Mechanisms of Axon Guidance Receptor Regulation and Signaling during Midline Crossing

During development, axon guidance receptors (AGRs) guide axons to their appropriate targets to form specific connections and establish functional neural circuits. AGRs are expressed on the cell surface in precise spatial and temporal patterns where they induce turning in axons by modulating the underlying actin cytoskeleton. However, the intracellular trafficking pathways that regulate AGR expression and the downstream effectors that serve to link AGRs to the cytoskeleton are still being elucidated. In Chapter 1, we provide new insights into the regulatory mechanisms and intracellular signaling pathways of AGRs that can diversify signaling outputs. In Chapters 2 and 5, we present data identifying the Wave regulatory complex (WRC) as a direct downstream effector of the AGRs Roundabout (Robo) and Frazzled (Fra). The WRC is a nucleation-promoting factor that drives Arp2/3-mediated branched actin polymerization. The WRC binds to a WRC-interacting receptor sequence (WIRS) in the cytoplasmic tails of Robo and Fra, and can serve as a direct link to the actin cytoskeleton. The WIRS-WRC interaction is important for both Robo’s and Fra’s function in the Drosophila nerve cord and also for vertebrate Robo1 and Dcc signaling. We thus demonstrate an essential and conserved role for the WIRS-WRC interaction in AGR signaling. In Chapters 3 and 4, we characterize the Ndfip-Nedd4 ubiquitin ligase pathway as a key mechanism for regulating Robo1 expression levels in the mammalian spinal cord. In Drosophila, the endosomal sorting receptor Commissureless (Comm) serves to downregulate Robo1 levels on commissural axons during midline crossing. As Comm is not conserved in vertebrates, the mechanism controlling Robo1 expression in the mammalian spinal cord is unknown. We show that Ndfip proteins recruit Nedd4 E3 ligases to promote the ubiquitylation and subsequent degradation of Robo1 receptors. Ndfip and Nedd4 proteins are expressed in the developing spinal cord and Ndfip and Nedd4 knockout mice show a significant reduction in midline crossing. In addition, loss of Ndfip proteins leads to the upregulation of Robo1 expression in pre-crossing commissural axons. We thus identify a long-sought mechanism that functions analogously to Drosophila Comm to regulate mammalian Robo1 and prevent premature Slit response during midline crossing. In Chapter 6, I explore the implications of these findings and discuss future research avenues