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Lola regulates midline crossing of CNS axons in Drosophila

Daniel Crowner, Knut Madden^*, Scott Goeke and Edward Giniger

Division of Basic Sciences, Program in Developmental Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave, N., Seattle, WA 98109, USA
^* Present address: Invitrogen, San Diego, CA, USA

View larger version (126K): [in a new window]   Fig. 1. lola limits midline crossing of CNS axons. Wild-type (A) and lola mutant (B,D-F) stage 16/17 embryos were fixed, stained with anti-Fasciclin II (mAb1D4) and visualized by peroxidase histochemistry. (C) A roboz14 embryo of the same stage is shown for comparison. Note multiple crossings of the CNS midline (arrows) by immunoreactive nerve tracts in all mutant panels, but not in the wild type. Midline is indicated by white triangle. (B) lola1A4; (C) roboz14; (D) lolacn03089; (E) lolaORB40; (F) lolaORE76. (B,D,E) Hypomorphic lola alleles; (F) A strong/null lola mutant. Note reduction of longitudinal axon tracts in hypomorphic lola alleles and nearly complete absence in the strong allele. Anterior is towards the top in all panels.

View larger version (142K): [in a new window]   Fig. 2. Cellular analysis of lola midline phenotype. Wild-type and mutant embryos were fixed and stained with the indicated antibodies. (A,B) Ventral views of the axonal projections of Apterous-positive interneurons, visualized by anti-ß-galactosidase staining of wild-type (A) or lolaORC46/ORE76 (B) embryos bearing a tau-lacZ fusion gene under control of a fragment of the apterous promoter. Note that ß-gal positive axons do not cross the midline (triangle) in wild type, but do so in the mutant (arrows). (C) Ventral view of an anti-Wrapper staining of a lolaORC46 mutant embryo. Note presence of immunopositive midline glial cells (arrow) in every segment. Anterior is towards the top in all three panels.

View larger version (80K): [in a new window]   Fig. 3. Slit protein levels are reduced in lola mutant embryos. A collection of lola mutant embryos and their non-mutant siblings was fixed, stained with anti-Slit and visualized by peroxidase histochemistry. (A) Wild type; (B) lola1A4. Identical camera settings were used for both embryos shown. Lateral view of the CNS midline of a late stage 16 embryo is shown in each panel; midline glial cells are prominently stained (arrows). Note that Slit immunoreactivity is present, but at reduced level, in glial cells in each segment in the mutant. Anterior is towards the left.

View larger version (82K): [in a new window]   Fig. 4. Robo protein levels are reduced in lola mutant embryos. (A) Outline of the two-color ratio method for quantification of immunofluorescence. lola mutant embryos and their non-mutant siblings were collected and fixed 10-11 hours after egg-laying (AEL) and doubly stained with mouse anti-Robo and rabbit anti-HRP. Antibodies were detected by incubating with the indicated fluorescent secondary antibodies and using the confocal microscope to collect a stack of image slices across the entire CNS. Integrated fluorescence intensities for each CNS were determined using NIH Image 1.62 to perform an average (i.e. summation) projection of the image stack; manually outlining the CNS in the projected image, and integrating the fluorescence signals for both chromophores within the outline. Fluorescence intensity comparisons were made between embryos from a single embryo collection and staining, with data collected from a single slide in a single confocal session. (B,D) Wild-type embryo; (C,E) homozygous lolaORC46 embryo. (B,C) Anti-HRP reference signal (visualized with Texas Red-conjugated secondary antibody); (D,E) Anti-Robo experimental signal (visualized with FITC secondary). For purposes of presentation, the intensities of the wild-type and mutant reference signals have been approximately matched and the cognate Robo signals adjusted accordingly. Quantification of the intensity ratios in this example show the Robo/HRP ratio to be decreased 40% in the mutant embryo when compared with the wild type.

View larger version (149K): [in a new window]   Fig. 5. lola interacts genetically with slit and robo. Embryos that were either (A) lolaORE120/lolaORE120, (B) slitIG107/+, (C) lolaORE120slitIG107/lolaORE120 or (D) lolaORE120roboz14/lolaORE120 were fixed, stained with anti-Fasciclin II and visualized by peroxidase histochemistry. Midline crossing of immunoreactive nerve bundles (arrows) was extremely rare in lolaORE120 embryos and in slit heterozygous embryos (or robo heterozygous, not shown), but very common in embryos that were simultaneously slit or robo heterozygous and lolaORE120 homozygous (see Table 2 for quantitation). All panels show ventral views of the CNS of stage 17 embryos; anterior is towards the top.

View larger version (103K): [in a new window]   Fig. 6. Ectopic lola induces ectopic slit. The splice variant lola 4.7 was placed under control of UASG and expressed throughout the developing neuroectoderm by crossing to GAL4-112A (Fuerstenberg and Giniger, 1998). slit expression was then assayed by in situ hybridization (A,B) or antibody staining (C-F). Ventral (E,F) or sagittal (A-D) views of stage 17 embryos are shown. In wild-type stage 17 embryos (A,C,E), Slit is expressed only in midline glial cells; expression of lola 4.7 throughout the neuroectoderm causes ectopic expression of slit mRNA (B) and protein (D,F), but only in additional midline cells.