Netrin-mediated signaling directs central neuron dendrite growth and connectivity in the developing vertebrate visual system

Netrins are a family of extracellular proteins that function as chemotropic guidance cues for migrating cells and axons during neural development. In visual system development, Netrin-1 has been shown to play a key role in retinal ganglion cell (RGC) axon growth and synaptogenesis where presynaptic RGC axons form partnerships with the dendrites of tectal neurons. However, the signals that guide the dendrites of tectal neurons and facilitate connections between these postsynaptic cells and RGC axons are yet unknown. This work in collaboration with other lab members has explored the dynamic cellular mechanisms by which netrin-1 influences visual circuit formation, particularly those that impact postsynaptic neuronal morphology, connectivity, and function during retinotectal wiring. Immunohistochemistry reveals that netrin-1 and its receptors DCC and UNC5 are expressed in the optic tectum at the developmental stage when circuit formation occurs. Time-lapse in vivo imaging of individual Xenopus laevis optic tectal neurons co-expressing tdTomato and PSD95-GFP revealed rapid remodeling and reorganization of dendritic arbors following acute manipulations in netrin-1 levels. Within four hours of treatment, tectal injection of recombinant netrin-1 or sequestration of endogenous netrin with an UNC-5 receptor ectodomain induced significant changes in the directionality and orientation of dendrite growth and in the maintenance of already established dendrites away from the tectal neuropil, demonstrating that relative levels of netrin are important for these functions. In contrast, altering DCC-mediated netrin signaling with function blocking antibodies induced postsynaptic specialization remodeling and changed growth directionality by turning stable dendrites. Reducing netrin signaling also decreased avoidance behavior in a visually guided task, suggesting that netrin is essential for emergent visual system function. In vivo 2 Photon imaging examining the cell autonomous effects of translation blocking morpholino oligonucleotides to netrin receptors UNC5 and DCC in single cell electroporated neurons revealed altered neuronal morphologies, decreases in branch length and number, and changes to the directionality of treated dendritic arbors. Together the findings in the following chapters suggest netrin mediated DCC and UNC5 signaling play a critical role in the normal development of vertebrate central neuron dendritic arbors during the establishment of the functional visual system