BACKGROUND: Pbx genes encode TALE class homeodomain transcription factors that pattern the developing neural tube, pancreas, and blood. Within the hindbrain, Pbx cooperates with Hox proteins to regulate rhombomere segment identity. Pbx cooperates with Eng to regulate midbrain-hindbrain boundary maintenance, and with MyoD to control fast muscle cell differentiation. Although previous results have demonstrated that Pbx is required for proper eye size, functions in regulating retinal cell identity and patterning have not yet been examined. RESULTS: Analysis of retinal ganglion cell axon pathfinding and outgrowth in pbx2/4 null embryos demonstrated a key role for pbx genes in regulating neural cell behavior. To identify Pbx-dependent genes involved in regulating retino-tectal pathfinding, we conducted a microarray screen for Pbx-dependent transcripts in zebrafish, and detected genes that are specifically expressed in the eye and tectum. A subset of Pbx-dependent retinal transcripts delineate specific domains in the dorso-temporal lobe of the developing retina. Furthermore, we determined that some Pbx-dependent transcripts also require Meis1 and Gdf6a function. Since gdf6a expression is also dependent on Pbx, we propose a model in which Pbx proteins regulate expression of the growth factor gdf6a, which in turn regulates patterning of the dorso-temporal lobe of the retina. This, in concert with aberrant tectal patterning in pbx2/4 null embryos, may lead to the observed defects in RGC outgrowth. CONCLUSIONS: These data define a novel role for Pbx in patterning the vertebrate retina and tectum in a manner required for proper retinal ganglion cell axon outgrowth.