The C-type lectin chondrolectin (chodl) represents one of the major gene products dysregulated in spinal muscular atrophy models in mice. However, to date, no function has been determined for the gene.Wehave identified chodl and other novel genes potentially involved in motor axon differentiation, by expression profiling of transgenically labeled motor neurons in embryonic zebrafish. To enrich the profile for genes involved in differentiation of peripheral motor axons,weinhibited the function ofLIM-HDs(LIMhomeodomainfactors) by overexpression of a dominant-negative cofactor, thereby rendering labeled axons unable to grow out of the spinal cord. Importantly, labeled cells still exhibited axon growth and most cells retained markers of motor neuron identity. Functional tests of chodl, by overexpression and knockdown, confirm crucial functions of this gene for motor axon growth in vivo. Indeed, knockdown of chodl induces arrest or stalling of motor axon growth at the horizontal myoseptum, an intermediate target and navigational choice point, and reduced muscle innervation at later developmental stages. This phenotype is rescued by chodl overexpression, suggesting that correct expression levels of chodl are important for interactions of growth cones of motor axons with the horizontal myoseptum. Combined, these results identify upstream regulators and downstream functions of chodl during motor axon growth.