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First published online October 12, 2006
doi: 10.1242/10.1242/dev.02605

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Segment-specific requirements for dorsoventral patterning genes during early brain development in Drosophila

Institute of Genetics, University of Mainz, D-55099 Mainz, Germany.

View larger version (127K): [in a new window]   Fig. 1. Expression of Vnd in the pNE and brain NBs of early wild-type embryos. (A-K) Vnd antibody staining at early stage 7 (A), stage 8 (B), stage 9 (C,D,E), stage 10 (F,G,H) and stage 11 (I,J,K). (F-K) Double labeling against Engrailed-lacZ (En) indicates part of the posterior border of brain neuromeres. (C,F,I) Flat preparations displaying the head ectoderm (anterior is upwards). (D,G,J) Higher magnifications of regions framed in C,F,I focusing on NBs; immuno-positive NBs are indicated by white letters, immuno-negative NBs are indicated by black letters. Broken black lines encircle domains of strong levels of Vnd; grey lines circle weak levels of Vnd. (E,H,K) Expression of Vnd and En in brain NBs (dark blue, strong Vnd; light blue, weak Vnd; brown, En). All NBs developing within the Vnd domain have been assigned as ventral NBs [for nomenclature of NBs, see Urbach et al. (Urbach et al., 2003)]. (A,B) Lateral view of anterior half of whole-mount embryos. Broken white line encircles the procephalic Vnd domain, encompassing about 66 cells (A, stage 7) and about 47 cells (B, stage 8), indicating that Vnd is already downregulated in part of the pNE cells (black arrows). The prospective protocerebral Vnd domain (black arrowhead) begins to separate from the trito-/deutocerebral Vnd domain (white arrowhead). (C-E) Vnd has mostly vanished from the anterioventral pNE of the deutocerebrum (DC) and posterioventral pNE of the protocerebrum (PC), and from the ventral NBs Dv3, Dv6 and Pcv1. (F-H) Vnd begins to vanish in the posterior DC and anterior tritocerebrum (TC; area around Dv4 in G). (I-K) Separate Vnd domains are located at the posterioventral border of the TC, DC and PC. as, en antennal stripe; hs, en head spot; is, en intercalary stripe; AN, MD, MX, LA, antennal, mandibular, maxillary, labial segment, respectively; CL, clypeolabrum; CF, cephalic furrow; FG, foregut.

View larger version (91K): [in a new window]   Fig. 2. Summary diagram of marker genes expressed in brain NBs of wild-type and vnd mutant embryos at stages 9 and 11. (A-D) The schemes represent NB maps of one hemibrain: anterior (a) is towards the top, posterior (p) is towards the bottom, dorsal (d) is leftwards, ventral (v) is rightwards. Numbers and distribution of NBs correspond to the situation in flat preparations of wild-type embryos at stage 9 (A,B,B') and stage 11 (C,D,D'). The color code indicates expression of the genes investigated in this study in individual brain NBs. Broken blue lines enclose the domain of Vnd expression and, accordingly, the population of ventral NBs; broken red lines mark neuromeric boundaries between TC, DC and PC. (A,C) Wild type (wt). (B,B',D,D') vnd embryos (vnd-); individual NBs encircled with broken black lines are absent to a certain degree. (B',D') The frequencies of their presence (in %) are indicated at stage 9 for all NBs, at stage 11 for all NBs in the TC, DC and all ventral and adjacent dorsal NBs in the PC. NBs (grey) develop within the Vnd domain. (B,D) Expression of a marker is indicated as missing if it is not detected in more than 90% observed cases. Hatched colors indicate ectopic gene expression. As the exact identity of NBs that ectopically express msh, ems and dac is not clear, hatched NBs represent potential candidates, of which only a subfraction will normally be found to express the respective marker. For further details, see text.

View larger version (173K): [in a new window]   Fig. 3. Defects in brain NB pattern in vnd mutants at stage 11. (A-F) Antibody double staining against Engrailed [En; En-lacZ (A,B), En-protein (C-F)] and Deadpan (Dpn; A-D) or Seven up-lacZ (Svp-lacZ; E,F) in wild type (A,B,E) or in vnd mutants (C,D,F). (A,B) All brain NBs have developed by stage 11. (B) Higher magnification of region framed in A. NBs delaminating from the en head spot (hs), the en antennal stripe (as) and the en intercalary stripe (is) are indicated. (C) Broken yellow line encloses ventral pNE, in which NB formation is abnormal. (D) Higher magnification of the region framed in C; Dv8, Tv4,5 are missing. (E,F) All ventral NBs (including the ventral Svp-lacZ-positive) are indicated in white or green, dorsal Svp-lacZ-positive NBs in black inscriptions. (F) In vnd mutants, ventral NBs indicated in green in E are missing. Segmental borders between the trito-(TC), deuto-(DC) and protocerebrum (PC) are marked by broken red lines. as, en antennal stripe; hs, en head spot; is, en intercalary stripe; AN, MD, MX, antennal, mandibular, maxillary segment, respectively; CL, clypeolabrum; FG, foregut. Anterior is upwards.

View larger version (122K): [in a new window]   Fig. 4. Increased apoptosis in the vnd mutant pNE. (A,B) Anti-Caspase 3 antibody staining in the head ectoderm at late stage 11 (lst11); blue staining along the midline in A indicates the blue balancer. (A) In heterozygotes (wild type; wt) a few scattered apoptotic cells are observed in the pNE. (B) In vnd mutants (vnd-), apoptotic cells are significantly more abundant in the pNE, especially in the ventral part (broken white line), and are also more frequent in the ventral neuroectoderm of the mandibular (MD), maxillary (MX) and labial (LA) segment. (C-E) NB layer in the DC of vnd embryos. A corresponding region is framed by a broken black line in B. (C) Mid-stage 11 (mst11); Caspase-3 is detected in a few ventral deutocerebral NBs (black asterisks). (D,E) Slightly later (lst11), degenerating ventral NBs can be identified at corresponding positions by morphological criteria [e.g. condensation and fragmentation of the nucleus and cytoplasm; Abrams et al. (Abrams et al., 1993)]. (D) Svp-lacZ/En staining suggests that NBs in position of Dv4/5 and Dv7 undergo apoptosis. (E) Ems/En labeling reveals that the degenerating NB (presumably Dv7) ectopically expresses Ems (compare with Fig. 5B). as, en antennal stripe; CL, clypeolabrum.

View larger version (99K): [in a new window]   Fig. 5. Defects in the brain NB pattern in vnd loss-of-function and gain-of-function embryos at stage 11, as revealed by marker genes specific for NB subsets. (A-X) Head flat preparations antibody labeled against Engrailed [En; En-lacZ (E,F,M,N) or En-protein (A-D,Q,R,W,X)] and Empty spiracles (Ems; A-D,Q,R) or Ladybird early (Lbe; E,F) or Eyeless (Ey; M,N,W,X), only Ey (O;P), Lbe (G,H,S,T) and Dachshund (Dac; I-L,U,V) in wild type (wt), vnd mutant (vnd-) and sca-vnd embryos as indicated; ventral views (anterior is upwards). (B,D,F,H,J,L,N,P,R,T,V,X) Higher magnifications at the level of NBs deriving from neuroectodermal regions (NE) framed in A,C,E,G,I,K,M,O,Q,S,U,W. (A,B) Ems is expressed in dorsal NBs in the PC (Ppd2,5,8; the latter two co-express En), DC (Dd3,6,8) and TC (Td6), and in the ventral Pcv5 and Dv3. (C) In vnd embryos, Ems is derepressed in the ventral trito-/deutocerebral NE and (D) in residual ventral/intermediate NBs (indicated with green inscriptions). Dorsal NBs are all identifiable; the ventral Pcv5 is missing. (E) In wild type, Lbe is downregulated in the pNE, (F) confined to the dorsal Td4 and Dd7, and to the ventral Pcv8 and Ppv3. (G) In vnd-, the Lbe domain in the TC and DC is ventrally slightly enlarged. (H) Conversely, Pcv8 and Ppv3 are missing. (I,J) In heterozygotes, Dac is found in about 10 dorsal and three ventral NBs (border indicated by the broken white line in J). Black arrow in I indicates Dac-positive Tv2, red arrow indicates an adjacent Dac cell in the MD. Blue midline staining in I indicates blue balancer. (K,L) In vnd-Dac is ectopically expressed in part of the ventral protocerebral pNE (black arrowhead in K) and approximately four descending NBs (black asterisks in L). Dac expression is absent in position of Tv2, and reduced in the MD. (M,N) Ey-positive NBs are indicated in the TC, DC and PC of wild type. (O,P) In vnd-, Ey is missing at positions of ventral (Dv6, Dv7, Pcv6) and intermediate (Td1, Td2) NBs, but is found in all dorsal NBs (e.g. Dd4 and Pcd2). There is faint ectopic Ey in the ventral intercalary NE (encircled by broken black line in P; the corresponding area is encircled in N). Similarly, ectopic Ey is expressed in ventral NE of the mandibular (MD), maxillary, (MX) and labial (LA) segments (O). (Q) In sca-vnd, Ems is largely repressed in the dorsal pNE of the TC, DC and PC, but not in the ventral pNE. In addition, En is absent from the en hs (corresponding region encircled with a broken line), and diminished in the en antennal stripe (as). (R) The ventral Pcv5 and Dv3 express Ems. The dorsal Dd5 lack En; Ppd5 and Ppd8 lack En and Ems. (S) Lbe is absent in the TC and DC but expanded in the PC. (T) Ppv3 and Pcv8, close to the border of the dorsal PC, express Lbe the strongest; additionally, about five dorsal NBs express ectopic Lbe (white arrowheads). (U) Dac is repressed in the ocular pNE and (V) in dorsal protocerebral NBs, but retained in ventral NBs (Pcv7,9). (W) Ey is repressed in the dorsal pNE of the DC and PC, and is absent in the TC, MD, MX and LA. (X) Ey expression is lacking in dorsal, but retained in most ventral, NBs in the PC and DC. CL, clypeolabrum; FG, foregut; OA, optic lobe anlagen.

View larger version (122K): [in a new window]   Fig. 6. vnd regulates expression of ind and msh in the pNE and brain NBs. Head flat preparations double stained with antibodies against Msh and En (A-L), or subjected to in situ hybridization against ind (M-X) of wild-type (wt; A-D,M-P), vnd mutant (vnd-; E-H,Q-T) and sca-vnd embryos (I-L,U-X); ventral views; stages as indicated. (B,D,F,H,J,L,N,P,R,T,V,X) Higher magnifications of the regions framed in A,C,E,G,I,K,M,O,Q,S,U,W, focusing on NBs and neuroectoderm (NE), respectively. (A,C) In wild type, Msh is found in the dorsal NE of the intercalary (IC), antennal (AN) and gnathal segments (MD, mandibular, MX, maxillary, LA, labial). (B,D) Dorsal NBs in the DC (B), and later in the TC/DC (D), express Msh. The ventral border of the Msh domain is indicated by a green broken line. It runs between the En-positive Dd5 and Dv8 (which does not express Msh). (E,G) In vnd-, Msh is expanded into the ventral pNE of the IC and DC. Ectopic Msh is not found in the PC. (F,H) Residual ventral NBs (white asterisks) in the TC and DC express ectopic Msh. Broken green line indicates the ventral border of the wild-type Msh domain (compare with B,D); the broken white line indicates ectopic Msh expression. (H) Ectopic Msh is found at lower levels in the en intercalary stripe (is; encircled with broken yellow line) owing to repression by ind, which is partly co-expressed (encircled with a broken yellow line in T). (I,K) In sca-vnd, Msh is largely repressed in the dorsal pNE. Low levels of Msh can be found in dorsalmost ectodermal patches in the head and trunk, and (J) sometimes in a dorsal NB in the DC (black asterisk). (L) Msh is almost completely repressed in NBs. (M,O) ind is expressed in three spots in the pNE of the intercalary (ind is, white arrowhead), antennal (ind as, red arrowhead) and ocular (ind oc, black arrowhead) segments. (N) The ind as gives rise to Dd1. (P) The ind is gives rise to Td1, Td2 and Td3. (Q,R) In vnd mutants, ind is and ind as are absent. ind is derepressed in ventral NE of the MD, MX and LA. (S,T) ind oc appears ventrally expanded; ind is becomes identifiable, but its overall size is reduced; ind as remains absent. (U,W) In sca-vnd, ind is largely repressed in the pNE. (V,X) Size of ind oc is diminished; ind is is absent, but ind as (and Dd1) is unaffected. (Y) Schemes summarizing the expression of ind and Msh in NBs of wild-type, vnd mutant and sca-vnd embryos at stage 11. Broken blue line encloses ventral NBs that, in wild type, persistently or transiently express Vnd until stage 11. Segmental borders are marked by broken red lines. as, en antennal stripe; hs, en head spot; is, en intercalary stripe; AN, IC, MD, MX, LA, antennal, intercalary, mandibular, maxillary, labial segment, respectively; CL, clypeolabrum; FG, foregut. Anterior is upwards.

View larger version (109K): [in a new window]   Fig. 7. Vnd represses glial fate in the early trito- and deutocerebrum. (A-H) Head flat preparations double stained with antibodies against the glial marker Repo and En in wild-type (wt; A,B), msh mutant (msh-; C,D), vnd mutant (vnd-; E,F) and sca-vnd embryos (G,H) at early stage 12, all in a ventral view. (B,D,F,H) Close-ups of regions framed in (A,C,E,G), respectively. (A,B) In the TC and DC, first glial cells develop in the dorsal pNE. In contrast, in the PC, some of the glial cells appear to develop from ventral NBs. Broken red lines indicate segmental boundaries. (C,D) In msh-, glial cells in the TC and DC are almost absent (one cryptic glial cell is detected at dorsalmost position in the DC; white arrowhead in D). Similarly, the number of glia cells in the MD, MX and LA is diminished (C). Glial cells are not affected in the PC. (E,F) In vnd-, the number of glial cells is significantly increased in the TC and DC. Most of the ectopic glial cells develop in the ventral TC and DC (F), as is indicated by their position relative to the en antennal stripe (as) and en head spot (hs). (G,H) In sca-vnd, glial cells are absent in the TC and DC, strongly resembling the phenotype in msh-. However, glia cell number is also reduced in the PC.

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