Glia initiate brain assembly through noncanonical Chimaerin-Furin axon guidance in C. elegans.

Brain assembly is hypothesized to begin when pioneer axons extend over non-neuronal cells, forming tracts guiding follower axons. Yet pioneer-neuron identities, their guidance substrates, and their interactions are not well understood. Here, using time-lapse embryonic imaging, genetics, protein-interaction, and functional studies, we uncover the early events of C. elegans brain assembly. We demonstrate that C. elegans glia are key for assembly initiation, guiding pioneer and follower axons using distinct signals. Pioneer sublateral neurons, with unique growth properties, anatomy, and innervation, cooperate with glia to mediate follower-axon guidance. We further identify a Chimaerin (CHIN-1)- Furin (KPC-1) double-mutant that severely disrupts assembly. CHIN-1 and KPC-1 function noncanonically, in glia and pioneer neurons, for guidance-cue trafficking. We exploit this bottleneck to define roles for glial Netrin and Semaphorin in pioneer- and follower-axon guidance, respectively, and for glial and pioneer-neuron Flamingo (CELSR) in follower-axon navigation. Taken together, our studies reveal previously undescribed glial roles in pioneer-axon guidance, suggesting conserved principles of brain assembly.