The establishment of neural circuits requires neurons to extend axons over considerable distances, using molecular cues in the embryonic environment to orient their growth cones. The textbook example of a guidance factor is netrin1, a member of the laminin superfamily first characterized in the spinal cord. Classic studies suggested that netrin1, produced by floor plate (FP) cells, acts by chemotaxis, diffusing over long distances to guide Dcc+ commissural axons towards the FP. Netrin1 was then implicated in guidance decisions throughout the nervous system. However, our studies have recently demonstrated that the key source of netrin1 is neural progenitor cells (NPCs) in the spinal ventricular zone. Rather than acting as a long-range diffusible gradient, our data supports a model in which NPC-derived netrin1 acts locally, forming a directional path for axons that also promotes axonal fasciculation. The bipolar geometry of NPCs permits them to deposit netrin1 at the basal (pial) margins of the spinal cord. This netrin1+ substrate then acts locally to direct fasciculated, ventral axon extension by haptotaxis, the directed growth of cells along an adhesive surface. However, netrin1 may mediate a more complex activity for axons beyond pure adhesion: netrin1 is then deposited onto Dcc+ axons, and these axons then grow precisely around the border of VZ, i.e. a netrin1- expressing domain. Ablating a small region of netrin1 expression causes axons deviate from their trajectory to follow the ectopic netrin1 boundary. We propose to call this collection of activities a ?hederal? growth boundary, from the analogy of a wall supporting the growth of ivy (genus: hedera) that is not itself penetrated by the ivy. Netrin1 can form a local growth substrate, and promote the fasciculated growth of axons around a netrin1-expressing domain. This reinterpretation of the mechanistic basis by which netrin1 functions, explains why little progress has been made using soluble netrin1 as a regenerative factor. To further this goal, we will assess [1] the mechanisms by which spinal NPCs establish a netrin1+ haptotactic substrate that promotes directed, fasciculated axonal growth in Aim 1, [2] whether NPC vs FP cells have distinct roles producing netrins in Aim 2 and [3] whether netrins have haptotactic activities in axon guidance in the forebrain in Aim 3.
Aim 1 : Determine the mechanism by which spinal NPCs establish netrin1 ?hederal? boundaries Hypothesis: Spinal NPCs traffic netrin1 to the pial surface to establish a haptotactic substrate. Netrin1 then transfers to Dcc+ axons to promote their fasciculated axon growth around netrin1-expressing regions.
Aim 2 : Determine the range role of FP-derived netrin1 in the spinal cord Hypothesis: FP-derived netrin1 acts over short range to fasciculate commissural axons crossing the midline Aim 3: Determine the role of NPC-derived netrin1/3 in the forebrain Hypothesis: Neural progenitors in the developing forebrain use the netrin family to establish growth boundaries that guide axons between the diencephalon and telencephalon.
We have recently reinterpreted the mechanism by which netrin1 acts to establish neural circuits in the spinal cord, showing that it acts by local haptotactic mechanisms, rather than long-range chemotaxis. Here we propose to examine the mechanistic basis by which neural progenitor cell-derived netrin1 is deposited both as a haptotactic substrate and on axons, to thereby direct fasciculated axon growth. This mechanistic understanding is critical for understanding how netrin1 can best be deployed as a regenerative factor.
