AbstractThe formation of the central nervous system depends on the coordinated development of neural and glial cell types that arise from a common precursor. Using an existing group of zebrafish mutants generated by viral insertion, we performed a “shelf‐screen” to identify genes necessary for astroglial development and axon scaffold formation. We screened 274 of 315 viral insertion lines using antibodies that label axons (anti‐Acetylated Tubulin) and astroglia (anti‐Gfap) and identified 25 mutants with defects in gliogenesis, glial patterning, neurogenesis, and axon guidance. We also identified a novel class of mutants affecting radial glial cell numbers. Defects in astroglial patterning were always associated with axon defects, supporting an important role for axon‐glial interactions during axon scaffold development. The genes disrupted in these viral lines have all been identified, providing a powerful new resource for the study of axon guidance, glio‐ and neurogenesis, and neuron‐glial interactions during development of the vertebrate CNS. Developmental Dynamics 239:2603–2618, 2010. © 2010 Wiley‐Liss, Inc.