In both the developing and mature nervous system, neurons can innervate multiple targets by sprouting secondary axon collaterals, or branches, from a primary axon shaft. Although positive and negative regulators of primary axonal growth cone guidance have been identified, little is known about the molecular mechanisms mediating axonal branching morphogenesis. In order to elucidate genes that may control axonal branching, we are performing a screen to identify mutants defective in either branch formation or elongation by using a reporter expressed in ADL, a chemosensory neuron. Unlike other amphid neurons, whose axons enter the nerve ring via the amphidial commissures, the axon of ADL projects into the nerve ring laterally, where it then branches into both a dorsal and a ventral process. These processes may represent one primary axon and one branch, or alternatively, the axon may bifurcate once it enters the nerve ring, giving rise to both processes. As a first step, we have examined the effects of known axon guidance genes on ADL axonal morphology. One molecule of particular interest is Slit, a secreted factor that has been shown to promote axon collateral formation and elongation of vertebrate sensory axons in vitro (K. Wang et al., Cell , in press). Slit may be a ligand for the SAX-3/Robo receptor, and
sax-3 mutants exhibit a variety of axonal branching morphologies, ranging from loss of both branches to multiple ectopic branches. Since
sax-3 deranges the nerve ring, at this point we cannot distinguish whether these represent primary branching defects, or secondary consequences of disruptions caused by
sax-3 guidance defects. We are currently using reverse genetic techniques to obtain a slit loss of function mutant in C. elegans . Interestingly, the
unc-6/unc-40 pathway may mediate ADL ventral branch formation. In
unc-6 or
unc-40 mutants, about 70% of ADL axons have an unbranched axon that extends dorsally in the nerve ring. This may be a primary branching defect since most other aspects of ADL guidance are normal. These results propose a role for the
unc-6/unc-40 and slit/sax-3 pathways as regulators of ADL axonal branching.