Commissural Axon Kinetics and the Role of Netrin in Early Brain Circuitry Development

As neurons begin to differentiate, they send out processes called axons to initiate the formation of functional nerve connections. A specialized structure at the end of an axon called the growth cone is believed to possess the impressive navigational and target recognition ability crucial for this process. The goal of the research presented in this thesis was to understand the cellular and molecular mechanisms that shape axon growth and guidance in vivo during early brain development using a multifaceted experimental approach. Towards this goal we employed the simple, well-characterized neuronal scaffold of the embryonic zebrafish brain in combination with cell labeling techniques and performed studies in three specific areas: (1) the dynamic behaviors of navigating growth cones to obtain information about their cellular interactions with each other and the local environment, (2) the action of specific proteins (netrin and its receptor DCC) known to be involved in axon guidance in order to determine their function in vivo, and (3) the mobility of GFP in growth cones as a way to gain insight into the dynamics of molecular species in these structures as they actively navigate. Critical to our studies was a stable transgenic gata2::GFP zebrafish line in which we found high level of GFP expression in early forebrain neuronal clusters allowing in vivo timelapse study of the growth cones that pioneer the postoptic commissural (POC) axon tract. Following the development of the POC also allowed us to investigate how early commissural growth cones behave at the midline. Timelapse analysis of POC axon kinetics revealed important insight into growth cone interactions with each other and their environment and showed that these have behavioral consequences. While it was known that commissural axons slow down while crossing the midline, our data showed that this is only true for the leader axons. Follower axons do not slow down unless the leader axon is ablated. Together this analysis revealed that in addition to specific molecular cues, axon-axon interactions are important for establishing early axon tract. This characterization of POC axon kinetics and growth cone behavior in turn, provided us with an assay for studying the specific role of netrin, primarily a midline attractant for commissural axons in the spinal cord and its receptor, deleted in colorectal cancer (DCC). Loss- and gain-of-function experiments in combination with timelapse imaging uncovered a novel function for netrin as a positional repellent cue for POC axons. Finally, prompted by the observed differences in leader and follower POC growth cones, we developed a new experimental approach to assay GFP mobility as a reporter for the diffusion rates of other molecular species inside growth cones in vivo. We found that diffusion rates in actively pioneering growth cones are significantly decreased compared to follower axons suggesting that diffusion rates might be linked to growth cone pathfinding. Collectively, the findings presented in this thesis constitute a framework that allows for an integrative approach of studying growth cone navigation in vivo. Basic integrative knowledge of this sort is expected to aid the development of medical therapies related to nerve injury and repair.