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doi: 10.1242/10.1242/dev.00276

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A re-evaluation of the contributions of Apterous and Notch to the dorsoventral lineage restriction boundary in the Drosophila wing

European Molecular Biology Laboratory, Meyerhofstr 1, 69117 Heidelberg, Germany

View larger version (70K): [in a new window]   Fig. 1. Cell interactions at the DV boundary. (A) Establishment of the signaling center and the DV affinity boundary. Ap (in red) induces Serrate (Ser) and Fringe (fng) expression in dorsal (D) cells and restricts Delta (D1) expression to ventral (V) cells. Ser signals to D cells and D1 to V cells to activate Notch along the DV boundary. Fng modifies Notch (N*) in D cells, thus making it sensitive to D1 but not to Serrate. Capricious (Caps) and Tartan (Trn) expression in D cells contribute to establishing the DV affinity boundary. Caps and Trn have been proposed to interact with an unknown partner (indicated by ?) expressed in D cells. (B) Maintenance of the signaling center and the affinity boundary. A positive-feedback loop between Wingless (Wg)-expressing cells along the DV boundary and Ser- and D1-expressing cells in adjacent cells maintain the signaling center along the DV boundary. Caps and Tartan are not asymmetrically expressed at late stages; thus, maintenance of the DV affinity boundary is independent of Caps and Tartan activity. (C) Late third instar wild-type wing disc labeled to visualize expression of Apterous (red) and Wingless (blue).

View larger version (107K): [in a new window]   Fig. 2. Fringe and the DV affinity boundary. Wing discs of the following genotypes labeled to visualize Wg protein (blue) and Gal4 protein (red). (A) apGal4/apGal4. (B) apGal4/apGal4; EP-fng. (C) apGal4/apGal4; UAS-fng. (D) apGal4/aprk568. (E) apGal4/aprk568; EP-fng. (F) apGal4/aprk568; UAS-fng. (G) apGal4/apUGO35. (H) apGal4/apugo35; EP-fng. (I) apGal4/apugo35; UAS-fng. (J) Wing disc of apGal4/aprk568; UAS-Ap labeled to visualize expression of Apterous (red) and Wingless (blue).

View larger version (83K): [in a new window]   Fig. 3. Cell affinities and sorting at the DV boundary. (A,D,G) Late third instar wing discs labeled to visualize expression of Engrailed protein (green) and an ap-lacZ reporter gene (anti-ß-gal, red). (B,E,H) Late third instar wing discs labeled to visualize expression of Wingless protein (blue) and ap-lacZ (red in B,E) or LMO (red in H). a, anterior; p, posterior; d, dorsal; v, ventral. (C,F,I) Drawing showing the relative locations of the AP compartment boundary (green) and the DV boundary (red). (A-C) Wild-type, (D-F) en-gal4; UAS-ap and (G-I) en-gal4; UAS-fng.

View larger version (49K): [in a new window]   Fig. 4. Cell affinities at the DV boundary. (A) Wild-type (lacZ), Chip or fringe (fng) mutant clones located in the D compartment and marked by the absence of the lacZ marker (red). Apterous (Ap+, red) expressing clone located in the V compartment. Wingless expression (blue). d, dorsal; v, ventral compartments. Note that wild-type clones were elongated with irregular borders, Chip clones and Apterous expressing clones were round with smooth borders, and fringe clones were intermediate in shape with irregular borders. (B) The 4 A/L2 ratio of D clones lacking GFP, Chip or fringe, or expressing LMO, and V clones expressing Apterous or Fringe. GFP, 0.4±0.1, n=35; dorsal Chip mutant, 0.7±0.1, n=15; dorsal LMO expressing, 0.8±0.1, n=16; ventral Apterous expressing, 0.8±0.1, n=19; dorsal fringe mutant, 0.5±0.1, n=25; ventral Fringe expressing, 0.5±0.1, n=14. Error bars indicate standard deviation. (C) Clones expressing Notchintra and ß-gal (red) in the D compartment. Wingless is shown in blue. Clones did not `round up' and had irregular borders.

View larger version (88K): [in a new window]   Fig. 6. Sorting out across the DV boundary. (A-C) Schematic representation of three types of clone behavior with respect to the DV boundary. Clones are outlined in black. Dorsal (d) cells are depicted in red; ventral (v) cells are depicted in gray. Notch activation and Wg expression is depicted in blue. (A) Clones respecting the DV boundary are elongated in shape with irregular borders, except when they abut the DV boundary. The endogenous DV boundary (between red and gray cells) corresponds to the stripe of Wg expression. (B) A clone of ventral cells that crossed the DV boundary into the D compartment. Under these conditions, the endogenous DV boundary does not correspond to the stripe of Wg expression. (C) A clone of ventral cells with D signaling properties that displaced the Wg stripe but did not cross completely into the D compartment. (D-I) Late third instar wing discs labeled to visualize expression of the ap-lacZ reporter gene (antibody to ß-gal, red) and Wingless (blue). ap-lacZ labels the D compartment. Dorsal (d) and ventral (v) compartments are indicated. (D) Clone of cells expressing GFP and born in the D compartment. (E) Clone of cells expressing. Ap (green) that was induced before the onset of ap-lacZ expression. Cells born in the dorsal compartment (red+green) or born in the ventral compartment (green) were located on the dorsal side of the Wg stripe and mixed with D cells. Thus, the clone crossed from the V into the D compartment (arrow). (F) Clone of cells expressing LMO (green) induced before the onset of ap-lacZ expression. Cells born in the dorsal (red+green) or the ventral (green) compartment were located on the ventral side of the Wg stripe and mixed with V cells. Thus, the clone crossed from the D into the V compartment (arrow). (G) Clone of D cells expressing LMO (green) induced after the onset of ap-lacZ expression (red+green) and located on the ventral side of the Wg stripe. (H) Ventral clone expressing Fng-myc labeled by the expression of Myc protein (green). The clone was born in the V compartment and displaced the Wg stripe toward ventral. Arrow in H indicates direction of crossing. (I) Dorsal clone mutant for fng labeled by the absence of the GFP marker (green). The clone was born in the dorsal compartment (red) and displaced the Wg stripe toward dorsal. (J) Dorsal clones expressing Fng-myc and LMO labeled by expression of LMO (green). (K,K') Dorsal clones expressing LMO and Necd (LMO in green). Note clones born in the dorsal compartment (arrowheads) remain at the dorsal side of the Wg stripe (purple)

View larger version (38K): [in a new window]   Fig. 5. Cell affinities at the DV boundary require Notch activation. (A) Clones expressing LMO (green). Cut is shown in purple. Dorsal clones were round and induced Cut expression in the clone (white) and in neighboring cells (purple). (B) Clones of cells expressing LMO and Fringe marked by the expression of GFP (green). Clones located in the dorsal compartment did not induce Wg expression (purple), were elongated and had irregular borders (arrow). (C,D) Clones of cells expressing LMO (green) and Necd. (C) Notch activation was blocked in the clone, as shown by absence of Cut expression. (D) When Notch was also blocked in the neighboring cells the shape of the clones became irregular and elongated. (E) Histogram indicating the 4 A/L2 ratio of clones lacking GFP; dorsal clones expressing LMO, LMO and fng, or LMO and Necd; ventral clones expressing Ap or Ap and Necd. GFP, 0.4±0.1, n=35; dorsal LMO expressing, 0.8±0.1, n=16; dorsal LMO and Fng expressing, 0.3±0.1, n=12; dorsal LMO and Necd expressing, 0.4±0.1, n=16 clones without Wg expression in neighboring cells; dorsal LMO and Necd expressing, 0.8±0.1, n=14 clones with Wg expression in neighboring cells; ventral Apterous expressing, 0.8±0.1, n=19; ventral Ap and Necd expressing, 0.8±0.1, n=12. Error bars indicate standard deviation. d, dorsal; v, ventral compartment.

View larger version (38K): [in a new window]   Fig. 7. Maintenance of the DV affinity boundary. (A-G) Dorsal cells are depicted in red, ventral in white. Notch activation is depicted in blue. Notch is activated in D and V cells. Apterous (Ap) activity in dorsal cells provides the underlying asymmetric cue. (B-D) Three models proposed to explain the activity of Notch and Ap in inducing a DV affinity difference. d* indicates a dorsal affinity state at the boundary; v* indicates a corresponding ventral affinity state. The model shown in B proposes that Fng acts on Notch and independently of Notch on another gene to induce the d* state (Rauskolb et al., 1999; O'Keefe and Thomas, 2001). According to this view, Fringe should be sufficient to induce a DV affinity difference. (C) Micchelli and Blair's model proposes that dorsal and ventral cells have the same cell affinity (x) in the absence of Notch activation. Thus, confrontation of boundary cells and cells in the wing blade should induce an affinity boundary. (D-G) We propose that Ap controls d and v affinity molecules, but that these require Notch activity to produce a sustained d* or v* affinity state. According to this model, Notch is required but cannot generate an affinity state on its own. Only interactions between cells of opposite compartments are able to induce an affinity boundary. (E) In the absence of Notch or Ap activity cell states d* and v* are not defined and the affinity boundary is not properly maintained. (F) Interaction between cell states d* or v* with cells of the opposite compartment is sufficient to induce an affinity difference. (G) fng mutant clones in the D compartment are not expected to confront d* and v* states and therefore cannot induce an affinity difference comparable with that produced by removing Ap activity (as in F). (H) A fng mutant clone in the D compartment (depicted in green) is expected to displace the Notch activity stripe (blue) around the clone. Notch induces symmetric growth, pushing the clone into the V compartment. Notch is not induced at the interface between the clone and the V compartment.