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In the Drosophila adult visual system, photoreceptor axons and their connecting interneurons are tied into a retinotopic pattern throughout the consecutive neuropil regions: lamina, medulla and lobula complex. Lamina and medulla are joined by the first or outer optic chiasm (OOC). Medulla, lobula and lobula plate are connected by the second or inner optic chiasm (IOC). In the regulatory mutant In(1)omb(H31) of the T-box gene optomotor-blind (omb), fibers were found to cross aberrantly through the IOC into the neuropil of the lobula complex. Here, we show that In(1)omb(H31) causes selective loss of OMB expression from glial cells within the IOC previously identified as IOC giant glia (ICg-glia). In the absence of OMB, ICg-glia retain their glial cell identity and survive until the adult stage but tend to be displaced into the lobula complex neuropil leading to a misprojection of axons through the IOC. In addition, adult mutant glia show an aberrant increase in length and frequency of glial cell processes. We narrowed down the onset of the IOC defect to the interval between 48 h and 72 h of pupal development. Within the 40 kb of regulatory DNA lacking in In(1)omb(H31), we identified an enhancer element (ombC) with activity in the ICg-glia. ombC-driven expression of omb in ICg-glia restored proper axonal projection through the IOC in In(1)omb(H31) mutant flies, as well as proper glial cell positioning and morphology. These results indicate that expression of the transcription factor OMB in ICg-glial cells is autonomously required for glial cell migration and morphology and non-autonomously influences axonal pathfinding.

Drosophila N-cadherin (CadN) is an evolutionarily conserved, atypical classical cadherin, which has a large complex extracellular domain and a catenin-binding cytoplasmic domain. We have previously shown that CadN regulates target selection of R7 photoreceptor axons. To determine the functional domains of CadN, we conducted a structure–function analysis focusing on its in vitro adhesive activity and in vivo function in R7 growth cones. We found that the cytoplasmic domain of CadN is largely dispensable for the targeting of R7 growth cones, and it is not essential for mediating homophilic interaction in cultured cells. Instead, the cytoplasmic domain of CadN is required for maintaining proper growth cone morphology. Domain swapping with the extracellular domain of CadN2, a related but non-adhesive cadherin, revealed that the CadN extracellular domain is required for both adhesive activity and R7 targeting. Using a target-mosaic system, we generated CadN mutant clones in the optic lobe and examined the target-selection of genetically wild-type R7 growth cones to CadN mutant target neurons. We found that CadN, but neither LAR nor Liprin-α, is required in the medulla neurons for R7 growth cones to select the correct medulla layer. Together, these data suggest that CadN mediates homophilic adhesive interactions between R7 growth cones and medulla neurons to regulate layer-specific target selection.

Molecular gradients have been postulated to control the topographic mapping of retinal axons in their central targets. Based initially on their expression patterns, and more recently on functional studies, members of the EphA subfamily of receptor tyrosine kinases and their ephrin-A ligands have been implicated in the guidance of retinal axons along the anterior–posterior axis of the chick optic tectum. The report that a receptor of the EphB subfamily, EphB2/Cek5/Nuk/Sek3, is expressed in a high ventral to low dorsal gradient in the developing chick retina and is present on ganglion cell axons suggests that it may be involved in the mapping of retinal axons along the corresponding dorsal–ventral axis of the tectum. To address this issue, we have determined the expression and distribution of ephrin-B1/LERK-2/Cek5-L and ephrin-B2/LERK-5/Htk-L/ELF-2, ligands for EphB2, in the developing chick retinotectal system using riboprobes, immunocytochemistry, and receptor affinity probes. Both ephrin-B1 and ephrin-B2 transcripts are expressed in a high dorsal to low ventral gradient in the developing retina, complementary to the distribution of EphB2. Ephrin-B1 and ephrin-B2 proteins are predominantly found in the developing plexiform layers, suggesting a role in the development of intraretinal connections. Neither protein is detected on ganglion cell axons. In tectum, ephrin-B1 transcripts are expressed in a high dorsal to low ventral gradient in the neuroepithelium and the protein is present along the processes of radial glia and is concentrated at their endfeet in the stratum opticum, at the time retinal axons are growing through it. This distribution of ephrin-B1 suggests that it influences retinal axon mapping along the dorsal–ventral tectal axis and may also be involved in intratectal development. In contrast, ephrin-B2 transcripts and protein are localized to the deeper retinorecipient laminae in the tectum at the time retinal axons begin to arborize in them, suggesting that this ligand may influence the laminar patterning of retinal axon terminations.