| During early nervous system development, chemotropic guidance cues command axons to grow across long distances to form new connections. Understanding the molecular mechanisms by which guidance cues can direct neuronal growth cone attraction or repulsion can offer insight into processes that control neural development and furthermore, may influence nervous system regeneration in the mature organism. In this work, we first introduce a robust zebrafish primary neuron culture system for functional nerve growth and guidance assays. Zebrafish is a widely used model organism in genetics and developmental biology research. They have been useful for studying embryonic development of the nervous system in vivo, but in vitro studies utilizing zebrafish have been limited, likely due to suboptimal conditions and the complexity of pioneering cell culture systems. We show that this novel primary neuron culture model system is useful for studies of neuronal development, chemotropic axon guidance, and mechanisms underlying inhibition of neural regeneration in vitro. In the second part of this work, we focus on sonic hedgehog protein (SHH), known to be an important morphogen in early neural patterning, and which has recently been shown to act at the ventral floor plate to guide commissural axons to the midline choice point in a transcriptionally-independent manner. However, the non-canonical signaling pathways that mediate attractive guidance by SHH remain poorly understood. Here, we show heterologous cross-desensitization of turning responses to SHH in the presence of netrin-1, another attractive cue at the floor plate, which may indicate shared cytosolic effectors between the two factors. For the first time, by live imaging, we demonstrate that analogous to netrin-1, a signaling pathway comprising of fast Ca2+ signals in the growth cone mediates attractive turning towards SHH. Chelation and depletion of intracellular Ca2+ stores abolished SHH induced turning responses. Furthermore, a gradient of combined subthreshold concentrations of SHH and netrin-1 induced robust growth cone attraction. Therefore, we identify a Ca 2+-dependent mechanism for SHH signaling in the growth cone and show complementary effects of SHH and netrin-1 on commissural axons. In this context, we suggest that at greater distances from the midline, subthreshold concentrations of SHH and netrin-1 are additive to initiate robust chemoattraction. In contrast, as extending growth cones reach the midline choice point, higher concentrations of SHH and netrin-1 induce heterologous cross-desensitization to silence the attractive signal and permit midline crossing. Such cross communication between multiple guidance cues may be a general feature of signal integration at the growth cone. |
