Central nervous system (CNS) assembly involves multiple cell-recognition events, including axon guidance and fasciculation. Such events have been studied extensively in model systems, yet how a connected brain emerges from these selective interactions remains enigmatic. Glia have been suggested to act as guideposts for axons en route to their final destination, however, a detailed molecular understanding of this glial function is not currently at hand. To understand how glia and neurons cooperate to form a CNS, we have been studying assembly of the C. elegans nerve ring (NR), a CNS neuropil consisting of ~170 axons enveloped by four astrocyte-like CEPsh glia and six mesodermal GLR glia. From a screen of fluorescent reporters expressed in subsets of embryonic neurons or glia, we determined that the NR is populated in an orderly manner, with specific glial processes extending early in conjunction with a small set of axons. Our ablation studies reveal these early cells are essential for insertion of diverse axon classes into the NR. We performed a forward genetic screen for mutants with amphid neuron entry defects resembling those in glia-ablated animals. Amphid neurons enter the NR late, allowing both early and late NR assembly factors to be identified. In addition to previously known guidance factors, we isolated a new mutant with a highly penetrant defect in NR axon entry. This mutant carries lesions in two genes, encoding a GTPase regulator and a proprotein convertase. While each lesion alone exhibits a mild NR defect (5-15%), combining both lesions blocks 70% of axons, of each of multiple neuron types, from entering the nerve ring. Surprisingly, full rescue of the double-mutant phenotype is only achieved by expressing either gene in both early-entering NR axons and glia. We performed live imaging to investigate the primary defects of this mutant during embryonic development. Moreover, we identified specific genetic interactions of each of these two new genes with known guidance signaling pathways. Importantly, the synergistic nature of the double mutant allowed us to perform a screen for a novel class of axon-guidance mutants that function redundantly. To date we have isolated over 20 mutants that fall into this category. Altogether, our studies suggest a pivotal role for glia in CNS formation and open the door to uncovering previously unrecognized axon guidance genes. Furthermore, the guidance roles of CEPsh glia are reminiscent of those played by radial glia in populating the vertebrate brain, and the mammalian homologs of the genes affected in our double mutant have known axon guidance roles in vertebrates. Our studies, therefore, may reveal conserved mechanisms promoting CNS assembly.
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