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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00608

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defective proventriculus is required for pattern formation along the proximodistal axis, cell proliferation and formation of veins in the Drosophila wing

Stefan Kölzer1, Bernhard Fuss2, Michael Hoch2 and Thomas Klein1,*

1 Institut für Genetik, Universität zu Köln, Weyertal 121, 50931 Köln, Germany
2 Institut für Zoologie, Abteilung Entwicklungsbiologie, Universität zu Bonn, 53115 Bonn, Germany



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  		Fig. 1. The wing phenotype of dveP1738-mutant flies. All wings are oriented proximal to the left and anterior to the top. (A) A wild-type wing. The arrowheads highlight the distance between the end of the medial costa and the anterior cross-vein. The longitudinal veins are numbered 2-5. The arrow indicates a small spot of vein material present in the proximal part of the wing blade. (B) A dveP1738-mutant wing. The arrows indicate regions where the longitudinal veins 2 and 5 are interrupted. Note that the distance between the arrowhead in the PW and that at the anterior cross-vein is strongly reduced in comparison with the normal wing. Furthermore, the wing is smaller than the wild-type wing. (C) A magnification of the PW of a wild-type wing. The anterior margin of the PW is subdivided into three easily distinguishable regions: the proximal, medial and distal costa. (D) The PW of a dveP1738-mutant wing. Most of the distal costa is deleted leaving only a very small remnant intact. Furthermore, the small spot of vein material, which can be observed in the adjacent wing blade (highlighted by the arrow in A and C), is deleted in the mutant wing, indicating that a small stripe of the adjacent wing pouch is also deleted. These observations indicate that Dve is required for the formation of a region encompassing most of the distal PW and a small part of the adjacent pouch. This region is highlighted by the pink box in A. The deletion causes the reduction of the distance between the anterior cross-vein and the PW.

 


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  		Fig. 3. Analysis of the proliferation defect of cells in dveP1738-mutant wing pouches. (A) An overlay of a wild-type (grey) and mutant (red) wing. The double-ended arrows show the distances between wing vein 3 and the anterior wing margin (labelled a), between vein 3 and vein 4 (labelled b), and between vein 4 and the posterior margin (labelled c). Distances are counted in cell numbers. The cell density was measured in three different areas (labelled d-f). The results are summarized in the table below (d-f, n=14; a and c, n=4; b, n=14 wings counted for each genotype). In the region between vein 3 and vein 4, no differences in the distance (b) and the cell density (e) were observed. These results confirm the observation that both wings are of the same size in this region, as seen by the overlay of the wings. By contrast, anterior to vein 3 and posterior to vein 4 the distances in the mutant wings are shorter (a,c). Furthermore, the cell density is similar in the anterior area (see d) and even slightly lower in the posterior area (see e) in the mutant. This indicates that the observed reduction in size of these areas in the mutant is caused by having fewer cells. (B-F) Clonal analysis of dveP1738. Clones were induced using hsFlp, and the wing imaginal discs were prepared 48 hours after heat shock. Discs are stained with anti-Dl and anti-ß-Gal antibodies. (B) dveP1738-mutant clones revealed by the absence of the GFP marker. (C) The same disc as in B showing the expression of ß-Gal. The expression of ß-Gal is complementary to that of GFP, showing loss of staining in the wild-type clones and stronger staining in the dveP1738-homozygous clones. (D) Expression of Dl (blue), GFP (green) and ß-Gal (red). The expression of Dl reveals the primordia of wing veins 3-5. The arrow indicates a wild-type clone with no obvious mutant counterpart. The arrowhead (B,D) points to a twin pair of clones in which the size of the mutant clone is dramatically reduced in comparison to its wild-type twin. (E) Expression of Dl in the disc also shown in B-D. The numbers highlight the primordia of the wing veins 3-5. A1-A3 labels the different areas in which the clones have been analysed. The results of this analysis are summarized in Table 1. (F) Another example of a dveP1738-mutant clone bearing wing imaginal disc, showing expression of Dl (blue), ß-Gal (red) and GFP (green). The arrows indicate a pair of clones that are separated by a band of heterozygous cells. This separation has been found in a few cases. Arrowheads indicate a pair of clones that are adjacent to each other.

 


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  		Fig. 2. Comparison of the wing region of a wild-type and dveP1738-mutant wing imaginal disc, in animals of the late third larval instar stage and of the early pupal stage. The discs are double stained with anti-ß-Gal (green) and anti-Wg (red) antibodies. (A) A wild-type wing imaginal disc of the late third larval instar stage. (C) A dve-mutant wing imaginal disc of the late third larval instar stage. Anterior is to the left; ventral to the bottom. The arrow (A,C) indicates the wg-expression domain along the DV boundary. ir, the inner ring-like expression domain of wg in the PW. The comparison of A and C reveals that the wing pouch of the dve-mutant wing disc is smaller. As a consequence the distance between the DV boundary and the inner ring-like domain of wg expression is reduced. (B,D) The defects become more obvious in wing imaginal discs of the early pupal stage. The dve-mutant wing (D) is smaller than its wild-type counterpart (B). The arrow highlights an indentation in the anterior margin of the mutant wing. Furthermore, the proximal fold, which is labelled by the asterisk (B,D) is smaller in the dve mutant (compare the distance between the two arrowheads in B and D).

 


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  		Fig. 4. Expression of dve during normal wing development. Wing imaginal discs were stained for Wg (red) and Dve (green). Anterior is to the left; ventral to the bottom. (A-C) A wing imaginal disc of the late second larval instar stage. Wg is expressed in a ventral area of the disc (B). At this time, Dve is not expressed (A,C). (D-F) A wing imaginal disc of the early third larval instar stage. Wg expression (E) resolves into a ring-like domain that is bisected by a stripe of Wg expression along the future DV compartment boundary (arrow). At this time, expression of Dve is initiated (D) and is observed in the region that is framed by the ring-like domain of Wg expression (F). (G-I) A wing imaginal disc of the late third larval instar stage. Wg is expressed in two ring-like domains and along the DV boundary (arrow and arrowhead in H). During this phase, Dve continues to be expressed in the area framed by the inner ring-like domain of wg expression (G,I). Note that Dve expression is suppressed at the DV boundary, with exception of a short stretch at the anterior side (arrowhead in G). Dve expression is further lowered in the region of the primordia of wing veins 3 and 4 (arrows in G). (J) No specific staining is observed in dveP1738-mutant wing imaginal discs of the third larval instar stage following anti-Dve antibody staining. This suggests that in dveP1738 is a null allele of the locus for wing development. (K) Expression of ß-Gal in the disc shown in J.

 


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  		Fig. 5. Comparison of the expression domain of dve with that of other genes involved in the patterning of the Drosophila wing along the PD axis. In C,D,F,G,I, anterior is to the left; ventral to the bottom. In A,B,E,H,J, discs of the early pupal phase are shown where the wing has everted, revealing the PD axis. In these images anterior is up; distal to the right. The everted wing helps to clarify the limits of the examined expression domains in relation to the expression of Wg. The arrow highlights the inner (IR) and the arrowhead the outer (OR) ring-like domain of Wg expression in the PW. (A) Persistent ß-galactosidase activity in adult flies carrying a P-lacZ insertion in the wg locus. The staining allows the determination of the structures that arise from the regions of Wg expression. It reveals that the ring-like domains of wg expression at the late third larval instar stage (arrow and arrowhead in Fig. 4H) label the anlagen of the proximal and medial regions of the PW (compare also with B). These observations suggest that the region between the IR and the actual wing pouch is the anlage of the distal region of the PW. (B) A dveP1738/+ wing imaginal disc of the early pupal phase, stained with anti-ß-Gal antibody, to reveal the expression of dve (green), and anti-Wg antibody, to reveal the expression of Wg (red). The expression domain of dve reaches close to the IR. (C) Expression of vg-QE (green), revealed by anti-ß-Gal, and Dve (red), revealed by anti-Dve antibody staining. The expression domain of Dve is larger than that of the vg-QE. (D,E) Expression of the vg-QE (green) relative to that of Wg (red). The expression domain of vg-QE is restricted to the wing pouch and a broad band of non-expressing cells separates it from the IR (arrow). (F) Anti-Nub (red) anti-Dve (green) double-antibody staining of a wing imaginal disc of the late third larval instar stage. The double staining reveals that the disc-like expression domain of Nub is larger and includes that of Dve. (G,H) Anti-Nub anti-Wg double staining. (G) A wing imaginal disc of the late third larval instar stage, showing Wg (green) and Nub (red) expression. (H) A wing imaginal disc in the early pupal phase stained with anti-Wg (red) and anti-Nub (green) antibodies. G and H reveal that the border of the Nub expression domain lies between the two ring-like domains of Wg expression. (I) Expression of fj, revealed by anti-ß-Gal staining (green), and Wg, revealed by anti-Wg antibody staining (red). fj is expressed in the wing pouch in a similar domain to Vg and is not expressed in the distal region of the PW. Thus, the expression domain is smaller than that of Dve. (J) A wing imaginal disc of the early pupal stage containing an rn-lacZ insertion to reveal the expression of rn. Anti-ß-Gal (green) anti-Wg (red) double staining reveals that the boundary of the rn expression domain is identical to that of the IR. Thus, the expression domain of rn is larger than that of Dve. (K) Summary of the comparison. The proximodistal extent of the expression domains are depicted as follows: Vg/Fj, turquoise; Dve, blue; Rn, red; the IR, pink; Nub, green; and the outer ring-like domain of Wg, mauve. The cartoon highlights the fact that the tested genes are expressed in ring-like (Wg) or disc-like (Dve, Nub, Fj and Vg) domains of different sizes. The size of the domain increases from Vg/Fj to Dve to Rn, and from the inner ring-like domain of Wg to Nub to the outer ring-like domain of Wg. The result of these different expression domains is the definition of concentric regions with different combinations of gene activities that probably define the different regions of the PW.

 


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  		Fig. 6. The regulation of the expression of dve. Anterior is to the left; ventral to the bottom. In all images, expression of Dve and Wg is revealed by antibody staining. (A-C) Vg is sufficient to initiate expression of dve in the wing area. (A) Expression of Dve (green) is lost in a vg83b27R-mutant wing disc, indicating that the function of vg is required for the induction of expression of dve. Red staining shows the expression of wg. In vg83b27R-mutant wing discs, only the weaker outer ring-like expression domain of Wg is present. The weak punctuate green staining is unspecific background staining. (B,C) A wing imaginal disc of the late third larval instar stage bearing Vg-expressing cell clones. The clones of UAS-vg expressing cells were induced with the help of the AyGal4-UAS-GFP chromosome during the second larval instar and are labelled by the green GFP marker in C. (B) Expression of Dve. The arrows indicate Vg-expressing clones located outside the normal Dve expression domain. (C) Pseudo-colour image of the same disc as in B, revealing the Vg-expressing cell clones in green and expression of Dve in red. The double staining reveals that Vg-expressing clones can induce ectopic expression of Dve in the PW (see arrows) and in the pleura (arrowhead). Note that Vg can induce expression of Dve in adjacent non-expressing cells (see clones highlighted by the arrows), indicating that the induction of Dve expression occurs in a non-autonomous manner. The ability of Vg to induce expression of Dve is restricted to certain regions of the wing, indicating that additional factors are required in other regions. (D-I) Negative regulation of dve expression by the Notch and wg pathways. (D) Expression of Dve in a wing imaginal disc where UAS-Nintra is activated by dpp-Gal4 in a medial band of cells perpendicular to the DV boundary (arrows). Expression of Dve is suppressed in the region where Nintra is expressed (highlighted by the arrows). (E) Expression of UAS-wg by dpp-Gal4 results in a similar suppression of the expression of Dve. (F-I) Expression of Dve in wing imaginal disc of the late third larval instar stage bearing arr2-mutant cell clones. (F,H) Dve expression. (G,I) Pseudo-colour image of the same wing discs as in F and H, respectively, including the green channel to reveal the mutant clones of mutant cells through the absence of GFP fluorescence. Expression of Dve is shown in red. The comparison of F,H with G,I shows that expression of Dve is elevated in arr2-mutant cells (arrows in F-I). The elevation is observable in cells of clones at the DV boundary (arrows in H,I) and also in mutant cells that are many cell diameters away from the Wg source at the DV boundary (arrows in F,G). (J-L) Expression of Dve and Wg in a nub1-mutant wing imaginal disc. (J) Expression of Wg in a nub-mutant wing imaginal disc of the late third larval instar stage. (K) Expression of Dve in the same disc as shown in J. (L) Merged view of both channels shown in J and K, showing Wg expression in red and Dve expression in green. The double staining reveals that expression of dve at the DV boundary is not suppressed in most of the regions (arrow in J-L). This suggests that Nub is required to suppress the expression of Dve at the DV boundary. (M) Expression of Dve (green) is not affected in a spadeflg-mutant wing imaginal disc. Red shows the expression of Wg and reveals that the inner ring-like domain of expression is lost. (N) Expression of Nub is unaffected in dveP1738-mutant wing imaginal discs.

 


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  		Fig. 7. The consequences of ectopic expression of dve. (A-D,F,G) Wing imaginal discs bearing clones of UAS-dve expressing cells in the wing area. The Dve-expressing clones were induced with help of the Flip-out method, using hsFlp and the AyGal4-UAS-GFP constructs, during the second larval instar stage and labelled by the green fluorescence of the GFP. Expression of all genes is detected by antibody staining. (A-C) Expression of dveP1738-lacZ and Nub in a wing imaginal disc bearing Dve-expressing clones. (A) Expression of dveP1738-lacZ. (B) Expression of Nub. (C) The same wing imaginal disc as shown in A and B, showing the Dve-expressing clones in green, expression of Nub in blue and expression of dveP1738-lacZ in red. Several effects are observed. First, clones in the dve expression domain but outside the wing blade cause the formation of folds around the clone (arrowheads in A-C). This suggests that elevation of Dve expression in the PW can locally enhance the proliferation in the surrounding cells. Secondly, with a low frequency, we find that expression of Dve can non-autonomously induce ectopic expression of dveP1738-lacZ and nub (see arrow in A-C). The ectopic Dve-expressing clone (above the arrow in C) is located at the hinge/notum boundary and expression of both genes is extended towards the clone. The pseudo-colour image shown in C reveals that the ectopic expression of Nub occurs in a larger domain than that of Dve, as is the case during normal development. (D) In a similar manner, we find that the expression of the inner ring-like domain of wg is correspondingly expanded. Expression of Wg is shown in red, the clones of Dve expressing cells are shown in green. The arrow indicates a clone near the inner ring-like expression domain of Wg. The clone induces an expansion of the inner ring-like expression domain of Wg in a similar manner to that shown for Nub and dveP1738-lacZ (see A-C). The third effect caused by the Dve-expressing clones is highlighted by the asterisks in A and C. Elevation of Dve expression suppresses the expression of ß-Gal, suggesting the existence of a negative feedback of Dve on its promoter. This negative influence of Dve on the activity of its promotor can also be observed when Dve is expressed with dpp-Gal, as shown in E. (E) Expression of UAS-dve with dpp-Gal4. Ectopic expression of dveP1738-lacZ and of Wg is shown in green and red, respectively. Expression of Dve suppresses the expression of dveP1738-lacZ (arrow). In addition expression of wg in the PW is interrupted in the domain of dpp-GAL4. (F,G) Forced expression of Dve at the DV boundary abolishes the expression of wg. (F) Expression of Wg. (G) Expression of Wg (blue) and dveP1738-lacZ (red) in a disc bearing Dve-expressing clones (green). A comparison of F with G reveals that wg expression is interrupted (arrows in F) in the Dve-expressing clones. (H) Expression of UAS-dve with sd-Gal4 abolishes expression of Wg in the wing. Arrow indicates the wing area, which is poorly developed and does not exhibit any expression of Wg.

 


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  		Fig. 8. The genetic hierarchy that controls pattern formation along the PD axis. For the sake of simplicity, the contribution of the AP patterning system is not included in the cartoon. (A) In the first step, the activity of Wg (red) and Dpp (not shown) define the position and extent of the wing field. Within this field, the Notch pathway induces the expression of Vg along the DV boundary. Wg and possibly Vg are required to induce the expression of Nub (blue) in a disc-like domain slightly larger than that of the early Wg domain (Ng et al., 1996Go; del Alamo Rodriguez et al., 2002Go). (B) Vg associates with Sd to form a bipartite transcription factor (Vg/Sd) that controls the expression of an unknown diffusible factor. This factor activates the expression of dve (light green) and rn (yellow) independently from each other and in domains of different sizes. The combined activity of Nub and Rn activate the inner ring-like domain of wg expression (dashed red circle). Dve, together with Nub and other factors, establishes the distal region of the PW, which is defined along the PD axis by the length of the medial and distal costal regions. Wg organizes the formation of the medial part of the PW. At the DV boundary, Vg/Sd activates the expression of Wg together with the Notch pathway. Wg suppresses the expression of Dve in cells near the DV boundary. (C) The cells at the DV boundary divide and the daughter cells that are displaced from the boundary form the wing pouch. Expression of Vg in the pouch cells is maintained by Wg, secreted from the DV boundary, and Dpp, secreted from the AP boundary (not shown). As a consequence, Vg is expressed in a gradient with the peak at the DV boundary. In the nascent pouch, Vg activates the expression of several patterning genes, such as Dll, and together with Wg, the genes of the achaete-scute complex, adjacent to the cells at the DV boundary (Neumann and Cohen, 1997Go; Zecca et al., 1996Go; Klein and Martinez-Arias, 1999Go), and fj. The activation of these targets is indicated by the three red arrows in the lower drawing of Step C. Fj is required for the establishment of a region within the pouch, and for the generation of planar polarity of the wing. The cartoon highlights the fact that all of the genes controlled by Vg/Sd are expressed in disc-like domains of different sizes. Their expression leads to concentric areas with different combinations of gene activities. These combinations are likely to establish different parts of the PW (see Step C).

 

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© The Company of Biologists Ltd 2003