Drawing lines in the Drosophila wing: initiation of wing vein development

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Drawing lines in the Drosophila wing: initiation of wing vein development Ethan Bier It has been proposed that wing veins in Drosophila form at boundaries between discrete sectors of cells that subdivide the anterior–posterior axis of the developing wing primordium. Recently, analysis of events underlying initiation of vein formation suggests that there is a general developmental mechanism for drawing lines between adjacent domains of cells, which is referred to as ‘for-export-only-signaling’. In this model, cells in one domain produce a short range signal to which they cannot respond. As a consequence of this constraint, cells lying in a narrow line immediately outside the signal-producing domain are the only cells that can respond to the signal by activating expression of vein-promoting genes. Addresses Section of Cell and Developmental Biology, Division of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0349; e-mail: [email protected] Current Opinion in Genetics & Development 2000, 10:393–398 0959-437X/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations A–P anterior–posterior dpp decapentaplegic EGF epidermal growth factor EGF-R EGF-receptor kni knirps knrl kni related ri radius incompletus salm spalt major

Introduction Wing veins are hollow, fluid-conducting tubes which form between the two apposed epithelial monolayers of the wing [1,2]. One set of veins (longitudinal veins) runs the length of the wing and another set of veins (cross-veins) run perpendicular to the longitudinal veins connecting them in various locations (Figure 1a). Wing vein cells are more darkly pigmented and densely packed than intervein cells, which comprise the regions between veins. In addition, vein cells survive into adulthood. In contrast, intervein cells adhere to one another via integrins [3,4], expand to assume broad flat pancake-like shapes [2] and die shortly after flies emerge as adults. Veins act as rigid supports in the wing which are necessary for flight [5,6]. In addition to acting as simple struts, veins in many insects determine the positions along which the wing will fold as it moves through the air during a wing beat [5]. As veins provide the only channels of living cells in the adult wing, sensory organs, which are required to coordinate wing beat motions [7], also form along them (e.g. in mosquitoes; Figure 1c). Because of their aerodynamic importance, variations in the spacing and number of veins in different insects (Figure 1a–e) are thought to be highly selected characteristics that are relevant to the evolution of

the diversity of flight modes used throughout the insect world [5,8]. Development of longitudinal wing veins in Drosophila melanogaster can be broken down into two broad periods [1,9–12]. In the first stage, during the third larval instar, vein formation is initiated in the wing imaginal disc — an isolated monolayer of cells. Gene expression in veins is initiated as a series of parallel stripes (Figure 1g, top panel) [11–13]. In the second phase of vein development, which takes place during early pupal stages, the monolayer of wing disc cells buds out (Figure 1g, middle panel) and folds into a bilayer along a line which will become the future margin of the wing (Figure 1g, bottom panel). The stripes of longitudinal vein primordia are bent back on themselves in a hairpin during this process and thereby give rise to vein cells on the prospective dorsal versus ventral surfaces of the wing. The stripes of dorsal and ventral vein cells communicate with one another during pupal development via various inductive signals in order to align precisely to generate a straight uninterrupted fluid tight tube of even diameter [10,14,15]. In addition, cross-veins form during the pupal period. In this review we focus on the first stage of vein development, in which longitudinal vein primordia are induced in a series of sharp stripes running along the edges of domains which subdivide the anterior–posterior (A–P) axis of the wing disc.

Veins form at boundaries along the A–P axis of the wing primordium One of the earliest markers for longitudinal veins is the rhomboid gene, which functions throughout various stages of development to promote localized activation of the EGF-receptor (EGF-R) [11,16–18]. rhomboid expression is initiated nearly simultaneously in all vein primordia during the middle of the third larval instar in straight sharp lines [11,12]. The onset of rhomboid expression in narrow stripes contrasts with the initiation of pair-rule gene expression in stripes along the A–P axis of early embryos. Pair-rule genes, which play critical roles in subdividing the A–P axis of the embryo into segments, are first expressed in broad domains and then refine progressively into sharp stripes one segment wide as borders between adjacent segments are established (reviewed in [19]). The fact that rhomboid expression in the wing disc is initiated in sharp stripes suggests that there are pre-existing well defined borders between discrete domains of cells at this developmental stage and that veins form along the edges of such domains.

The L3 and L4 veins form along the borders of the central organizer of the wing A–P patterning in the wing is initiated by the engrailed gene, which encodes a homeobox-containing transcription

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Insect wings and development. (a) A wild-type Drosophila wing with the major longitudinal veins L1–L6 and margin (M) indicated. (b) A syrphid fly wing. Note that a vein (P3) runs between L3 and L4. A vein does not normally form in this position in the Drosophila wing. In mutants with ectopic veins, however, we propose that a vein can form along this ‘paravein’ border. (c–e) Mosquito (c), psycodid (d) and cranefly (e) wings have more primitive vein patterns than Drosophila. (f) A diagram of an archetypal wing with twice the number of veins as Drosophila. The relationship between veins (L0–L6) and proposed paraveins (P0–P7) in Drosophila to the standard nomenclature for

veins in all insects (in parentheses) is indicated. (g) Overview of vein development in Drosophila. Top panel: vein formation is initiated in a monolayer of wing disc cells during the mid-third larval instar along boundaries between discrete territories of cells. Middle panel: the wing disc buds out during early prepupal development, leading to the apposition of dorsal and vein primordia of the two future wing surfaces. Bottom panel: the adult wing derives from a bilayer of cells comprised of vein and intervein material. All veins have both dorsal and ventral components that must be strictly aligned to create an uninterrupted fluid-conducting tube.

factor expressed in the posterior portion of the wing primordium (reviewed in [20]). Engrailed defines the fates of posterior cells which form a discrete lineage compartment as they do not intermix with cells from the anterior portion of the wing primordium [21]. Engrailed functions to initiate A–P patterning in the wing in part by activating expression of the hedgehog gene, which encodes a signal capable of diffusing several cell diameters (Figure 2a). Engrailed also blocks the response to Hedgehog signaling in cells expressing engrailed. The result of posterior compartment cells producing Hedgehog but not responding to it is that only a narrow stripe of adjacent anterior compartment cells can respond to the signal. This type of signaling, which is restricted to neighboring cells, will be referred to as ‘forexport-only signaling’. Hedgehog signaling activates a variety of genes in the narrow strip of responsive cells, or the ‘central organizer’, including decapentaplegic (dpp), which encodes a long-range patterning morphogen; patched, which encodes a component of the Hedgehog receptor;

vein, which encodes an EGF-related ligand [22,23]; and knot, which encodes a transcription factor [24,25••]. A variety of evidence indicates that the L3 and L4 veins form along the anterior and posterior borders respectively of the central organizer cells expressing patched. Evidence for the anterior border of the patched expression domain defining the position of the L3 vein is that mutant patchedclones of cells located between the L2 and L3 veins are circumnavigated by an L3-like vein (i.e. it is marked with sensory organs normally found only on L3) [26,27]. The posterior edge of the patched domain coincides with the A–P compartment border, along which the L4 primordium forms within the posterior compartment. In addition, double-staining experiments indicate that the anterior edge of the patched expression domain abuts the L3 primordium [28]. Consistent with the view that Hedgehog signaling is responsible for determining the spacing between L3 and L4, increasing levels of Hedgehog results in an anterior

Drawing lines in the Drosophilia wing: initiation of wing vein development Bier

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Figure 2 Veins form at boundaries along the A–P axis of the wing. Veins form at boundaries along the A–P axis of the wing. (a) Engrailed (En), which is expressed in the posterior compartment (green domain), activates expression of Hedgehog (Hh) and at the same time prevents posterior compartment cells from responding to Hh. Some Hh diffuses anteriorly, where it can activate expression of genes in the A–P organizer (blue domain) including knot (kn), vein (vn) and decapentaplegic (dpp). (b) Vn diffuses from the A–P organizer and activates EGFreceptor (EGF-R) signaling in neighboring cells, which will become the L3 and L4 vein primordia. Organizer cells cannot respond to Vn, in part, because kn suppresses expression of EGF-R. Dpp also diffuses from the organizer and functions as a morphogen to activate target genes in a thresholddependent fashion. (c) Moderate levels of Dpp emanating from the organizer activate expression of the spalt major (salm) gene (pink domain). The L2 vein (dark blue line) forms just anterior to the domain of salm expression. (d) Salm activates expression of a hypothetical short-range secreted signal (X) and also suppresses the response to signal X. Only cells abutting the salm-expression domain can respond to this short-range signal by inducing expression of the kni and knrl genes in the L2 primordium. kni and knrl then organize gene expression in and around the L2 primordium.

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displacement of L3 (relative to L4 and the A–P compartment border), whereas decreasing Hedgehog levels causes a posterior shift of L3 [29,30,31•]. A form of for-export-only signaling may be responsible for establishment of the L3 and L4 vein primordia (Figure 2b). In this past year, Jym Mohler et al. have shown that the knot gene, which is allelic to collier [24,25••], plays a role in suppressing vein development between L3 and L4. knot, like patched is activated by Hedgehog signaling between the L3 and L4 veins [25••]. The vein gene is also expressed between the L3 and L4 primordia [23]. vein plays a role in L3 and L4 development as revealed by vein mutants, which have gaps in these two veins. García-Bellido and co-workers used mosaic analysis to show that vein function is required only in anterior cells in order to induce formation of the L4 vein [32], which forms in the adjacent posterior compartment. This critical observation demonstrates that the Vein ligand acts on neighboring posterior cells to induce L4 development and is likely to be functioning as a forexport-only signal. Given that vein expression is restricted to cells between L3 and L4, it is also likely that this gene contributes to inducing L3 development in neighboring

vein-nonexpressing anterior neighbors. Interestingly, expression of EGF-R (which is likely to be activated by Vein), is strongly downregulated between L3 and L4 during the time these primordia become established [33]. Mohler et al. have shown that one mechanism by which knot may act to suppress vein development in the intervein region between L3 and L4 is by repressing Egf-r expression because mis-expression of knot results in a corresponding pattern of Egf-r downregulation [25••]. Consistent with knot functioning to suppress L3 and L4 vein development, ubiquitous knot expression selectively eliminates these two veins. In addition, forced expression of knot in a central stripe of cells which is slightly broader than normal (i.e. that overlaps the L3 primordium), results in an anterior displacement of L3, similar to that observed in response to elevated Hedgehog signaling. Mohler also shows that some features of the knot mutant phenotype can be mimicked by forcing expression of Egf-r between L3 and L4 [25••]. A model for L3 and L4 development consistent with existing data (Figure 2b) [25••] is that the effect of Hedgehog signaling on vein development is mediated by the combined activities of the knot and vein genes. The Vein signal

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promotes vein development by activating EGF-R in cells abutting the central organizer whereas knot suppresses vein formation between L3 and L4, at least in part, by repressing Egf-r expression. The coordinated actions of vein and knot result in for-export-only signaling in which only cells adjacent to those expressing vein and knot can respond to Vein.

The L2 vein is initiated along the anterior border of the spalt major expression domain One of the key genes expressed in the central organizer of the wing that mediates the long-range effects of Hedgehog is dpp. Dpp is believed to either diffuse or be transported across the wing disc and activate various target genes in a threshold-dependent fashion (reviewed in [20]). One Dpp target gene, activated by moderate levels of Dpp signaling [34,35], is spalt major (salm), which encodes a transcription factor [36] expressed in a broad central domain of cells in the wing (Figure 2c). Double-labeling experiments indicate that the L2 primordium forms along the anterior edge of the salm expression domain [28]. Consistent with the anterior salm border playing an instructive role in L2 formation, it has been observed that ectopic L2 veins form within clones lacking function of salm and that these veins closely follow the mutant salm clone borders [28]. The ectopic L2 veins in these clones therefore resemble the normal L2 vein in that they are composed of cells not expressing salm which directly abut salm-expressing cells. These results suggest a for-exportonly form of signaling, in which salm-expressing cells produce a short-range signal to which they cannot respond (Figure 2d). As salm encodes a transcription factor [36], it may activate expression of a short-range secreted factor and also repress expression of a gene(s) required for initiating vein development. Consistent with the idea that salm can repress response to an L2-promoting signal, ubiquitous expression of salm eliminates the L2 primordium [28].

Vein-organizing genes activate vein-specific genetic programs Longitudinal veins share some basic properties such as expression of rhomboid and downregulation of the key intervein gene blistered, which is equivalent to Drosophila Serum Response Factor [37]. There are also genes expressed in some veins but not others. For example, the Delta gene is expressed in all veins but L2 [13], the caupolican and araucan genes are expressed only in the odd-numbered veins [38], and the proneural achaete and scute genes are expressed only in L3 — the only longitudinal vein other than margin in Drosophila having sensory organs [39]. On the basis of these and other gene-expression differences, each vein can be uniquely identified. In addition, gene-expression patterns are altered in a vein-specific fashion in various early acting vein mutants. For example, in radius incompletus (ri) mutants, expression of all vein markers is lost in L2, whereas in abrupt mutants, all L5 vein genes fail to be initiated [31•]. These observations indicate that the development of each vein is directed by a vein-specific genetic program.

One potential basis for vein-specific gene expression programs is that different genes might be activated at vein-inducing boundaries which control vein-specific patterns of gene expression in particular locations. There is good evidence that the neighboring knirps (kni) [40] and knis-related (knrl) genes [41], which encode related proteins steroid-hormone receptor family, perform such a vein-organizing function for the L2 primordium. Analysis of ri mutants, which lack expression of vein markers in L2 and fail to form an L2 vein, indicates that ri is a cis-acting allele of the kni/knrl locus, which eliminates expression of the kni and knrl genes specifically in the L2 primordium [42•]. Results from genetic epistasis experiments are consistent with kni/knrl mediating the vein-inducing effect of the anterior salm boundary (Figure 2d) [42•]. For example, when salm clones are generated in ri mutants, ectopic L2 veins do not form within the clones. In addition, ubiquitous misexpression of salm, which obscures the normal salm anterior border, eliminates kni/knrl expression in L2. Analysis of ri loss-of-function mutants as well as mutants misexpressing kni or knrl revealed several regulatory activities of the kni/knrl genes in L2 which are consistent with these genes controlling expression of various genes in and around the L2 primordium [42•]. These functions include: activation of rhomboid and repression of blistered in the L2 primordium, autoactivation (e.g. kni can activate knrl), repression of salm expression — which presumably helps sharpen the anterior border of salm — and activation of a putative lateral inhibitory activity to suppress vein development in adjacent cells. There is also indirect evidence for an L3 vein-organizing activity. In wild-type wings, a variety of vein markers are expressed in L3, which are expressed in stripes of differing width. Importantly, double-labeling experiments indicate that the centers of each of these vein markers coincide in a sharp line which lies just anterior to the patched expression domain [31•]. Expression of these markers also remains tightly linked under experimental manipulations that shift the position of the L3 primordium either anteriorly or posteriorly (e.g. a coordinated shift of all L3 markers is observed in which the nested patterns of gene expression are strictly maintained) [31•]. An explanation for this strong degree of coupling of L3 gene expression patterns is that there is an L3 vein-organizing gene which controls the expression of each of these genes. Gene expression in the L5 primordium may also be controlled by a vein-organizing gene. The abrupt gene is the candidate for the L5 vein organizing gene since expression of all known L5 genes is lost in abrupt mutants [31•]. There are several important unanswered questions regarding vein initiation. One of the most interesting questions is whether vein organizing genes such as kni/knrl or abrupt determine vein identity (e.g. L2 versus L5 fates) or act in a more limited fashion to promote vein over intervein fates. There are also some important missing links in the genetic pathways controlling vein development. For example, what

Drawing lines in the Drosophilia wing: initiation of wing vein development Bier

is the hypothetical signal produced by salm-expressing cells which induces L2 formation in adjacent cells? In the case of the L3 and L4 veins, are there vein-organizing genes that mediate the effect of knot and vein in establishing these vein primordia? Answers to these and other leading questions should be forthcoming in the near future.

Evolution of the varieties of vein patterns Primitive insects have approximately twice the number of veins as Drosophila (Figure 1d–f) [43,44]. As wings are thought to have evolved only once in insects (e.g. in the pterygota, reviewed in [10]), an interesting question is how did the variety of different vein patterns arise during evolution from the putative common ancestor of pterygotes? It has been generally envisioned that fusion of vein primordia took place during the evolution of insects with fewer than the full primitive pattern of veins [43]. An alternative explanation to the vein fusion hypothesis, is that all insects, including those such as Drosophila with a simplified vein pattern, have the same number of vein-inducing boundaries associated with A–P patterning (e.g. 12–14 boundaries), but that in these derived insects, a subset of these boundaries (which we have termed ‘paraveins’) are silenced with respect to vein initiation [31•] (compare diagram of primitive vein pattern in Figure 1f to that of Drosophila in Figure 1g, lower panel). Several lines of evidence support the silenced paravein view. First, in many Drosophila mutants that have extra veins there is a strong tendency for the ectopic veins to run longitudinally between normal veins [45]. Second, there are gene-expression boundaries in positions running between vein primordia in Drosophila (e.g. the posterior border of the salm expression domain runs between L4 and L5) [28]. In the case of the paravein proposed to run between L3 and L4, a common gene-expression boundary exists in wing primordia of Drosophila and the more primitive syrphid fly [31•] and in the syrphid fly a vein forms along this border (Figure 1b). Finally, the paravein hypothesis provides a ready explanation for an otherwise puzzling feature of insect phylogeny which is that in many different groups of insects in which the founding member of that group clearly had a reduced vein pattern (e.g. the same reduced vein pattern is present in the great majority of insects in that group), there are exceptional species which display the primitive vein pattern. This last fact would be difficult to explain in terms of vein fusion models but can be explained according to the paravein hypothesis by supposing that the hypothetical genetic systems which silence vein formation in paraveins could be mutated to result in the atavistic re-emergence of the primitive vein pattern in which all vein and paravein boundaries induce veins.

Conclusions We have summarized evidence indicating that veins form at boundaries between discrete territories of cells which subdivide the A–P axis of the developing wing primordium. A proposed general model for initiation of vein formation at

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these boundaries is that cells in one domain produce a shortrange signal to which they themselves cannot respond. The consequence of this for-export-only signaling is that only cells outside of that domain, but within range of the signal, can respond by activating expression of a vein-specific organizing gene, which controls gene expression in and around that vein. In addition to veins forming at boundaries, it has been proposed that there is an additional set of boundaries (paravein boundaries) which run between veins in Drosophila and serve as vein-inducing borders in primitive insects which have twice the number of veins as Drosophila.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest 1.

Waddington CH: The genetic control of wing development in Drosophila. J Genet 1940, 41:75-139.

2.

Fristrom D, Wilcox M, Fristrom J: The distribution of PS integrins, laminin A and F-actin during key stages in Drosophila wing development. Development 1993, 117:509-523.

3.

Wilcox M, DiAntonio A, Leptin M: The function of PS integrins during Drosophila wing morphogenesis. Development 1989, 107:891-897.

4.

Zusman S, Patel-King RS, Ffrench-Constant C, Hynes RO: Requirement for integrins during Drosophila development. Development 1990, 108:391-402.

5.

Brodsky AK: The Evolution of Insect Flight. New York: Oxford University Press; 1994.

6.

Dickinson MH, Lehmann FO, Sane SP: Wing rotation and the aerodynamic basis of insect flight. Science 1999, 284:1954-1960.

7.

Dickinson MH: Comparison of encoding properties of campaniform sensilla on the fly wing. J Exp Biol 1990, 151:245-261.

8.

Gulan PJ, Cranston PS: The Insects: an Outline of Entomology. London: Chamman and Hall; 1994.

9.

Díaz-Benjumea FJ, García-Bellido A: Genetic analysis of the wing vein pattern of Drosophila. Wilhelm Roux’s Arch Dev Biol 1990, 198:336-354.

10. García-Bellido A, de Celis JF: Developmental genetics of the venation pattern of Drosophila. Annu Rev Genet 1992, 26:277-304. 11. Sturtevant MA, Roark M, Bier E: The Drosophila rhomboid gene mediates the localized formation of wing veins and interacts genetically with components of the EGF-R signaling pathway. Genes Dev 1993, 7:961-973. 12. Sturtevant MA, Bier E: Analysis of the genetic hierarchy guiding wing vein formation in Drosophila. Development 1995, 121:785-801. 13. Kooh PJ, Fehon RG, Muskavitch MA: Implications of dynamic patterns of Delta and Notch expression for cellular interactions during Drosophila development. Development 1993, 117:493-507. 14. García-Bellido A: Inductive mechanisms in the process of wing vein formation in Drosophila. Wilhelm Roux’s Arch Dev Biol 1977, 182:93-106. 15. Yu K, Sturtevant MA, Biehs B, François F, Padgett RW, Blackman RK, Bier E: The Drosophila decapentaplegic and short gastrulation genes function antagonistically during adult wing vein development. Development 1996, 122:4033-4044. 16. Díaz-Benjumea FJ, García-Bellido A: Behavior of cells mutant for an EGF receptor homologue of Drosophila in genetic mosaics. Proc R Soc Lond B 1990, 242:36-44. 17.

Noll R, Sturtevant MA, Gollapudi R, Bier E: New functions of the Drosophila rhomboid gene during embryonic and adult development are revealed by a novel genetic method, enhancer piracy. Development 1994, 120:2329-2338.

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18. Bier E: Localized activation of RTK/MAPK pathways during development. Bioessays 1998, 20:189-194.

ing vein mutants indicates that different genetic programs control the formation of each vein primordium.

19. Small S, Levine M: The initiation of pair-rule stripes in the Drosophila blastoderm. Curr Opin Genet Dev 1991, 1:255-260.

32. González-Gaitán M, Capdevila MP, García-Bellido A: Cell proliferation in the wing imaginal disc of Drosophila. Mech Dev 1994, 46:183-200.

20. Lawrence PA, Struhl G: Morphogens, compartments, and pattern: lessons from Drosophila? Cell 1996, 85:951-961.

33. Guichard A, Biehs B, Sturtevant MA, Wickline L, Chacko J, Howard K, Bier E: rhomboid and Star interact synergistically to promote EGF-R/MAPK signaling during Drosophila wing vein development. Development 1999, 126:2663-2676.

21. Morata G, Lawrence P: Control of compartment development by the engrailed gene. Nature 1975, 255:614-617. 22. Schnepp B, Grumbling G, Donaldson T, Simcox A: vein is a novel component in the Drosophila Epidermal Growth Factor Receptor pathway with similarity to the neuregulins. Genes Dev 1996, 10:2302-2313. 23. Simcox A, Grumbling G, Schnepp B, Bennington-Mathias C, Hersperger E, Shearn A: Molecular, phenotypic, and expression analysis of vein, a gene required for growth of the Drosophila wing disc. Dev Biol 1996, 177:475-489. 24. Vervoort M, Crozatier M, Valle D, Vincent A: The COE transcription factor Collier is a mediator of short-range Hedgehog-induced patterning of the Drosophila wing. Curr Biol 1999, 9:632-639. 25. Mohler J, Seecoomar M, Agarwal S, Bier E, Hsai J: Activation of knot ·· (kn) specifies the 3-4 intervein region in the Drosophila wing. Development 2000, 127:55-63. The knot gene is expressed between the strip of Hedgehog-responsive cells between the L3 and L4 vein primordia and functions in these cells to suppress vein development. The suppression of vein development by knot is mediated in part by repression of Egf-r expression between the L3 and L4 primordia. A for-export-only signaling model is proposed to account for the presented data wherein knot expressing cells, which also express the EGF-related signal Vein, cannot respond to Vein as a consequence of Egf-r downregulation. 26. Phillips RG, Roberts IAH, Ingham PW, Whittle JRS: The Drosophila segment polarity gene patched is involved in a position-signalling mechanism in imaginal discs. Development 1990, 110:105-114. 27. Nestoras K, Lee H, Mohler J: Role of knot (kn) in wing patterning in Drosophila. Genetics 1997, 147:1203-1212. 28. Sturtevant MA, Biehs B, Marin E, Bier E: The spalt gene links the A/P compartment boundary to a linear adult structure in the Drosophila wing. Development 1997, 124:21-32. 29. Johnson RL, Grenier JK, Scott MP: patched overexpression alters wing disc size and pattern: transcriptional and post-translational effects on hedgehog targets. Development 1995, 121:4161-4170. 30. Strigini M, Cohen SM: A Hedgehog activity gradient contributes to AP axial patterning of the Drosophila wing. Development 1997, 124:4697-4705. 31. Biehs B, Sturtevant MA, Bier E: Boundaries in the Drosophila wing · imaginal discs organize vein-specific genetic programs. Development 1998, 125:4245-4257. Double label experiments with different vein markers expressed in stripes of varying width reveal that all such stripes have coincident centers in wild-type and mutant wing discs in which the L3 primordium is shifted in position relative to L4. Examination of vein and intervein markers in a panel of early act-

34. Nellen D, Burke R, Struhl G, Basler K: Direct and long range action of a Dpp morphogen gradient. Cell 1996, 85:357-368. 35. Lecuit T, Brook WJ, Ng M, Calleja M, Sun H, Cohen SM: Two distinct mechanisms for long range patterning by Decapentaplegic in the Drosophila wing. Nature 1996, 381:387-393. 36. Wagner-Berhomboidlz JT, Wilson C, Gibson G, Schuh R, Gehring WJ: Identification of target genes of the homeotic gene Antennapedia by enhancer detection. Genes Dev 1991, 5:2467-2480. 37.

Montagne J, Groppe J, Guillemin K, Krasnow MA, Gehring WJ, Affolter M: The Drosophila Serum Response Factor gene is required for the formation of intervein tissue of the wing and is allelic to blistered. Development 1996, 122:2589-2597.

38. Gomez-Skarmata JL, del Corral RD, de la Calle-Mustienes E, Ferre-Marco D, Modolell J: araucan and caupolican, two members of the novel iroquois complex, encode homeoproteins that control proneural and vein-forming genes. Cell 1996, 85:95-105. 39. Romani S, Campuzano S, Macagno ER, Modolell J: Expression of achaete and scute genes in Drosophila imaginal discs and their function in sensory organ development. Genes Dev 1989, 3:997-1007. 40. Nauber U, Pankratz MJ, Kienlin A, Seifert E, Klemm U, Jäckle H: Abdominal segmentation of the Drosophila embryo requires a hormone receptor-like protein encoded by the gap gene knirps. Nature 1988, 336:489-492. 41. Oro AE, Ong ES, Margolis JS, Posakony JW, McKeown M, Evans RM: The Drosophila gene knirps-related is a member of the steroidreceptor gene superfamily. Nature 1988, 336:493-496. 42. Lunde K, Biehs B, Bier B: The knirps and knirps-related genes · organize development of the second wing vein in Drosophila. Development 1998, 125:4145-4154. The knirps and knirps-related genes are expressed in coincident narrow stripes in the L2 primordium. The knirps/knrl genes, which are required to meditate the effect of salm in inducing formation of the L2 primordium, organize the expression of several genes involved in L2 development in and about the L2 primordium. 43. Colless DH, McAlpine DK: Chapter 34 — Diptera. In The Insects of Australia. Carlton, Victoria: Melbourne University Press; 1970. 44. Nijhout HF: The Development and Evolution of Butterfly Wing Patterns. Washington DC: Smithsonian Institution Press; 1991. 45. Thompson JN Jr: Studies on the nature and function of polygenic loci in Drosophila. II. The subthreshold wing vein pattern revealed in selection experiments. Heredity 1974, 33:389-401.