Dissecting Ret receptor signaling in space-filling dendrite patterning in Drosophila

保守的受体酪氨酸激酶Ret（在转染过程中重排）在神经系统的发育、维持和疾病中起重要作用。尽管Ret已被大量研究，但其在神经系统发育中的功能和信号还不完全清楚。我们最近的研究表明，Ret在果蝇感觉神经元的一个子集的树突生长、动力学和粘附中起着重要作用。果蝇幼体周围神经系统是研究体内树突发育分子机制的有力模型，具有高度定型的神经元形态和可获得的共聚焦活体成像显微镜。这些神经元的感觉树突生长在表皮细胞层和细胞外基质（ECM）之间的二维环境中。最复杂的IV类（C4da）神经元以完整而非冗余的方式覆盖整个幼虫体壁，它们的发育严重依赖于Ret功能。我们已经证明，Ret是C4da神经元树突- ecm粘附所必需的，它与整合素形成功能复合物，并通过小GTPase Rac1发出下游信号。然而，Ret依赖的树突生长和动力学依赖于其他迄今尚未确定的分子线索。通过候选筛选和全基因组分析Ret缺陷C4da神经元，我们发现了连接Ret功能与TGFß信号传导的新配体和下游衔接蛋白。因此，我们假设果蝇Ret是一种tgf ß样受体，介导C4da神经元树突生长，以响应一种新的细胞外配体。我们将通过基因损失和功能分析，细胞和生化分析来研究这些新的相互作用。首先，我们的目标是通过生成CRISPR/Cas9介导的敲除和敲入细胞系来分析新的假定的Ret配体在C4da神经元树突发育中的功能。我们将广泛研究其在C4da神经元树突生长中的发育表达和功能。其次，我们将在体内和体外研究Ret和TGFß以及受体酪氨酸激酶信号传导之间的联系，以研究配体-受体相互作用和相关的信号传导途径。最后，我们将分析已鉴定的细胞内接头蛋白在Ret依赖性C4da神经元树突发育中的作用，以获得下游信号传导过程的分子和细胞视角。综上所述，我们的研究将解决一个新的Ret依赖信号机制所需的空间填充树突生长。

The conserved receptor tyrosine kinase Ret (Rearranged during transfection) plays a major role in nervous system development, maintenance and disease. Although Ret has been heavily studied, its function and signaling in nervous system development are only incompletely understood in vivo. We have recently shown that Ret plays a major role in dendrite growth, dynamics and adhesion in a subset of Drosophila sensory neurons. The Drosophila larval peripheral nervous system has been a powerful model to study molecular mechanisms of dendrite development in vivo, featuring highly stereotyped neuronal morphologies and accessibility to confocal live imaging microscopy. Sensory dendrites of these neurons grow in a 2-dimensional environment between an epidermal cell layer and the extracellular matrix (ECM). The most complex class IV (C4da) neurons cover the entire larval body wall with their dendrites in a complete yet non-redundant manner, and their development critically relies on Ret function. We have shown that Ret is required for dendrite-ECM adhesion in C4da neurons by forming a functional complex with integrins and downstream signaling by the small GTPase Rac1. However, Ret dependent dendrite growth and dynamics are relying on other so far unidentified molecular cues. By candidate screening and genome wide analysis of Ret deficient C4da neurons we have identified novel ligand and downstream adaptor proteins linking Ret function to TGFß signaling. We therefore hypothesize that Drosophila Ret is a TGFß-like receptor mediating C4da neuron dendrite growth in response to a novel extracellular ligand. We will investigate these novel interactors by genetic loss and gain of function analyses, cellular and biochemical assays. First, we aim to analyze the function of the novel putative Ret ligand in C4da neuron dendrite development by generating CRISPR/Cas9 mediated knockout and knock-in lines. We will extensively investigate its developmental expression and function in C4da neuron dendrite growth. Second, we will address the link between Ret and TGFß and receptor tyrosine kinase signaling in vivo and in vitro to investigate ligand-receptor interaction and the relevant signaling pathway. Lastly, we will analyze the role of the identified intracellular adaptor proteins in Ret dependent C4da neuron dendrite development to get molecular and cellular insight into the downstream signaling processes. Taken together, our studies will address a novel Ret dependent signaling mechanism required for space-filling dendrite growth.