Cno is necessary for MG migration and for commissural axon ensheathment and subdivision. (A-F′) sim-Gal4 UAS-CD8::GFP Drosophila embryos stained with BP102 (magenta) in a wild-type (A-C′) or cno² mutant (D-F′) background. Stage and orientation are shown in the upper right corner. (A,A′,D,D′) At stage 15, the MG of wild-type embryos completely enwrap and separate both commissures (A). At this stage, cno² mutant embryos show failures in MG migration (arrowhead) and commissure ensheathment (arrows) (D). Schematics of wild-type (A′) and cno² mutant (D′) segments at stage 15. (B,E) At stage 17, wild-type MG extend projections into the commissures, further subdividing individual axon bundles (arrows in B). In cno² mutants this process fails (arrows in E). (C,C′,F,F′) Ventral views of stage 16 embryos. In wild-type embryos, both commissures appear well separated at this stage (arrows in C,C′). In cno² mutants, anterior and posterior commissures are hardly separated, almost forming a continuous commissure (arrows in F,F′). (G) Quantitation of cno²/cno² mutant phenotypes at the midline. (H,I) Sagittal views of wild-type (H) and slit-Gal4>>UAS-cno (x2) (I) embryos stained for HRP (a general neuronal marker) and Wrapper (as a MG marker). MG are very frequently detected in between segments (arrows in I; compare with H). Scale bars: 10 μm.

Cno colocalizes with Wrapper and Nrx-IV. (A-B″) sim-Gal4 UAS-CD8::GFP Drosophila stage 16 embryos. Confocal micrographs of one CNS segment in a sagittal (A-A″) or ventral (B-B″) view. Cno (magenta) colocalizes with Wrapper (blue) (arrows in A-B″). In a sagittal view, the colocalization of the two proteins in the MG projections that subdivide the commissural axons is apparent (arrows in A-A″). (C,C′) Ventral cord of an Nrx-IV-GFP stage 16 embryo showing colocalization of Cno (magenta) and Nrx-IV (GFP) at the midline, where the MG are in close contact with both commissures (Elav, in blue, labels all neuronal cell bodies). Scale bars: 10 μm.

cno genetically interacts with wrapper and Nrx-IV. (A-B′) Two segments of Nrx-IV⁴³⁰⁴, +/+, cno2 Drosophila transheterozygotes (A,A′) or wrapper¹⁷⁵/+; cno2/cno2 embryos (B,B′) are shown in a ventral view. HRP is in magenta (A,B) or gray (A′,B′) and Slit (MG) is in green (A,B). The asterisks indicate the space between the anterior commissure (ac) and the posterior commissure (pc). Strong defects in MG enwrapping (A,B) and commissure separation (A′,B′) are shown. (C) Quantitation of cno-Nrx-IV and cno-wrapper genetic interactions. Scale bars: 10 μm.

Cno forms a complex in vivo with Wrapper and Nrx-IV. UAS-cno-GFP Drosophila embryo lysates were subject to immunoprecipitation (IP) with anti-GFP antibody and probed on immunoblots (IB) with anti-Wrapper and with anti-GFP (as an IP control). Nrx-IV-GFP embryo lysates were subject to IP with anti-GFP and probed on immunoblots with anti-Cno, anti-Wrapper (as a positive control) and with anti-GFP (as an IP control).

Cno subcellular localization at MG is not altered in Nrx-IV and wrapper mutants. (A-C′) Ventral views of three segments are displayed. In wild-type Drosophila embryos (A,A′), Cno (magenta) is detected at the anterior (ac) and posterior (pc) commissures. BP102 is shown in green. In Nrx-IV⁴³⁰⁴ (B,B′) and in wrapper¹⁷⁵ (C,C′) mutant embryos, Cno was frequently missing from commissures (arrows) or detected between them (arrowheads). (D-F′) High magnifications of one segment in a sagittal view. Slit (as a marker of MG) is in green, HRP (neurons) is in blue and Cno is in magenta. In wild-type embryos (D,D′), Cno is highly concentrated at the interface between MG and both ac and pc. Notice also the presence of Cno at the MG projections within the commissures (arrows). The magenta arrow points to strong Cno expression independent of the MG. In Nrx-IV⁴³⁰⁴ (E,E′) and wrapper¹⁷⁵ (F,F′) mutants, MG fail to completely enwrap commissures (arrowheads). Cno at the remaining MG still accumulates at contacts between MG and commissural axons (arrows in E-F′). Scale bars: 10 μm.