The UNC-6/Netrin guidance cue directs dorsal-ventral axon pathfinding and is expressed in ventral cord motor neurons. Classically, UNC-6 was thought to form a ventral-to-dorsal gradient that was interpreted dynamically by growth cones. Our previous VD growth cone imaging studies indicated that the mechanism is likely more complex, involving discrete aspects of growth cone polarity coupled with regulation of growth cone protrusion. This polarity/protrusion model parts from the classical gradient model in important ways. First, growth cone polarity is separable from growth cone protrusion (Gujar et al., 2019). Second, UNC-40/DCC and UNC-5 receptors both have roles in growth cones that grow dorsally away from UNC-6 (Norris and Lundquist, 2011; Norris et al., 2014). To further test the polarity/protrusion model, we constructed a membrane-anchored UNC-6 molecule by adding a C-terminal transmembrane domain through genome editing called
unc-6(
lq154). This mutant is predicted to lack diffusible UNC-6. The AVM neuron is born laterally and extends an axon ventrally toward UNC-6. As there is no contact between AVM and UNC-6-expressing cells, diffusible UNC-6 is predicted to be required to guide the AVM axon.
unc-6(
lq154) animals display axon guidance defects to the same extent as
unc-6(
ev400) null mutants, consistent with the idea that no diffusible UNC-6 is produced. In contrast, VD/DD axon guidance defects of
unc-6(
lq154) were less severe than those of
unc-6(
ev400), suggesting that
unc-6(
lq154) retains some function in VD/DD axon guidance.
unc-6(
ev400) VD growth cones were unpolarized, whereas polarity in VD growth cones near the ventral surface in
unc-6(
lq154) was normal. Growth cones further from the ventral surface, in the dorsal half of the animal, were unpolarized in
unc-6(
lq154). This result suggests that initial growth cone polarity is normal in
unc-6(
lq154), but polarity is lost as the growth cones migrate dorsally. Possibly, a close-range or contact-mediated interaction of UNC-6 and UNC-5 polarizes the growth cone, but longer-range diffusible UNC-6 is required to maintain this polarity. Preliminary studies indicate that hypomorphic
unc-5 alleles, which are predicted to affect only the long isoforms of UNC-5, show a similar phenotype. This leads to the notion that the short
unc-5B isoform mediates close-range polarity, possibly a contact-mediated event, whereas the long isoforms might mediate maintenance of polarity over longer distances requiring diffusible UNC-6.