Notch-mediated modulation of Sonic hedgehog signaling in neural fate specification and differentiation

项目摘要 在发育中的生物体中，复杂组织和器官系统的形成取决于以下能力： 分裂的干细胞和祖细胞以适当整合存在于胚胎中的细胞外信号 环境这些信号的联合作用反过来又指导不同的过程，如祖细胞分化， 维持、分化和分配特定的细胞命运。理解基础的关键一步 因此，预防出生缺陷的方法和手段在于确定不同的信号通路如何机械地 交叉，允许激活一个信号传导途径以影响对其他信号的反应。此外，委员会认为， 组合活动有可能从有限的范围扩大可能的成果范围， 发展信号。我们在脊髓发育中的研究发现了Notch的一个意想不到的作用， 受体信号传导调节细胞对组织形态发生素Sonic hedgehog（Shh）的反应。两 Notch通路的激活和失活改变了神经祖细胞的背腹侧记录， 神经元和神经胶质细胞命运的相应变化（Kong et al. Dev Cell，2015）。在追踪 在这些效应背后的机制，我们发现Notch信号影响Shh的运输， 受体Patched 1（Ptch 1）和关键的下游Shh效应器Smoothened（Smo）到初级纤毛，导致 下游Shh通路活动的变化。重要的是，Notch的这种作用可以在多个细胞中看到。 包括小鼠和人类神经祖细胞，成纤维细胞和骨骼肌成肌细胞，这表明它可能是 这是哺乳动物细胞的一般特征。总的来说，我们的研究揭示了一个新的和令人惊讶的近端作用， 在Shh形态发生蛋白梯度的解释中形成缺口，从而影响细胞命运 保持战略定力然而，Notch影响Shh信号蛋白运输的方式， 仍然未知。我们的初步数据表明，Notch的这些作用最有可能是转录的， 介导的，提出了Notch调控的靶基因是什么，以及它们如何影响肿瘤细胞的问题。 运输Shh通路成分，可能还有其他信号蛋白，初级纤毛？此外， 这种Notch介导的途径的缺陷导致影响纤毛运输的先天性缺陷， 称为纤毛病变我们提出的研究将解决这些问题，首先通过确定转录 Notch及其下游效应子Hes 1的靶点与纤毛运输的变化一致， 通过开发一个平台来研究Shh中这些新发现的Notch靶基因的功能 通过CRISPR/Cas9介导的缺失进行信号传导。通过这种方法，我们将：a）揭示 神经命运选择中Notch和Shh转导途径之间的功能交叉，和B） 鉴定Shh信号传导和蛋白质运输到初级纤毛的新调节剂。

PROJECT SUMMARY The formation of complex tissue and organs systems in developing organisms depends on the ability of dividing stem and progenitor cells to properly integrate extracellular signals present in the embryonic environment. The combined actions of these signals in turn direct diverse processes such as progenitor maintenance, differentiation, and assignment of specific cell fates. A key step towards understanding the basis of and means for preventing birth defect thus lies in defining how different signaling pathways mechanistically intersect, permitting the activation of one signaling pathway to influence responses to other signals. Moreover, the combinatorial activities have the potential to extend the range of outcomes possible from a limited range of developmental signals. Our studies in the developing spinal cord have identified an unexpected role for Notch receptor signaling in modulating the response of cells to the tissue morphogen Sonic hedgehog (Shh). Both activation and inactivation of the Notch pathway alters the dorsoventral register of neural progenitors, leading to corresponding changes in neuronal and glial cell fates (Kong et al. Dev Cell, 2015). In tracking down the mechanism behind these effects, we discovered that Notch signaling influences the trafficking of the Shh receptor Patched1 (Ptch1) and the key downstream Shh effector Smoothened (Smo) to primary cilia, leading to changes in downstream Shh pathway activities. Importantly, this role for Notch can be seen in multiple cell types including mouse and human neural progenitors, fibroblasts, and skeletal myoblasts, suggesting it may be a general feature of mammalian cells. Collectively, our studies reveal a novel and surprisingly proximal role for Notch in shaping the interpretation of the Shh morphogen gradient and thereby impacting cell fate determination. The means by which Notch influences the trafficking of Shh signaling proteins, however, remains unknown. Our preliminary data suggest that these actions of Notch are most likely transcriptionally mediated, raising the questions of what are the target genes regulated by Notch, and how do they impact the trafficking of Shh pathway components, and possibly other signaling proteins, to primary cilia? Moreover, do defects in this Notch-mediated pathway contribute to congenital defects affecting ciliary transport, collectively termed ciliopathies? Our proposed studies will address these questions, first by identifying the transcriptional targets of Notch and its downstream effector Hes1 that coincide with changes in ciliary trafficking, and second by developing a platform for investigating the function of these newly identified Notch target genes in Shh signaling through CRISPR/Cas9-mediated deletions. With this approach, we will: a) reveal the nature of the functional intersection between the Notch and Shh transduction pathways in neural fate selection and b) identify new regulators of Shh signaling and protein trafficking to primary cilia more generally.