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

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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.