Netrin Guides Spinal Commissural Axons to the Midline at Long Range Through the Receptors DCC and Neogenin

During neurodevelopment, commissural axons are guided to the ventral midline in a remarkably precise and stereotyped way. Netrin, a secreted laminin-related protein, provides the major attractive cue for midline guidance. It is thought to act at long-range, functioning either in solution (chemotaxis) or bound to surfaces (haptotaxis). A gradient of Netrin-1 along the dorsal-ventral axis of the spinal cord is thought to attract commissural axons to the midline, with the floor plate being a major source of diffusible Netrin. However, this view has recently been challenged. To address this controversy and determine what role, if any, floor plate-derived Netrin-1 plays in commissural axon guidance, we quantitatively examined the phenotypes of mice specifically lacking floor plate Netrin-1 expression. We observed that the loss of floor plate-derived Netrin-1 cause commissural axons to improperly project through the ventral motor column and resulted in fewer commissural axons that cross the ventral midline. The precrossing guidance defects observed at-a-distance from the floor plate supports the operation of Netrin as a long-range chemotropic factor. To complement these studies, we investigated the differential roles for Netrin-1 receptors, Dcc and Neo1. How these two Netrin receptors collaborate during midline crossing has yet to be fully examined. Using transgenic embryos that express Cre-recombinase within discrete spinal interneuron populations in combination with fluorescent reporter lines, we show that midline guidance of all commissural interneuron populations wholly depends on Netrin-1 signaling through the Dcc and Neo1 receptors. However, the genetic deletion of Dcc more severely perturbed midline guidance of the dorsal commissural neuron population compared to ventral interneurons, which predominantly express Neo1. The two populations differ in Dcc and Neo1 expression, both in terms of abundance and splice isoforms, and one of these differences could account for their differential dependence on Dcc for midline attraction. To gain better insight into other genes that regulate midline guidance of commissural neurons, I generated a novel Robo3Cre/+ mouse line to use in combination with fluorescent reporter lines to purify commissural neurons from embryonic spinal cord. Transcriptome analysis of isolated commissural neurons identified RGMb, a Neo1-specific ligand that is highly expressed by dorsal commissural neurons that could modulate Netrin signaling. Additionally, I profiled the transciptome of embryonic floor plate and characterized several floor plate-specific secreted proteins that were homologous to known guidance cues. The commissural neuron and floor plate transcriptomes will provide an invaluable starting point for testing the role of candidate guidance factors. The body of work performed here has reaffirmed the long-range nature of Netrin’s attractive effect, showed that distinct neuronal populations express unique levels and isoforms of Dcc and Neo1 to achieve a common guidance outcome, and identified candidate factors that may collaborate with Dcc/Neo1 and Netrin in midline guidance