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First published online 25 July 2007
doi: 10.1242/dev.02876

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Respective roles of the DRL receptor and its ligand WNT5 in Drosophila mushroom body development

Nicola Grillenzoni^*, Adrien Flandre^*, Christelle Lasbleiz and Jean-Maurice Dura

Institut de Génétique Humaine, CNRS UPR 1142, 141, rue de la Cardonille, 34396 Montpellier Cedex, France.

View larger version (59K): [in this window] [in a new window]   Fig. 1. MB defects in the drl LOF mutants are already present in newly hatched Drosophila first instar larvae and persist through metamorphosis. MBs are visualized in GAL4-OK107 UAS-mCD8-GFP individuals. (A,B,C) Wild-type brains, (D,E,F) drl LOF brains. Asterisks indicate the vertical lobes either present in the wild-type situation or reduced in the mutant situation. The midline is shown by a dashed line. The midline crossing is displayed at all stages in drl LOF mutant brains. In the displayed sample, the drl LOF mutant adult brain shows a disappearance of the ' vertical lobes (compare A with D). In late drl LOF mutant third instar larvae, the MB vertical larval lobes are absent in most of the analyzed brains (12 out of 15) (compare B with E). Much less reduction of the MB vertical larval lobes was observed in newly hatched first instar larvae (compare C with F). Scale bars: 50 µm in A and B; 20 µm in C.

View larger version (86K): [in this window] [in a new window]   Fig. 2. MB mitotic clones induced in Drosophila early first instar larvae. In a heterozygous (lio2/+) genetic background, lio2/lio2 MB clones display wild-type morphology (compare A with B), meaning that the defects observed in the lio2/lio2 genetic background are non-cell-autonomous. In a lio2/drlR343 genetic background (C), labeling of a single MB neuroblast lineage reveals that the mutant axons, once having crossed the midline, follow the contralateral ß lobe (arrow). The dashed line indicates the midline. The genotype is GAL-C155, hs-FLP, UAS-mCD8-GFP; tubP-GAL80 FRT40A/FRT40A in A, GAL-C155, hs-FLP, UAS-mCD8-GFP; tubP-GAL80 FRT40A/lio2 FRT40A in B and GAL-C155, hs-FLP, UAS-mCD8-GFP; tubP-GAL80 drlR343 FRT40A/lio2 FRT40A in C. Scale bars: 50 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 3. Specificity of the anti-LIO antibody. Wild-type (A) and lio2/drlR343 (protein null) mutant (B) Drosophila third instar larval brains were double stained with anti-LIO (green) and anti-FASII (red). Panels A and B are reconstructions of three confocal sections of 1 µm. No crossreaction with another protein is detected in null individuals. Some background staining may occur in the null individuals, but its localization and aspect are clearly unrelated to the true labeling seen in the wild type. The wild-type left hemisphere is somehow twisted, and its MB is not in the same focal plan, but it appears normally in subsequent confocal sections. Scale bars: 100 µm.

View larger version (81K): [in this window] [in a new window]   Fig. 4. DRL expression in the Drosophila stage 16 embryonic brain. (A,D) Confocal reconstructions of the same brain, A being dorsal to D. (B,C,E,F) Double-channel images derived from A and D. In the stage 16 wild-type embryonic brain, two commissural bundles express the DRL protein (red), one located posteriorly and slightly dorsally (A,B,C), the other being in the anterior part of the commissure (D,E,F). These bundles do not express the FASII marker (blue, C and F) and are HRP-positive (green, B and E), confirming their neuronal identity. (G-I) The LIO (red) pattern relative to the embryonic MBs (green GAL4-OK107 line), going from a dorsal to a ventral view; no colocalization is observed. Scale bar: 10 µm.

View larger version (136K): [in this window] [in a new window]   Fig. 5. drl LOF mutation induces no apparent defects in the embryonic Drosophila brain. Stage 16 brains of drlRed2/+ (A) and drlRed2/drlR343 (B) embryos labeled with anti-ß-galactosidase (red) and anti-HRP (green) antibodies. No apparent differences were observed between the two genetic backgrounds. Wild-type brain (C,E, with C being dorsal to E) and lio2/drlR343 brain (D,F, with D being dorsal to F) from stage 16 embryos labeled with an anti-FASII antibody. The FASII-positive fibers do not display any apparent morphological defects in the lio2/drlR343 mutant compared to wild type (compare C with D and E with F). Scale bars: 10 µm.

View larger version (80K): [in this window] [in a new window]   Fig. 6. DRL expression pattern in the Drosophila third instar larval brain. (A-E) Reconstructions of ten confocal sections spaced by 1 µm, with A being the most frontal section and E the most caudal. (F) A single confocal section at the level of the interhemispheric glial cells. The DRL protein (green) is expressed in the third instar larval brain in a complex pattern characterized by a lack of colocalization with MB axons well labeled by FASII (red in B). Different populations of cells, presumably neurons, send fibers crossing the brain commissure (arrow in B). About four cells (two are marked with arrowheads in F), which are glial cells (compare to A-C in Fig. 8) located in the posterior part of the brain, display cytoplasmic processes that wrap around the MB lobes (see arrow in C). DRL expression is also observed in the optic lobes (arrowhead in A-E) and in the incoming photoreceptor axons (asterisk in A-C). In the thoracico-abdominal part of the CNS, DRL protein is detected in commissural neurons (small arrows in C-E). Scale bar: 100 µm.

View larger version (8K): [in this window] [in a new window]   Fig. 7. Rescue of drl LOF MB defects using different Drosophila GAL4 lines. All the lines analyzed are in the lio2/drlR343 genetic background. In the x-axis label, both the GAL4 line used and the absence (-) or presence (+) of the UAS-drl construct are specified. n, the number of brains analyzed. On the y-axis, the percentage of wild-type brains is shown. MB morphology was assessed using the anti-FASII antibody. P-values were obtained after 2 test comparisons. Only the GAL4-c155 was able to rescue; the other GAL4 lines (247, OK107 and 442) were unable to rescue the drl MB phenotypes. These results indicate that the drl MB defects are linked to the lack of expression of the drl RTK in neuronal cells extrinsic to the MBs.

View larger version (74K): [in this window] [in a new window]   Fig. 8. Expression patterns of different enhancer-trap lines and REPO in the Drosophila third instar brain. Each row shows the merged and single channels of the same brain; the inset in each panel is a magnification of the interhemispheric region. (A-C) The lio1 insertion drives the expression of nuclear ß-galactosidase in four REPO-positive interhemispheric cells. (D-F) Colocalization of two REPO-positive cells with nuclear ß-galactosidase driven by the GAL4-C155 line is shown. (G-I) GAL4-7B does not drive expression of nuclear ß-galactosidase in REPO-positive cells. Scale bar: 50 µm.

View larger version (49K): [in this window] [in a new window]   Fig. 9. MB defects of Drosophila Wnt5 LOF mutants are rescued by Wnt5+ expression in the MBs. (A-C) Composite confocal images of MB phenotypes visualized with the GAL4-c739 line combined with UAS-mCD8-GFP. (A) In the Wnt5 null mutation, there is a complete arrest of axonal growth at the level of the peduncle, although Kenyon cells appear to be unaffected. The genotype is Wnt5400/Y; GAL4-c739 UAS-mCD8-GFP/+. B (excluding Kenyon cells) and C (including Kenyon cells) are images from the same brain and show a complete rescue of the MB mutant phenotype. The genotype is Wnt5400/Y; GAL4-c739 UAS-mCD8-GFP/+; UAS-Wnt5+/+.

View larger version (12K): [in this window] [in a new window]   Fig. 10. Rescue of Drosophila Wnt5 LOF MB defects using different MB GAL4 lines. All the lines analyzed are in the Wnt5400/Y genetic background. In the x-axis label, both the GAL4 line used and the absence (-) or presence (+) of the UAS-GFP and UAS-Wnt5+ constructs are specified. n, the number of brains analyzed. The y-axis shows the percentage of wild-type brains. MB morphology was assessed using the anti-FASII antibody, or directly with GFP fluorescence when possible. P-values were obtained after 2 test comparisons. The three MB GAL4 lines (OK107, 247, c739) were able to rescue the Wnt5 MB phenotype. These results indicate that the Wnt5 MB defects are linked to the lack of expression of the WNT5 protein in intrinsic MB neurons.

View larger version (12K): [in this window] [in a new window]   Fig. 11. Genetic interaction between drl and Wnt5 during MB development in Drosophila. Different genotypes were assessed for the production of a drl-like phenotype. MB morphology was assessed using the anti-FASII antibody. These results indicate that reduced expression of lio+/drl+ associated with overexpression of Wnt5+ gradually leads to a drl-like phenotype. lio/+, lio2 GAL4-c739/+. 1 UAS, GAL4-c739/+; UAS-Wnt5+/+. 2 UAS, GAL4-c739/UAS-Wnt5+; UAS-Wnt5+/+. lio/1 UAS, lio2 GAL4-c739/+; UAS-Wnt5+/+. lio/2 UAS, lio2 GAL4-c739/UAS-Wnt5+; UAS-Wnt5+/+. lio/drl, lio2 GAL4-c739/drlR343. n, the number of brains analyzed. +, wild-type; +/-, moderate midline crossing; -, complete midline crossing.