Mechanisms of Dcc Receptor Signaling in Neuronal Morphogenesis

PROJECT SUMMARY Determining how neurons are assembled into functional circuits will provide insight into developmental disorders of the nervous system and may suggest therapeutic approaches to promote nerve regeneration. To navigate to their correct targets, axons must precisely modulate their responses to extracellular cues. For example, to cross the midline commissural axons must first respond to attractive cues while preventing the response to repulsive cues. After entering the midline, axons switch their response to allow them to be repelled away from the midline to establish connections that are essential for coordinated sensory and motor behavior. Netrin attracts commissural axons to the midline in both invertebrates and vertebrates through receptors of the Deleted in Colorectal Carcinoma (DCC) family (encoded by frazzled (fra) in the fly). We have shown that Fra (and likely DCC) signals in two ways to direct axon guidance across the midline. First, Fra responds to its canonical Netrin ligand to promote local cytoskeletal rearrangements and second, Fra acts independently of Netrin to directly regulate gene transcription. There are three major knowledge gaps in our understanding of Fra and Dcc signaling mechanisms that this proposal will address: 1) the molecules that coordinate Netrin- dependent regulation of the actin cytoskeleton during axon attraction in vivo are incompletely defined, 2) many of the factors that control the transcriptional activity of Fra (and Dcc) are unknown, 3) the downstream target genes that are regulated by Fra's transcription factor function and how they contribute to Fra and Dcc- dependent processes are largely unknown. Three complementary sets of preliminary findings underpin our proposed research. First, analysis of pathogenic variants of human DCC (hDCC) have led to the discovery of an essential and conserved role of the Wave Regulatory Complex (WRC) in Netrin-dependent axon attraction. Second, we have completed an in vivo proteomic screen for Fra interacting proteins and have identified and validated several novel interacting proteins and pathways that are likely to contribute to Fra-dependent signaling. Third, we have performed RNA sequencing in embryonic neurons isolated from Fra loss of function and gain of function conditions and have identified a set of 15 reciprocally regulated transcriptional targets. This preliminary data provides the foundation and rationale for the experiments that we are proposing to 1) define how Fra/Dcc signaling recruits and regulates the WRC during axon guidance, 2) define how the COP9 complex works with Fra to regulate transcription and 3) define the contribution of Fra's transcriptional targets to axon and dendrite targeting. Our research will identify new molecular components of both Netrin-dependent and Netrin-independent Fra/Dcc signaling and may inform future studies of these signaling pathways in both neuronal and non-neuronal contexts and may offer insights into pathological conditions in humans that are caused by aberrant DCC function.