生物学）和Ryan Leib博士（蛋白质组学） 斯坦福大学质谱核心设施主任和我的科学顾问委员会成员）。一 延长拨款也将使我能够在罗柏芝博士的指导下进一步接受培训， 分析小鼠胚胎心脏的发育缺陷总之，培训和指导我将 在延长的K99期间收到的信息将使我具备过渡到研究所需的知识 导致心脏异位和先天性心脏缺陷的分子机制。最终这 培训将是至关重要的，在我过渡到一个独立的调查，并帮助我建立一个充满活力的 细胞信号传导和发育生物学领域的研究计划。

PROJECT SUMMARY/ABSTRACT Hedgehog (Hh) signaling is essential for the development of many tissues and organs. Hh ligands are morphogens and thus direct cell fate decisions in a manner dependent on signaling strength. Precise regulation of this signaling strength is critical for embryonic development, as even modest disruptions in the signaling amplitude can result in severe birth defects. Many studies have shown that signaling strength is influenced by both morphogen concentration and duration of morphogen exposure. However, an equally important layer of regulation remains unrecognized and understudied: How does a target cell regulate its sensitivity to a morphogen? The central focus of my postdoctoral research has been to study the mechanisms that modulate a cell’s sensitivity to extracellular morphogens. Initially, I used genome-wide CRISPR screens to discover three novel Hh signaling attenuators: Mosmo, Megf8, and Mgrn1. Through support from the K99, I showed that these three proteins form a membrane-tethered ubiquitin ligase complex (the MMM complex) that suppresses a cell’s sensitivity to Hh morphogens by clearing the Hh effector Smoothened (SMO) from the cell surface and primary cilium. Using knockout mouse models, I also showed that the MMM complex is essential for proper left-right patterning and embryonic heart development. Collectively, my work has begun to (1) unravel a new mechanism through which target cells modify their responses to extracellular cues by altering the landscape of proteins at the cell surface and (2) shed light on the mechanisms that underlie the complex genetics of heterotaxy and congenital heart defects. COVID-19 related research restrictions and university closures severely delayed my career plans and personal development. While my research progress reflects the successful completion of Aims 2 and 3 in my original proposal, COVID-19 research restrictions severely delayed progress on Aim1, which sought to determine the mechanism through which the MMM complex regulates protein trafficking events. A funding extension would allow me to develop critical new skills in mass spectrometry and advanced microscopy to understand how the MMM complex regulates the trafficking of proteins to the cell surface and primary cilium. I will accomplish this with training from my mentor Dr. Rajat Rohatgi (biochemistry), my co-mentor Dr. Tim Stearns (cilia biology), and Dr. Ryan Leib (the proteomics director of the Stanford mass spectrometry core facility and member of my scientific advisory committee). A funding extension would also allow me to further train under Dr. Cecilia Lo, to become more proficient at analyzing embryonic mouse hearts for developmental defects. In summary, the training and mentorship I will receive during the extended K99 period will equip me with the knowledge necessary to transition into the study of the molecular mechanisms that contribute to heterotaxy and congenital heart defects. Ultimately, this training will be critical in my transition into an independent investigator and help me establish a vibrant research program in the fields of Cell Signaling and Developmental Biology.