Signaling receptor turnover by endocytic pathways in the patterning of the vertebrate nervous system

脊椎动物神经系统的模式是细胞多样性产生和大脑连接的关键事件。Sonic Hedgehog(Shh)信号被认为是一种“形态原”梯度，根据Shh信号沿背腹轴的浓度阈值指定不同的细胞类型。默认模型表明Shh是一种信号分子，在腹侧神经系统中以从高到低的梯度表达。它与细胞表面的一种称为Patched1(Ptch1)的受体结合，启动一系列细胞内事件，最终导致转录激活和细胞命运获得。正常情况下，在缺乏配体的情况下，ptch1阻止称为Smoothens的辅助受体的激活，这反过来又是通过Gli转录因子激活Shh途径所必需的。因此，ptch1作为Shh途径激活的负调节因子，Shh与ptch1的结合解除了这种抑制。然而，尚不清楚Shh信号如何在发育中的神经管等生长组织中实现分级反应。有待确定的是，ptch1如何帮助在发育中的脊椎动物神经系统中对Shh形态原场产生分级反应。 Hedgehog配体与ptch1的结合通过内吞途径触发它们的内化，最终到达溶酶体，在溶酶体中ptch1被降解。因此，设置Shh信号输出范围的一种方法是通过内吞作用控制ptch1的稳定水平。在果蝇和哺乳动物细胞中，已知泛素连接酶以ptch1为目标降解并允许Shh信号的激活。然而，ptch1内化和靶向内吞和溶酶体小泡的调控机制尚不清楚。在对新的ptch1相互作用蛋白的筛选中，我们发现了与胞内穿梭有关的蛋白，这些蛋白可能调节ptch1对细胞内小泡的靶向。这有望促进ptch1的降解，并增强发育中的神经系统中依赖Shh的腹侧模式。我们的目标是：(1)通过在细胞培养模型系统中的分析，验证这些假定的ptch1相互作用蛋白在内吞加工中的作用；以及(2)评估这些蛋白在神经发育过程中的过度表达和击倒是否可以改变腹侧模式以响应Shh信号。 这项拟议的研究解决了神经生物学中的一个基本问题：对形态原信号的分级反应是如何控制的？我们在易处理的模型系统中使用体外和体内方法相结合的方法来解决这个问题，提供了一个吸引人的基础细胞和发育生物学的培训机会。总之，这项研究将极大地促进我们对Shh信号的理解，并导致对模式是如何在脊椎动物发育的神经系统中产生的基本见解。

The patterning of the vertebrate nervous system is a key event in the generation of cell diversity and the wiring of the brain. It is thought that Sonic Hedgehog (Shh) signaling acts as a “morphogen” gradient specifying distinct cell types based on the concentration threshold of Shh signaling along the dorso-ventral axis. The default model states that Shh is a signaling molecule expressed in a gradient of high to low in the ventral nervous system. It binds to a receptor called Patched1 (Ptch1) at the cell surface in initiation an intracellular series of events that ultimately leads to transcriptional activation and cell fate acquisition. Normally, in the absence of ligand, Ptch1 prevents the activation of a co-receptor called Smoothened, which in turn is required for the activation of Shh pathway through the Gli transcription factors. Ptch1 therefore acts as a negative regulator for Shh pathway activation and the binding of Shh to Ptch1 relieves this inhibition. However, it is unclear how a graded response to Shh signaling is achieved in growing tissues such as the developing neural tube. What remains to be determined is how Ptch1 helps create a graded response to the Shh morphogen field in the developing vertebrate nervous system. The binding of Hedgehog ligands to Ptch1 triggers their internalization through the endocytic pathway and ultimately to the lysosome, where Ptch1 is degraded. Thus, one way to set the output range of Shh signaling is to control the steady-state levels of Ptch1 through endocytosis. In Drosophila and mammalian cells, it is known that ubiquitin ligases target Ptch1 to degradation and allow for the activation of Shh signaling. However, it is not known how Ptch1 internalization and targeting to endocytic and lysosomal vesicles is regulated. In a screen for novel Ptch1-interacting proteins, we identified proteins implicated in endocytic shuttling that may regulate the targeting of Ptch1 to intracellular vesicles. This is expected to promote Ptch1 degradation and enhance Shh-dependent ventral patterning in the developing nervous system. Our objectives are: (1) validate the role of these putative Ptch1-interacting proteins in endocyctic processing using assays in cell culture model systems; and (2) evaluate whether the over-expression and knockdown of these proteins during neural development can alter ventral patterning in response to Shh signaling. The proposed research addresses a fundamental question in neurobiology: how is the graded response to morphogen signaling controlled? We use a combination of in vitro and in vivo approaches in tractable model systems to address this question, providing an engaging training opportunity in basic cellular and developmental biology. Altogether, this research will significantly advance our understanding of the Shh signaling and lead to fundamental insights into how pattern is generated in the developing vertebrate nervous system.