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doi: 10.1242/10.1242/dev.00319

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A mosaic genetic screen for genes necessary for Drosophila mushroom body neuronal morphogenesis

John E. Reuter^1,*, Timothy M. Nardine^1,*, Andrea Penton¹, Pierre Billuart¹, Ethan K. Scott¹, Tadao Usui², Tadashi Uemura^2,3 and Liqun Luo^1,

¹ Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA
² Laboratory of Molecular Genetics, Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
³ Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan

View larger version (31K): [in a new window]   Fig. 1. (A) Schematic of mushroom body development [adapted, with permission, from Lee et al. (Lee et al., 1999)]. The three colors represent three classes of MB neurons born at different developmental windows as indicated. (B) Schematic of the genetic scheme for MARCM-based mosaic screen on chromosome arm 2R. (C) Three types of labeled MARCM clones of MB neurons can be generated by heat-shock induction of FLP recombinase. If mitotic recombination occurs in a dividing neuroblast (Nb), and if the regenerating neuroblast loses the repressor for marker expression, a labeled neuroblast clone is generated (boxed). If the ganglion mother cell (G) loses the repressor, a two-cell clone is generated (not shown). If mitotic recombination occurs in a dividing ganglion mother cell, one postmitotic neuronal progeny (N) loses the repressor resulting in a single-cell clone (arrowhead).

View larger version (53K): [in a new window]   Fig. 2. Mutations that affect cell number, size, and membrane protein distributions. All images are two-dimensional projections of confocal z-stacks showing mushroom body neuroblast clones homozygous for wild type (A), wkp1 (B), 26D19 (C), 29B70 (D) and amz1 (E). MARCM clones were induced in newly hatched larvae, fixed and stained for a membrane-targeted mCD8-GFP in wandering third instar larvae. Insets in A-D are high-magnification single confocal sections that show the details of the cell body staining. Image in B is overexposed to show the very weak labeling of axons and dendrites by the mCD8-GFP marker. The long arrow in E indicates an abnormally large cell. As the mushroom body is a three-dimensional structure, the dorsal lobe is sometimes almost perpendicular to the X-Y plane because of variation in mounting (see E). In this and all subsequent figures, samples are oriented such that dorsal is upwards and the midline is towards the right. Unless otherwise mentioned, arrowheads point to the calyx, which is the dendritic field of MB neurons. Brackets define the axonal peduncle before branching into the lobes. Horizontal and vertical arrows indicate the dorsal and medial lobes, respectively.

View larger version (61K): [in a new window]   Fig. 3. roadblock (robl) mutants affect axonal transport and neuroblast proliferation. (A,B) Neuroblast clones homozygous for roblMB examined in wandering third instar larva (A) and in adult (B). Red staining in B represents FasII immunoreactivity, which labels strongly the last-born class of /ß neurons. The dorsal axons in roblMB neuroblast clones (green) are therefore ' axons, as they are not stained with FasII. (C) Wild-type mushroom body neuroblast clone double labeled with FasII (red). (D) A roblB neuroblast clone examined in wandering third instar larva. (E) A roblZ neuroblast clone examined in adult. (F,G) Axons of neurons in adult medial lobe from single-cell clones of wild type (F) or roblZ (G). Long arrows in all images indicate accumulations of intense mCD8-GFP staining most probably because of accumulation of cargoes along the axons. Short oblique arrow in E indicates the cell body region of this neuroblast clone.

View larger version (88K): [in a new window]   Fig. 4. short stop mutants cause multiple defects including neuronal polarity. (A,B) neuroblast clones homozygous for shot examined in larva (A) and adult (B). Long arrows point to overextended processes from the calyx. Short oblique arrow in A indicates axon termination in the peduncle as the intensity of mCD8-GFP staining progressively decreases as axons enter the lobe. Both images are overexposed to show the weak axonal lobes (composed of only the early-born neurons; the horizontal arrow indicates where the dorsal lobes should be) and the long arrow indicates the overextension from the calyx. Insets show the normal exposure of the cell body region. (C,D) Neuroblast clones with single staining of mCD8-GFP (C1,D1), Nod-ßgal (C2,D2) and double labeling (C3,D3) for shot3 (C) and wild type (D). In wild type, Nod-ßgal is confined to the cell bodies, calyx and sometimes the proximal part of the peduncle. In shot3 neuroblast clones, Nod-ßgal often intensely labels the entire peduncle and sometimes can be seen in the axonal lobes and distal end of the axons (double arrows in C, middle and right panels). Overextensions from the dendritic field (long arrow) are also strongly labeled with Nod-ßgal. (E) Quantification of the Nod-ßgal mislocalization phenotype. n=23 for wild type and n=14 for shot3.

View larger version (72K): [in a new window]   Fig. 5. flamingo regulates dendritic extension. (A-C) Larval neuroblast clones homozygous for fmiMB (A) and fmiE59 (B) have overextending processes from the calyx (long arrows) that are labeled with Nod-ßgal (C, and high magnification in the inset). (D) Adult fmiE59 neuroblast clones also have processes that overextend from the calyx (long arrow). In addition, reduction of cell number is evident, as is the lack of the dorsal lobe. (E,F) Clonal expression of UAS-fmi (genotype y,w, hs-FLP, UAS-mCD8-GFP/Y; FRTG13, fmiE59/FRTG13, tubP-GAL80; UAS-fmi/+; GAL4-OK107/+) rescues the phenotypes of dendritic overextension and cell number reduction in both larva (E) and adult (F).

View larger version (94K): [in a new window]   Fig. 6. Flamingo overexpression results in axon retraction. (A) Adult mushroom body axon lobes as visualized by pan-MB expression of mCD8-GFP (green) using GAL4-OK107; double labeled with FasII (in red) (B) Overexpression of Fmi using GAL4-OK107 results in the loss of the dorsal lobe (horizontal arrow). (C-F) Representative images of mushroom bodies overexpressing Fmi at different developmental stages as indicated. APF, after puparium formation. (G) Quantification of dorsal lobe phenotypes. n=50, 8, 16, 12, 50 for the five stages quantified. A is adapted from Billuart et al. (Billuart et al., 2001). Copyright (2001), with permission from Elsevier Science.

View larger version (151K): [in a new window]   Fig. 7. Phenotypic analysis of heron and kali. (A) Larval MB neuroblast clones homozygous for hrn2 results in process extension from the calyx (long arrow). (B) Dendritic overextension persists in hrn clones when examined in adult (long arrow). Nod-ßgal is distributed in a subset of overextending processes. (C,D) Neuroblast clones homozygous for kali1 extend processes in all directions from the calyx that are positive for Nod-ßgal (long arrows). (D) A partial confocal z-stack that removed axonal lobes overlapping the overextended dendrites in the x-y plane.