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Embryonic and larval development of the Drosophila mushroom bodies: concentric layer subdivisions and the role of fasciclin II

¹ Institute of Biological Sciences, and Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba 305-8572, Japan
² National Institute for Basic Biology, and PRESTO, Japan Science and Technology Corporation Okazaki, Aichi 444-8585, Japan

View larger version (90K): [in a new window]   Fig. 1. The larval and adult mushroom bodies. (A) Third instar larval brain stained with anti-FAS II (magenta) and GFP (green) driven by a MB GAL4 line, 201Y. (B) Adult brain stained for FAS II (magenta) and dnc-lacZ (green). and ', adult dorsal lobes; ß, ß' and , adult medial lobes; cx, calyx; DL, dorsal lobe; KCs, Kenyon cells; ML, medial lobe; OL, optic lobe; Ped, peduncle; VNC, ventral nerve cord. Reconstruction of optical sections. Scale bars: 50 µm.

View larger version (88K): [in a new window]   Fig. 2. Embryonic development of the mushroom body (MB) axonal tracts. (A-C) Single optical sections showing the embryonic MB tracts. Lateral views. The embryonic MB tracts are labeled with UAS-tau-lacZ (green) driven by 238Y-GAL4. The major brain tracts are visualized with anti-FAS II antibody (magenta). (A) Stage 14. Arrowheads indicate an interface cell that co-expresses FAS II and 238Y on its surface. MB neuroblasts are indicated with stars. (B) Stage 15. The 238Y MB neurons extend thin pioneer axons (arrows) along the FAS II-expressing cells. (C) Stage 16. The MB tracts converge at LPT. FAS II expression on the nearby cells is down regulated by this stage while the growing MB tracts (arrows) become more prominent. (D,E) Late stage 16. Reconstructed from optical sections. Lateral (D) and dorsal (E) views. The 238Y signal (green) is selectively enhanced to show the embryonic MBs. Major FAS II tracts also are shown (white). The embryonic brain hemisphere and its core neuropil are outlined with brown line. ACT, antennocerebral tract; BN, Bolwig’s nerve; DCT, dorsal commissural tract; fc, frontal commissure; LPT, lateral protocerebral tract; MPT, medial protocerebral tract; OL, optic lobe; P1, P4l, P4m, P5l, fiber tract founder clusters (Nassif et al., 1998) Ped, peduncle; VCT, ventral commissural tract. Scale bars: in A, 10 µm for A-C; 10 µm in D,E.

View larger version (131K): [in a new window]   Fig. 3. Differential expression of MB markers in the first instar brain. (A,D,G,J) Expression of dnc-lacZ is revealed by an anti-ß-GAL antibody (green/white). (B,E,H,K) GFP expression driven by 201Y (green/white). (C,F,I,L) GFP expression driven by c739 (green/white). (A-C) Slightly oblique dorsal views of MBs showing the Kenyon cells labeled with an anti-DAC antibody (magenta/white). Stars indicate the positions of the MB neuroblasts. (D-F) Lateral views showing the peduncle and lobes. Major axonal tracts, lobes and peduncle are labeled with an anti-FAS II antibody (magenta/white). Note that FAS II is expressed in the lobes and the distal part of the peduncle. (G-I) Single optical sections of the medial lobes. Marker expression is shown on the right in single channel. (J-L) Single optical sections of the dorsal lobes. Marker and FAS II expressions are shown on the right in single channels. Similar internal staining was obtained for peduncle sections (not shown). Scale bars: in A, 20 µm for A-C; in D, 20 µm for D-F. ACT, antennocerebral tract; cx, calyx; DL, dorsal lobe; h, heel; ML, medial lobe; Ped, peduncle.

View larger version (118K): [in a new window]   Fig. 4. Differential expression of MB markers in the second instar brain. (A,D,G,J) Expression of dnc-lacZ (green/white). (B,E,H,K) GFP expression driven by 201Y (green/white). (C,F,I,L) GFP expression driven by c739 (green/white). (A-C) Dorsal views of the Kenyon cell clusters labeled with an anti-DAC antibody (magenta/white). Stars indicate the positions of the MB neuroblasts. (D-F) Lateral views. The peduncles and lobes are labeled with an anti-FAS II antibody (magenta/white). (G-I) Single optical sections of the peduncles and the medial lobes. Marker expression is shown on the right in single channel. (J-L) Single optical sections of the dorsal lobes. Marker and FAS II expressions are shown on the right in single channels. Note similar internal staining for the peduncles and the lobes. Scale bars: in A, 25 µm for A-C; in D, 25 µm for D-F. ACT, antennocerebral tract; cx, calyx; DL, dorsal lobe; h, heel; ML, medial lobe; Ped, peduncle.

View larger version (132K): [in a new window]   Fig. 5. Differential expression of MB markers in the third instar brain. (A,D,G) Expression of dnc-lacZ (green/white). (B,E,H) GFP expression driven by 201Y (green/white). (C,F,I) GFP expression driven by c739 (green/white). (A-C) Dorsal views of the Kenyon cell clusters of wandering third-instar larvae labeled with an anti-DAC antibody (magenta/white). Single optical sections. Stars indicate the positions of the MB neuroblasts. (D-F) Lateral views showing the lobes and the distal part of the peduncles. Reconstruction of optical sections. (G-I) Single optical sections of the peduncles. The lobes and the peduncle are labeled with an anti-FAS II antibody (magenta/white). Note similar internal staining for the peduncles and the lobes. Expression of 201Y and c739 was monitored with UAS-GFP. FAS II expression is weak in the innermost axons labeled with c739 and absent in the core. (J) Lateral view of a third instar MB double labeled with anti-Synaptotagmin (SYT; magenta/white) and anti-ß-gal (green/white) antibodies. Reconstruction of optical sections. (K) High-magnification view of the dorsal lobe and heel showing a patchy distribution of the dnc-lacZ terminals in J. Single optical section. (L) A high power view of the dendritic arborization (J) of the dnc-lacZ neurons in the calyx. Note the extensive arborization of the dnc-lacZ neurons around the globular synaptic terminals (arrowheads). Single optical sections. Scale bars: in A, 25 µm for A-C; in D, 50 µm for D-F; 50 µm in J; 20 µm in K,L. ACT, antennocerebral tract; cx, calyx; DL, dorsal lobe; h, heel; ML, medial lobe; Ped, peduncle.

View larger version (160K): [in a new window]   Fig. 6. Characterization of the core fibers. (A) Lateral view of wandering third instar MB labeled with an anti-FAS II antibody (magenta/white) and phalloidin (green/white). (B,C) MB axonal fibers around the calyces. MBs are labeled with UAS-mCD8::GFP (green/white) driven by 201Y and phalloidin (magenta/white). Single optical sections of wandering third instar MBs. (D-I) GFP reporter staining in wandering third instar MBs. GAL4 drivers are (D-F) elav-GAL4, (G,H) OK107 and (I) 201Y. OK107 is expressed moderately in the MB neuroblasts and GMC, and strongly in the differentiated Kenyon cells. The reporters are (D,F-I) UAS-mCD8::GFP (green/white) and (E) UAS-GFP (green/white). (D,G) Dorsal views of Kenyon cells labeled with anti-DAC antibody (magenta); stars indicate the positions of the neuroblasts. (E,F,H,I) Lateral views of the lobes and the distal part of the peduncle labeled with anti-FAS II antibody. Note the core staining with the elav-GAL4 and OK107 drivers. Insets are cross sections of peduncles. Scale bars: in A, 20 µm for A,E,F,H,I; 20 µm in B,C; in D, 20 µm for D,G. ACT, antennocerebral tract; cx, calyx; DL, dorsal lobe; h, heel; ML, medial lobe; Ped, peduncle.

View larger version (97K): [in a new window]   Fig. 7. Sequential generation of the MB neurons and their projections. (A,D) Neuroblast clone (green/white) induced in the first instar. (B,E) Two-cell clone induced in the first instar. (C,F) Neuroblasts clone induced at the beginning of the third instar. Clones are labeled with elev-GAL4 and UAS-mCD8::GFP. (A-C) Dorsal views of the Kenyon cells labeled with anti-EY (blue). Late third instar stage. Stars indicate the location of the four-MB neuroblasts. The position of the GFP marked neuroblast is labeled with a yellow star. (D-F) Lateral views showing the peduncle, dorsal and medial lobes. Insets in D-F are optical cross sections of the peduncle. The peduncle and lobes are labeled with anti-FAS II (magenta). Arrow in the inset indicates the core. DL, dorsal lobe; cx, calyx; ML, medial lobe; Ped, peduncle. Scale bars: in A, 40 µm in A-C; in D, 40 µm for D-F. Genotypes: GAL4c155, hs-FLP/X or Y; G13, UAS-mCD8::GFP/G13, tubP-GAL80.

View larger version (105K): [in a new window]   Fig. 8. Structural MB defects in loss-of-function fas II mutants. (A) Axonal projections of embryonic MB primordium in fas IIeb112 mutant (protein null). Lateral view of the embryonic brain at late stage 16. FAS II (magenta) and 238Y (green). Single optical section. The embryonic MBs are labeled with UAS-tau-lacZ and 238Y-GAL4. Arrows indicate the growing MB axons. (B) Overview of an embryonic MB primordium in fas IIeb112. Reconstruction of optical sections. Same embryo as in A. Neurons are labeled with anti-HRP antibody (white). 238Y signal (green) is selectively enhanced. (C,D) Third instar larval MBs double labeled with anti-FAS II (magenta/white) and anti-DIF (green/white), which stains MB structures (Cantera et al., 1999). (C) fas IIe76 hypomorphic (10%) mutant. Note the thin dorsal lobe (arrowhead) and fusion of the medial lobes. (D) fas IIe86 hypomorphic (50%) mutant. Note the small dorsal lobe (arrowhead) and the medial lobes (arrow). Calyces are markedly expanded with aberrant accumulation of the FAS II protein. (E,F) fas IIeB112 mutant clones at the late third instar stage (green/white). Mitotic recombination was induced in the first instar stage. (E) Dorsal view of the Kenyon cells labeled with anti-EY (blue). Stars indicate neuroblasts. (F) Lateral view showing the peduncle, dorsal and medial lobes labeled with anti-FAS II (magenta/white). Inset shows a cross section of the peduncle. The core fibers are not labeled owing to the driver/reporter combination used for the generation of the mosaic clones. Genotype used: hs-FLP, tubP-GAL80, FRT19A/ fas IIeB112, FRT19A; 201Y/ UAS-GFP-T2. cx, calyx; DL, dorsal lobe; ML, medial lobe; Ped, peduncle. Scale bars: 10 µm in A,B; in C, 50 µm for C,D; 20 µm in E,F.

View larger version (99K): [in a new window]   Fig. 9. Structural MB defects caused by overexpression of FAS II. Lateral views of larval MBs at wandering third instar stage. GAL4 drivers are (A) elav-GAL4, (B-D) OK107, (E) 201Y and (F) 238Y. Reporters are UAS-mCD8::GFP in all cases (green). Insets show cross sections of peduncles with phalloidin staining (magenta). Arrowhead in A, malformed thin dorsal lobe. (B) MB with type 1 defects (Table 2). Note the blunt medial lobe as compared with the wild-type medial lobe in F, which terminates in three blobs. (C) MB with type 2 defects. Note the malformed thin dorsal lobe (arrowhead). (D) MB with type 3 defects. Note the malformed medial lobe (arrowhead) and expansion of the dorsal lobe. Scale bar: 20 µm. DL, dorsal lobe; ML, medial lobe; Ped, peduncle.

View larger version (32K): [in a new window]   Fig. 10. (A) The MB primordia in the embryonic brain. Frontal view of the embryonic brain at late stage 16. Only major tracts are shown. Optic lobes are not included. (B,C) Layer organization of the second and third instar larval MBs. Dorsal images of Kenyon cell clusters and cross sections of the peduncle and lobes. Corresponding subdivisions are shown in same colors. Relative expression levels of various MB markers are summarized in the table. The second instar MBs can be concentrically subdivided in two layers surrounding the core. With increase in the numbers of the Kenyon cells and their projections, the third instar MBs can be subdivided into three layers surrounding the core. Note that the distoproximal concentric subdivisions of each of the four Kenyon cell clusters topologically correspond to the unified concentric subdivisions in the lobes and the peduncle. The core consists of a bundle of newly formed axon fibers that contain densely packed actin filaments. AF, actin filaments; ACT, antennocerebral tract; DCT, dorsal commissural tract; DNC, dnc-lacZ; KCs, Kenyon cells; LPT, lateral protocerebral tract; MPT, medial protocerebral tract; P1, P4l, P4m, and D/T, fiber tract founder clusters (Nassif et al., 1998); Ped, peduncle; VCT, ventral commissural tract.