Molecular cues in pathfinding of axial motoneurons in the developing zebrafish

Establishing neuromuscular specificity is an important step during development to ensure proper motor function. Motor axons may travel over relatively long distances from their positions in the CNS to muscle targets in the periphery. The zebrafish model has been useful for investigating the process of motor axon pathfinding. The embryonic motor system of the zebrafish is relatively simple and has been well studied. Both forward and reverse genetics have been used to study axon pathfinding of zebrafish trunk motor axons. Our study of two motor axon mutants, stumpy and topped, has led to the identification of two molecules that guide trunk motor axons to their muscle targets.;stumpy mutants exhibit a phenotype wherein motor axons stall for prolonged periods at intermediate targets prior to them reaching their final target. Positional cloning mapped the mutation to the zebrafish homolog of the collagenXIXa1 (colXIX). colXIX is expressed at known intermediate targets during the time of axon outgrowth. Knocking down ColXIX using morpholinos (MO) phenocopies stumpy. Also stumpy mutants were rescued by knocking down ColXIX and adding back mouse colXIX RNA. This suggests that ColXIX functions to enable growth cones to navigate intermediate target during development.;Semaphorin 5A (Sema5A) was initially identified as a candidate gene for the topped mutant but this does not appear to be a case. However, Sema5A appears to play a role in motor axon pathfinding. Sema5A is expressed in the myotome during the time of axon outgrowth. Knocking down Sema5A using MOs specifically affects the Caudal Primary (CaP) motor axon, inducing a delay in its extension to its muscle target as well as axon branching. This MO phenotype can be rescued by adding back rat sema5A RNA. Sema5A has been previously shown to act as a bifunctional cue in the rat habenula. In zebrafish, we saw that adding back RNA encoding the sema domain alone rescued the branching phenotype in sema5A morphants. Conversely, adding back RNA encoding the thrombospondin repeat (TSR) domain alone into sema5A morphants exclusively rescued delay in ventral motor axon extension. These data show that Sema5A is a bifunctional axon guidance cue for vertebrate motor axons in vivo.;The addition of ColXIX and Sema5A to the list of molecules that are involved in this seemingly simple pathfinding process demonstrates that numerous factors and pathways may be involved in establishing precise neuromuscular connections. Both these molecules have been shown to function in other contexts in the nervous system thus, understanding the roles these molecules play in axonal pathfinding can reveal novel mechanisms involved in wiring the nervous system.