The Drosophila Jing zinc finger plays a role in the survival of specialized cells at the midline of the central nervous system (CNS) and of tracheal cells. A screen for gain-of-function (GOF) enhancers of jing GOF in the eye identified the Drosophila homolog of the human disease gene alpha-thalassemia/mental retardation X-linked (ATR-X). The ATR-X gene encodes an SNF2 family Helicase/ATPase protein with chromatin remodeling activity. DATR-X has a very similar genetic structure but is smaller than its vertebrate counterparts due to the absence of a zinc finger domain. DATR-X transcripts and protein are widely expressed throughout embryogenesis and enriched in the CNS. DATR-X localizes to nuclei in Drosophila embryos and its N-terminus is sufficient for its nuclear localization. Alterations in DATR-X and Jing levels specifically in CNS neurons or glia by over-expression and RNA interference (RNAi), suggested that DATR-X and jing have a common function, as they both affect repulsion of longitudinal glia (LG), neurons and longitudinal axons from the CNS midline. With a variety of CNS cell type markers and polyclonal anti-DATR-X antibodies, the function ofDATR-X within specific subtypes of neurons (MP2 pioneer neurons) and glia (LG) were examined due to their essential involvement in axon guidance. Analysis of DATR-X deficient embryos and those with targeted RNAi-induced reductions points to DATR-X as an essential player in the formation of longitudinal axons and glial survival and positioning in the developing Drosophila embryonic CNS. Furthermore, truncated DATR-X proteins missing the Helicase C domain interfered with longitudinal glial repulsion, but at a reduced level compared with wild-type DATR-X. Together, these results establish that proper DATR-X levels and function in both glial and neuronal nuclei are required for the construction of precise axonal architecture in Drosophila embryos.