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Wing disc development in the fly: the early stages
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Wing disc development in the fly: the early stages Thomas Klein The establishment of the wing anlage in Drosophila is dependent on the presence of two organizing centers located at the boundaries of the dorsoventral and anteroposterior compartments. How these boundaries are defined was not understood until recently. Furthermore, nothing was known about how the hinge region of the wing is defined. Recent data have now started to provide some insight in the molecular processes required for the definition of the major boundaries and subdivision of the wing anlage into the hinge and blade region. Addresses Institut für Genetik, Universität zu Köln, Weyertal 121, 50931 Köln, Germany; e-mail: [email protected] Current Opinion in Genetics & Development 2001, 11:470–475 0959-437X/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations ap apterous A-P anteroposterior Dpp Decapentaplegic D-V dorsoventral EGF-R epidermal growth factor receptor en engrailed Hh Hedgehog hth homothorax iro-C iroquois gene complex Su(H) Suppression of Hairless tsh teashirt vg vestigial vgQE vg quadrant enhancer Wg Wingless
Introduction Multicellular organisms often form body wall appendages for specialized functions such as locomotion or flying. Recent studies on the development of the Drosophila wing are providing an insight into the genetic and molecular network orchestrating initiation, subdivision and patterning of these appendages. The knowledge gathered from these studies has provided the vertebrate field with starting points to identify molecules important for the development of vertebrate limbs (see Tickle and Münsterberg, this issue [pp 476–481], for a review of vertebrate limb development). It transpires that the molecular basis for the formation of vertebrate limbs is remarkably similar to that of the Drosophila wing [1]. In this review, I focus on recent work that has started to clarify some of the less well understood aspects of Drosophila wing development. The wing in Drosophila develops from one of the imaginal discs from which most of the adult body is assembled. Imaginal discs are monocellular epithelial layers that consist of undifferentiated, proliferating cells. The wing imaginal disc comprises ~20 cells when it is formed during embryonic development. These cells proliferate during
the three larval stages to generate a disc of ~75,000 cells in the late third instar (~96h after hatching). The disc is basically a single cell layered epithelium, thus pattern formation occurs in a two-dimensional layer. This presents a problem, one also shared by a painter: incorporating the third dimension in a two dimensional sheet. It is solved by organizing the wing primordium in a concentric way with the distal structures (wing blade and margin) in the center and the proximal structures (hinge) at the periphery (Figure 1). By the late third instar, the wing primordium is established and one can identify its major elements, hinge, blade and margin, with the help of appropriate molecular markers (see Figure 1). However, the processes leading to its formation start at the beginning of the third larval instar (~48h after hatching) and are controlled by two major patterning centers, that are established at the boundaries of the dorsoventral (D-V) and anteroposterior (A-P) compartments. Several genetic screens have identified important genes controlling wing development: chief among them engrailed (en), apterous (ap), vestigial (vg), and the genes that play a role in the Notch (N) Decapentaplegic (Dpp), Wingless (Wg) and Hedgehog (Hh) signaling pathways. The function of these genes and signaling pathways during establishment of the wing anlage (hinge, blade and margin) has been covered amply in other reviews and is summarized briefly in Figure 2. In the past year, another signaling pathway could be added to the list, the epidermal growth factor receptor (EGF-R) pathway [2••,3••]. This pathway had only been implicated in very late patterning processes in the wing, such as vein development, but recent findings show that it has several important functions during early wing disc development that help define distinct cell populations. These are important because the organizing centers are established at the boundaries of these populations. I discuss the function of the EGF-R pathway below and shortly summarize some recent work that has started to illuminate the definition and subdivision of the hinge region of the wing.
Early subdivision of the wing disc: notum versus wing Mutations in the gene wg suggest that the first step of wing development is to define a region in the wing disc where the wing will form. Flies mutant for wg display a very severe wing phenotype, namely a complete loss of all wing structures replaced by a tiny duplication of notal structures (wing to notum transformation) [4,5]. Conversely, ectopic expression of wg at the correct time leads to the ectopic induction of wing structures in the notum [6,7]. This places wg at the top of the genetic hierarchy that controls wing development and suggests that it is involved in defining
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Figure 1 The adult structures formed by the wing disc of Drosophila. Orientation is anterior to the left and ventral to the bottom. (a) Major elements of the wing anlage highlighted by Wg expression in a late third instar wing disc. Wg is expressed in two ring-like domains in the hinge region and along the dorsoventral compartment boundary (D-V boundary) dividing the wing blade. The inner ring-like domain frames the wing blade. The domain along the D-V boundary corresponds to the area in which the wing margin will form. Wg is also expressed in a broad band in the dorsal part of the disc, which will become the notum of the fly. As the disc is a two-dimensional structure, the third dimension must be integrated in this level with help of the two existing axes. The anlage of the wing is therefore organized in a concentric way with the distalmost point in the center of the anlage and the proximal parts located around it. During pupation, the wing evaginates and the dorsal and ventral part of the wing adhere together. The evagination transforms the two dimensional anlage into (b) a three-dimensional structure (in which wg expression in an adult fly is depicted). The persisting pattern of wg expression reveals the adult structures in whose anlage it is expressed at late third larval instar. All the major structures of the wing are recognizable: the blade comprising the dorsal and ventral layer of the pouch adhering to each other, the margin running around the blade at the boundary of the dorsal and ventral part of the pouch and the hinge that connects the wing blade to the body.
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the area where the wing will form. In analogy to vertebrate terminology, I will here call this area the wing field. The wing field is defined during the second larval instar (24–48h after hatching) at a time when wg is expressed in the ventral region of the disc (Figure 2a). The expression of wg is controlled by the Hh signaling pathway, which is also required to establish the A-P organizing center [8]. The wing disc does not only form the wing — it also forms half of the notum (see Figure 1). Recent work now reveals that the formation of this part of the disc is dependent on the activity of the EGF-R signaling pathway [3••] (Figure 3a). During this process, the pathway is activated by the secreted neuregulin-like signaling protein Vein [3••,9,10]. Vein is expressed in the dorsal part of the disc and loss of its function during the second larval instar leads to the loss of all notal structures, a phenotype which is complementary to that of wg mutants [3••,9]. Conversely,
ectopic activation of the Vein/EGF-R pathway in the wing field prevents wing formation and promotes formation of notal structures [2••,3••]. Vein is restricted to the dorsal part of the wing disc by the suppressive influence of wg, which suppresses the expression of Vein, in the ventral region of the disc [3••]. Conversely, wg expression extends dorsally if the EGF-R pathway is interrupted in the disc, suggesting that the pathway suppresses wg expression in the dorsal part of the disc [2••,3••]. This antagonistic relationship between the Wg and EGF-R pathways helps to divide the early disc into notum and wing regions. It explains why a wing → notum duplication occurs in the absence of wg function: In wg mutant wing discs, Vein is expressed ectopically in ventral regions and promotes the development of notal fates there [3••]. It is not yet clear whether the wing fate is the default state of the wing disc and the function of wg is solely to prevent activation of the Vein/EGF-R pathway in the ventral region of the disc.
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Establishment and maintenance of the Ap and En expression domains in the wing disc One important principle that has emerged from study of the development of the Drosophila wing is that the formation of the wing primordium is organized at boundaries of different cell populations. At these boundaries, organizing centers are established, which control the distalization and further pattern formation of the wing field through secretion of long-range signaling molecules such as Dpp and Wg (Figure 2). For wing development, the main centers lie at the boundaries of the A-P and future D-V compartments, which are determined by the activity of the selector proteins Ap and En. Therefore, these boundaries are also the boundaries between En-expressing and En-non-expressing cells, A-P boundary, and that between Ap-expressing and non-expressing cells, D-V boundary, respectively (see Figure 2). How are the expression domains of En and Ap established? Studies of imaginal disc formation in the embryo showed that the wing and leg discs have a common precursor which later separate as a result of the dorsal segregation of the wing disc [11,12]. This common precursor is established at the A-P boundary within the mesothoraxic segment and consists of En-expressing and non-expressing cells [12]. Therefore, it seems that the A-P boundary is inherited from
The establishment of the wing primordium: expression of Ap in a wing disc of a late second instar larva. Ap expression overlaps ventrally with that of wg, which is expressed in the ventral part of the disc. Activity of wg defines the wing field in the ventral part of the disc and the Ap domain divides the field in two parts. The box highlights the region of overlap of the two expression domains. In this region, genes important for the establishment of the distal wing fates, such as the intermediate and distal hinge, blade and margin are activated along a small stripe straddling the Ap-expression boundary. Expression of these genes is induced by interactions between cells of the two cell populations mediated by the Notch pathway. Ap controls expression of the genes encoding the Notch ligands Serrate and Delta and its regulator, the fringe gene product. As a consequence, the Notch pathway is only active at the D-V boundary, where it induces the expression of target genes. The region is drawn schematically in A1. Subdivision of the wing field starts with the induction of vestigial expression through the Notch pathway. vg is controlled by the vg boundary enhancer (vgBE), which is activated by the Su(H) protein [16]. Stabilization of vg expression requires the activity of wg and therefore only takes place where the Ap and Wg expression domains overlap [7,19] (D2). Vg is a nuclear protein that associates with the DNA-binding factor Scalloped to form a heterodimer transcription factor required for gene activation specific for the distal part of the wing [24,25]. The activity of Vg imposes the wing blade fate on cells expressing it [16]. (D3) In a third step, Notch/Su(H) signaling and Vestigial collaborate to induce the expression of genes that are required for the establishment and patterning of the margin along the D-V boundary, among them wg [26,27]. As a consequence of the restriction of Notch/Su(H) signaling to the D-V boundary, the initial primordium of the wing blade is, at this stage, a stripe straddling the D-V interface. (A4) After the establishment of the expression of wg and vg in the wing primordium, the wing blade begins to grow: the expression of vg is maintained in the cells of the blade that lie outside of the domain of Notch/Su(H) signaling through the activity of another enhancer of vg, the vg quadrant enhancer (vgQE; gray oval in A4) [16]. This enhancer requires Vg and Wg as well as the Dpp protein for its activity [16,28]. Dpp is produced in a stripe along the A-P boundary and diffuses from its source in an A-P direction. Its expression is induced by the Hh signal [8]. Hh is a short-range signaling molecule produced in the posterior compartment of the disc under control of the selector protein Engrailed (En) [8]. Wg produced at the D-V boundary and Dpp produced at the A-P boundary diffuse from their sources and, together with Vestigial, control vg expression in the wing blade through the vgQE. From the dependence of the vgQE on Vg previously activated by the vgBE, it follows that all wing pouch cells are progeny of the cells determined at the D-V boundary through the activity of Notch [16]. The activity of the vgBE and the expression of wg at the wing margin continues to be controlled by Notch/Su(H) signaling and is therefore restricted to the D-V boundary. Notch also suppresses the activity of the vgQE, which is therefore not active at the D-V boundary (A4). During the time of early wing development, the expression of wg is changing rapidly and expands along the A-P direction. This expansion is dependent on the Notch activity at the D-V boundary [6] and results in enlargement of the wing field. Through the activity of Hth described in the text, the periphery of the wing field becomes the hinge region. In this region, the expression of Vg is suppressed. For a description of the further subdivision of the hinge region see Figure 4.
the embryo and is maintained in the wing and leg discs through later stages. The common disc precursor also includes Wg-expressing cells at the ventro-anterior position [12], but because the wing disc arises from the dorsal part of the common precursor, the wing disc does not inherit wg-expressing cells and therefore initially has no obvious D-V boundary. Nevertheless, a defined Ap expression domain can be observed during the first larval instar stage (T Klein,
Wing disc development in the fly: the early stages Klein
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Figure 3 The functions of the Vein/EGF-R pathway during early wing disc development. (a) Wg expression in a second instar wing disc. Wg is expressed in the ventral part of the disc. Vein expression (outlined schematically) is restricted to the dorsal part of the disc through the antagonistic influence of Wg diffusing from the ventral part. At the same time, Vein/EGF-R activity restricts Wg expression to the ventral part. High activity of Vein establishes the notal fate and maintains its own expression through an autoregulatory loop. (b) A second instar disc displaying the expression pattern of Ap. Vein diffuses from its dorsal source and activates Ap expression in a broader domain than itself, which overlaps with the wg expression domain. As described in Figure 2, this overlap is crucial for the distalization of the wing field. In addition, Vein also helps to maintain the expression of En in the posterior of the wing disc. How this is achieved is not known. The dashed lines in (a) and (b) show the outline of the discs.
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unpublished observation), suggesting that polarity along the D-V axis is established shortly after the larva has hatched. Recent experiments now show that the Vein/EGF-R pathway is also required for the establishment of the ap expression domain [3••] (Figure 3b). Vein, which is expressed in the dorsal part of the disc, diffuses ventrally and generates a gradient of EGF-R activity that provides at least two thresholds for gene activation: high activity helps to maintain the expression of vein itself, whereas lower levels activate ap expression [3••]. As a consequence, ap expression occurs in a broader domain than that of Vein and is found more ventrally, overlapping with the wg expression domain (Figure 3b). This overlap is very important for the establishment of distal fates and further development of the wing anlage (Figure 2). The results show that whereas the EGF-R pathway is initially required to prevent wing development to begin with, it subsequently plays an important role in the proximo-distal subdivision of the wing field through activation of Ap expression. The expression of Ap divides the wing field into two cell populations and creates an important boundary required for further development. The maintenance of Ap expression is not dependent on the EGF-R pathway and it is at present not understood. Unexpectedly, however, the Vein/EGF-R pathway is involved in maintenance of the en expression pattern. Several alleles of vein and other genes encoding members of the EGF-R pathway, such as pointed, when homozygous cause duplications of A-P specific pattern elements in the posterior half of the wing [2••,9,13]. The duplications, although located in the posterior compartment, develop anterior traits, such as the bristles of the anterior margin,
suggesting that the cells have adopted anterior fates. Analysis of the mutant wing discs, revealed that en expression is partly lost, resulting in the establishment of an extra A-P organizing center, which induces the pattern duplication [2••,9]. These observations show that the EGF-R pathway is required to maintain the expression of En and hence the integrity of the posterior compartment. In summary the EGF-R pathway is required to initiate the Ap expression domain and to maintain that of En. Therefore the activity of the EGF-R pathway is crucial for the definition of the major cell populations in the wing field, the boundaries of which establish the organizing centers.
Formation of the hinge region The wing is subdivided along its proximal axis into the hinge and blade. Although a lot is known about the mechanisms that form and pattern the wing blade, relatively little was known about the processes involved in the development of the hinge. Recent work has begun to shed some light on these processes. Two transcription factors, encoded by the genes homothorax (hth) and teashirt (tsh), are required for proper development of the hinge [14••,15••]. Both factors are expressed in all cells of the wing disc during the second larval instar stage, before becoming restricted to the hinge region during the third larval instar [14••,15••]. The expression of hth is regulated by wg and tsh [14••,15••]. tsh and hth together seem to be required to suppress pouch formation in the proximal region of the wing area and lateral regions of the notum by inhibiting the activation of one of the enhancers
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the genetic network controlling initiation of appendage formation might be conserved throughout phyla.
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Molecules involved in subdivision of the hinge region. The picture shows the wg expression pattern of a late third instar wing disc. Wg is expressed in the intermediate and distal part of the hinge. The dorsal proximal hinge the hinge forms at the expression boundary of the Iro-C genes. It seems that interactions at this boundary are required for the formation of this part of the hinge and the boundary between notum and wing. Expression of the Iro-C genes is initiated with the help of the Vein/EGF-R pathway. Wg is required for the formation of the intermediate part of the hinge, which forms even in the absence of Ap function and therefore in the absence of further distalization of the primordium. For the formation of the distal hinge, a Vg-controlled signal is required emerging from the wing pouch. One important target of this unknown signal is probably wg, which is essential for the development of this region.
of the pouch-determining gene vg [14••,15••]. This enhancer is called the vg quadrant enhancer (vgQE) and activates vg-expression in the wing pouch [16] (see Figure 2). Ectopic expression of vg suppresses the expression of hth in the wing hinge and loss of vg in the wing pouch induces ectopic hth expression [14••,15••]. These mutual antagonistic relationships between hth and vg seem to initially help subdivide the wing field in the proximodistal direction into the hinge and blade regions. Interestingly, the vertebrate homologs of hth, Meis1 and Meis2, appear to have a similar role in establishing proximal fates in vertebrate limbs [17]. This supports the notion that
The hinge region is further subdivided into at least three parts: proximal, intermediate and distal (Figure 4). The molecules involved are largely unknown but recent work has identified some pieces of this puzzle. One report has revealed that the transcription factors encoded by the iroquois gene complex (iro-C) are required for the proper formation of the most proximal dorsal part of the hinge and definition of the border between notum (body wall) and wing [18••]. The results suggest that a signaling center is established at the expression boundary of the iro-C genes, which directs the development of the dorsal proximal hinge region [18••]. The iro-C genes are activated by the EGF-R pathway [3••] and could account for the requirement of the pathway in formation of the notum. Other experiments, however, suggest that the Vein/EGF-R pathway has an independent function in the formation of the notum prior to the expression of the iro-C genes [3••]. Several ectopic expression experiments indicate that wg activity is required nonautonomously for induction of the intermediate fate. This is based on ectopic expression studies of wg in the notum [7,19]. Recently, another study has revealed that formation of the distal hinge region depends on an unknown signal(s) emitted from the wing blade that is under the control of Vg [7,20••]. One important target of this signal is Wg, whose activity is essential for the development of this region [21]. Hence the distinct regions of the hinge appear to be established through interactions between different cell populations. The genes that have been identified either help to define these cell populations (Vg, Iro-C proteins) or are involved in communication between them (Wg).
Conclusions These observations provide an insight into poorly understood aspects of Drosophila wing development. They have started to clarify how the wing disc is subdivided into its major domains: notum and wing. They also indicate how the wing field becomes polarized in the D-V direction through initiation of ap expression by the EGF-pathway. The functional duality of the EGF-R pathway in wing development is an elegant principle for pattern formation. The question that still remains to be solved, however, is how the D-V polarity of the wing disc is initiated. The question is now shifted from how ap expression is defined to how Vein expression is defined. In vertebrates, two homologs of Ap, Lhx2 and Lmx-1, are involved in limb development in a similar fashion to Ap [22]. Given the high degree of conservation in the genetic network controlling the development of the Drosophila wing and vertebrate limb, it will be interesting to determine if the EGF-R pathway plays is involved in the regulation of these genes. It has been revealed that genes encoding members of the EGF-R pathway are expressed at the right time in the limb of the chick [23].
Wing disc development in the fly: the early stages Klein
Update After completion of this manuscript, a work by Kumar and Moses [29] was published describing that the EGF-R and Notch pathways act antagonistically as homeotic determinants of the eye antennal disc. They show that hyperactivation of the EGF-R pathway or downregulation of the Notch pathway leads the complete transformation of the eye into an antenna suggesting an antagonistic relationship of between the two pathways during the eye versus antenna decision in the eye antennal disc. As loss of Notch function in the wing disc leads to a wing to notum transformation (Couso and Martinez-Arias [30]), a similar antagonistic relationship between the Notch and EGF-R signalling pathways could exist in the wing disc. It will be very interesting to test the role of the Notch pathway and its relation to the EGF-R and Wg pathways during the wing versus notum decision in the wing imaginal disc.
Acknowledgements I would like to thank Robert Wilson and Alfonso Martinez-Arias for critical comments on the manuscript. The work of the author is supported by the Deutsche Forschungsgemeinschaft.
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Johnson RL, Tabin CJ: Molecular models for vertebrate limb development. Cell 1997, 90:979-990.
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Baonza A, Roch F, Martin-Blanco E: DER signaling restricts the boundaries of the wing field during Drosophila development. Proc Natl Acad Sci USA 2000, 97:7331-7335. This paper reports the function of the EGF-R pathway in the maintenance of En expression and restriction of Wg expression to the ventral region of the wing imaginal disc.
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Wang SH, Simcox A, Campbell G: Dual role for Drosophila epidermal growth factor receptor signaling in early wing disc development. Genes Dev 2000, 14:2271-2276. The paper describes the function of the EGF-R pathway during early subdivision of the wing disc. It identifies Vein as the ligand and shows that the pathway is required for initiation of Ap expression. It further demonstrates that the Vein/EGF-R pathway is required for the expression of the iro-C genes and to antagonize Wg expression.
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11. Goto S, Hayashi S: Specification of the embryonic limb primordium by graded activity of decapentaplegic. Development 1997, 124:125-132. 12. Cohen SM: Imaginal disc development. In Development of Drosophila. Edited by Martinez-Arias A, Bate M. New York: Cold Spring Harbor Laboratory Press; 1993:747-849. 13. Scholz H, Deatrick J, Klaes A, Klambt C: Genetic dissection of pointed, a Drosophila gene encoding two ETS-related proteins. Genetics 1993, 135:455-468. 14. Azpiazu N, Morata G: Function and regulation of homothorax in the •• wing imaginal disc of Drosophila. Development 2000, 127:2685-2693. The authors of this paper describe the function of Hth during wing development. They show that Hth is required for hinge development and that Vg suppresses the expression of Hth in the blade region. 15. Casares F, Mann RS: A dual role for homothorax in inhibiting wing •• blade development and specifying proximal wing identities in Drosophila. Development 2000, 127:1499-1508. The authors describe the function of Hth and Tsh during wing development. They show the mutual antagonistic relations between Vg and Hth/Tsh. They further show that Wg and Tsh are required for the expression of Hth in the hinge region. They further show that Hth is absolutely required for hinge development. 16. Kim J, Sebring A, Esch JJ, Kraus ME, Vorwerk K, Magee J, Carroll SB: Integration of positional signals and regulation of wing formation and identity by Drosophila vestigial gene. Nature 1996, 382:133-138. 17.
Mercader N, Leonardo E, Azpiazu N, Serrano A, Morata G, Martinez C, Torres M: Conserved regulation of proximodistal limb axis development by Meis1/Hth. Nature 1999, 402:425-429.
18. Diez del Corral R, Aroca P, JL Gm-S, Cavodeassi F, Modolell J: The •• Iroquois homeodomain proteins are required to specify body wall identity in Drosophila. Genes Dev 1999, 13:1754-1761. This work shows the importance of the genes of the iro-C gene complex for the development of the proximal dorsal hinge. It further shows that the genes are required to define a boundary between the notum and the wing through interactions at the boundary of the expression domain of the iro-C genes 19. Klein T, Martinez-Arias AM: The vestigial gene product provides a molecular context for the interpretation of signals during the development of the wing in Drosophila. Development 1999, 126:913-925. 20. Liu X, Grammont M, Irvine KD: Roles for scalloped and vestigial in •• regulating cell affinity and interactions between the wing blade and the wing hinge. Dev Biol 2000, 228:287-303. A demonstration of the non-autonomous requirement for Vg and Sd in the development of the distal part of the hinge. 21. Neumann CJ, Cohen SM: Distinct mitogenic and cell fate specification functions of wingless in different regions of the wing. Development 1996, 122:1781-1789. 22. Rincon-Limas DE, Lu CH, Canal I, Calleja M, Rodriguez-Esteban C, Izpisua-Belmonte JC, Botas J: Conservation of the expression and function of apterous orthologs in Drosophila and mammals. Proc Natl Acad Sci USA 1999, 96:2165-2170. 23. Dealy CN, Scranton V, Cheng HC: Roles of transforming growth factor-alpha and epidermal growth factor in chick limb development. Dev Biol 1998, 202:43-55.
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Ng M, Diaz-Benjumea FJ, Vincent JP, Wu J, Cohen SM: Specification of the wing by localized expression of wingless protein. Nature 1996, 381:316-318.
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Klein T, Arias AM: Different spatial and temporal interactions between notch, wingless, and vestigial specify proximal and distal pattern elements of the wing in Drosophila. Dev Biol 1998, 194:196-212.
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Wing disc development in the fly: the early stages Thomas Klein The establishment of the wing anlage in Drosophila is dependent on the presence of two organizing centers located at the boundaries of the dorsoventral and anteroposterior compartments. How these boundaries are defined was not understood until recently. Furthermore, nothing was known about how the hinge region of the wing is defined. Recent data have now started to provide some insight in the molecular processes required for the definition of the major boundaries and subdivision of the wing anlage into the hinge and blade region. Addresses Institut für Genetik, Universität zu Köln, Weyertal 121, 50931 Köln, Germany; e-mail: [email protected] Current Opinion in Genetics & Development 2001, 11:470–475 0959-437X/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations ap apterous A-P anteroposterior Dpp Decapentaplegic D-V dorsoventral EGF-R epidermal growth factor receptor en engrailed Hh Hedgehog hth homothorax iro-C iroquois gene complex Su(H) Suppression of Hairless tsh teashirt vg vestigial vgQE vg quadrant enhancer Wg Wingless
Introduction Multicellular organisms often form body wall appendages for specialized functions such as locomotion or flying. Recent studies on the development of the Drosophila wing are providing an insight into the genetic and molecular network orchestrating initiation, subdivision and patterning of these appendages. The knowledge gathered from these studies has provided the vertebrate field with starting points to identify molecules important for the development of vertebrate limbs (see Tickle and Münsterberg, this issue [pp 476–481], for a review of vertebrate limb development). It transpires that the molecular basis for the formation of vertebrate limbs is remarkably similar to that of the Drosophila wing [1]. In this review, I focus on recent work that has started to clarify some of the less well understood aspects of Drosophila wing development. The wing in Drosophila develops from one of the imaginal discs from which most of the adult body is assembled. Imaginal discs are monocellular epithelial layers that consist of undifferentiated, proliferating cells. The wing imaginal disc comprises ~20 cells when it is formed during embryonic development. These cells proliferate during
the three larval stages to generate a disc of ~75,000 cells in the late third instar (~96h after hatching). The disc is basically a single cell layered epithelium, thus pattern formation occurs in a two-dimensional layer. This presents a problem, one also shared by a painter: incorporating the third dimension in a two dimensional sheet. It is solved by organizing the wing primordium in a concentric way with the distal structures (wing blade and margin) in the center and the proximal structures (hinge) at the periphery (Figure 1). By the late third instar, the wing primordium is established and one can identify its major elements, hinge, blade and margin, with the help of appropriate molecular markers (see Figure 1). However, the processes leading to its formation start at the beginning of the third larval instar (~48h after hatching) and are controlled by two major patterning centers, that are established at the boundaries of the dorsoventral (D-V) and anteroposterior (A-P) compartments. Several genetic screens have identified important genes controlling wing development: chief among them engrailed (en), apterous (ap), vestigial (vg), and the genes that play a role in the Notch (N) Decapentaplegic (Dpp), Wingless (Wg) and Hedgehog (Hh) signaling pathways. The function of these genes and signaling pathways during establishment of the wing anlage (hinge, blade and margin) has been covered amply in other reviews and is summarized briefly in Figure 2. In the past year, another signaling pathway could be added to the list, the epidermal growth factor receptor (EGF-R) pathway [2••,3••]. This pathway had only been implicated in very late patterning processes in the wing, such as vein development, but recent findings show that it has several important functions during early wing disc development that help define distinct cell populations. These are important because the organizing centers are established at the boundaries of these populations. I discuss the function of the EGF-R pathway below and shortly summarize some recent work that has started to illuminate the definition and subdivision of the hinge region of the wing.
Early subdivision of the wing disc: notum versus wing Mutations in the gene wg suggest that the first step of wing development is to define a region in the wing disc where the wing will form. Flies mutant for wg display a very severe wing phenotype, namely a complete loss of all wing structures replaced by a tiny duplication of notal structures (wing to notum transformation) [4,5]. Conversely, ectopic expression of wg at the correct time leads to the ectopic induction of wing structures in the notum [6,7]. This places wg at the top of the genetic hierarchy that controls wing development and suggests that it is involved in defining
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Figure 1 The adult structures formed by the wing disc of Drosophila. Orientation is anterior to the left and ventral to the bottom. (a) Major elements of the wing anlage highlighted by Wg expression in a late third instar wing disc. Wg is expressed in two ring-like domains in the hinge region and along the dorsoventral compartment boundary (D-V boundary) dividing the wing blade. The inner ring-like domain frames the wing blade. The domain along the D-V boundary corresponds to the area in which the wing margin will form. Wg is also expressed in a broad band in the dorsal part of the disc, which will become the notum of the fly. As the disc is a two-dimensional structure, the third dimension must be integrated in this level with help of the two existing axes. The anlage of the wing is therefore organized in a concentric way with the distalmost point in the center of the anlage and the proximal parts located around it. During pupation, the wing evaginates and the dorsal and ventral part of the wing adhere together. The evagination transforms the two dimensional anlage into (b) a three-dimensional structure (in which wg expression in an adult fly is depicted). The persisting pattern of wg expression reveals the adult structures in whose anlage it is expressed at late third larval instar. All the major structures of the wing are recognizable: the blade comprising the dorsal and ventral layer of the pouch adhering to each other, the margin running around the blade at the boundary of the dorsal and ventral part of the pouch and the hinge that connects the wing blade to the body.
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the area where the wing will form. In analogy to vertebrate terminology, I will here call this area the wing field. The wing field is defined during the second larval instar (24–48h after hatching) at a time when wg is expressed in the ventral region of the disc (Figure 2a). The expression of wg is controlled by the Hh signaling pathway, which is also required to establish the A-P organizing center [8]. The wing disc does not only form the wing — it also forms half of the notum (see Figure 1). Recent work now reveals that the formation of this part of the disc is dependent on the activity of the EGF-R signaling pathway [3••] (Figure 3a). During this process, the pathway is activated by the secreted neuregulin-like signaling protein Vein [3••,9,10]. Vein is expressed in the dorsal part of the disc and loss of its function during the second larval instar leads to the loss of all notal structures, a phenotype which is complementary to that of wg mutants [3••,9]. Conversely,
ectopic activation of the Vein/EGF-R pathway in the wing field prevents wing formation and promotes formation of notal structures [2••,3••]. Vein is restricted to the dorsal part of the wing disc by the suppressive influence of wg, which suppresses the expression of Vein, in the ventral region of the disc [3••]. Conversely, wg expression extends dorsally if the EGF-R pathway is interrupted in the disc, suggesting that the pathway suppresses wg expression in the dorsal part of the disc [2••,3••]. This antagonistic relationship between the Wg and EGF-R pathways helps to divide the early disc into notum and wing regions. It explains why a wing → notum duplication occurs in the absence of wg function: In wg mutant wing discs, Vein is expressed ectopically in ventral regions and promotes the development of notal fates there [3••]. It is not yet clear whether the wing fate is the default state of the wing disc and the function of wg is solely to prevent activation of the Vein/EGF-R pathway in the ventral region of the disc.
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Establishment and maintenance of the Ap and En expression domains in the wing disc One important principle that has emerged from study of the development of the Drosophila wing is that the formation of the wing primordium is organized at boundaries of different cell populations. At these boundaries, organizing centers are established, which control the distalization and further pattern formation of the wing field through secretion of long-range signaling molecules such as Dpp and Wg (Figure 2). For wing development, the main centers lie at the boundaries of the A-P and future D-V compartments, which are determined by the activity of the selector proteins Ap and En. Therefore, these boundaries are also the boundaries between En-expressing and En-non-expressing cells, A-P boundary, and that between Ap-expressing and non-expressing cells, D-V boundary, respectively (see Figure 2). How are the expression domains of En and Ap established? Studies of imaginal disc formation in the embryo showed that the wing and leg discs have a common precursor which later separate as a result of the dorsal segregation of the wing disc [11,12]. This common precursor is established at the A-P boundary within the mesothoraxic segment and consists of En-expressing and non-expressing cells [12]. Therefore, it seems that the A-P boundary is inherited from
The establishment of the wing primordium: expression of Ap in a wing disc of a late second instar larva. Ap expression overlaps ventrally with that of wg, which is expressed in the ventral part of the disc. Activity of wg defines the wing field in the ventral part of the disc and the Ap domain divides the field in two parts. The box highlights the region of overlap of the two expression domains. In this region, genes important for the establishment of the distal wing fates, such as the intermediate and distal hinge, blade and margin are activated along a small stripe straddling the Ap-expression boundary. Expression of these genes is induced by interactions between cells of the two cell populations mediated by the Notch pathway. Ap controls expression of the genes encoding the Notch ligands Serrate and Delta and its regulator, the fringe gene product. As a consequence, the Notch pathway is only active at the D-V boundary, where it induces the expression of target genes. The region is drawn schematically in A1. Subdivision of the wing field starts with the induction of vestigial expression through the Notch pathway. vg is controlled by the vg boundary enhancer (vgBE), which is activated by the Su(H) protein [16]. Stabilization of vg expression requires the activity of wg and therefore only takes place where the Ap and Wg expression domains overlap [7,19] (D2). Vg is a nuclear protein that associates with the DNA-binding factor Scalloped to form a heterodimer transcription factor required for gene activation specific for the distal part of the wing [24,25]. The activity of Vg imposes the wing blade fate on cells expressing it [16]. (D3) In a third step, Notch/Su(H) signaling and Vestigial collaborate to induce the expression of genes that are required for the establishment and patterning of the margin along the D-V boundary, among them wg [26,27]. As a consequence of the restriction of Notch/Su(H) signaling to the D-V boundary, the initial primordium of the wing blade is, at this stage, a stripe straddling the D-V interface. (A4) After the establishment of the expression of wg and vg in the wing primordium, the wing blade begins to grow: the expression of vg is maintained in the cells of the blade that lie outside of the domain of Notch/Su(H) signaling through the activity of another enhancer of vg, the vg quadrant enhancer (vgQE; gray oval in A4) [16]. This enhancer requires Vg and Wg as well as the Dpp protein for its activity [16,28]. Dpp is produced in a stripe along the A-P boundary and diffuses from its source in an A-P direction. Its expression is induced by the Hh signal [8]. Hh is a short-range signaling molecule produced in the posterior compartment of the disc under control of the selector protein Engrailed (En) [8]. Wg produced at the D-V boundary and Dpp produced at the A-P boundary diffuse from their sources and, together with Vestigial, control vg expression in the wing blade through the vgQE. From the dependence of the vgQE on Vg previously activated by the vgBE, it follows that all wing pouch cells are progeny of the cells determined at the D-V boundary through the activity of Notch [16]. The activity of the vgBE and the expression of wg at the wing margin continues to be controlled by Notch/Su(H) signaling and is therefore restricted to the D-V boundary. Notch also suppresses the activity of the vgQE, which is therefore not active at the D-V boundary (A4). During the time of early wing development, the expression of wg is changing rapidly and expands along the A-P direction. This expansion is dependent on the Notch activity at the D-V boundary [6] and results in enlargement of the wing field. Through the activity of Hth described in the text, the periphery of the wing field becomes the hinge region. In this region, the expression of Vg is suppressed. For a description of the further subdivision of the hinge region see Figure 4.
the embryo and is maintained in the wing and leg discs through later stages. The common disc precursor also includes Wg-expressing cells at the ventro-anterior position [12], but because the wing disc arises from the dorsal part of the common precursor, the wing disc does not inherit wg-expressing cells and therefore initially has no obvious D-V boundary. Nevertheless, a defined Ap expression domain can be observed during the first larval instar stage (T Klein,
Wing disc development in the fly: the early stages Klein
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Figure 3 The functions of the Vein/EGF-R pathway during early wing disc development. (a) Wg expression in a second instar wing disc. Wg is expressed in the ventral part of the disc. Vein expression (outlined schematically) is restricted to the dorsal part of the disc through the antagonistic influence of Wg diffusing from the ventral part. At the same time, Vein/EGF-R activity restricts Wg expression to the ventral part. High activity of Vein establishes the notal fate and maintains its own expression through an autoregulatory loop. (b) A second instar disc displaying the expression pattern of Ap. Vein diffuses from its dorsal source and activates Ap expression in a broader domain than itself, which overlaps with the wg expression domain. As described in Figure 2, this overlap is crucial for the distalization of the wing field. In addition, Vein also helps to maintain the expression of En in the posterior of the wing disc. How this is achieved is not known. The dashed lines in (a) and (b) show the outline of the discs.
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unpublished observation), suggesting that polarity along the D-V axis is established shortly after the larva has hatched. Recent experiments now show that the Vein/EGF-R pathway is also required for the establishment of the ap expression domain [3••] (Figure 3b). Vein, which is expressed in the dorsal part of the disc, diffuses ventrally and generates a gradient of EGF-R activity that provides at least two thresholds for gene activation: high activity helps to maintain the expression of vein itself, whereas lower levels activate ap expression [3••]. As a consequence, ap expression occurs in a broader domain than that of Vein and is found more ventrally, overlapping with the wg expression domain (Figure 3b). This overlap is very important for the establishment of distal fates and further development of the wing anlage (Figure 2). The results show that whereas the EGF-R pathway is initially required to prevent wing development to begin with, it subsequently plays an important role in the proximo-distal subdivision of the wing field through activation of Ap expression. The expression of Ap divides the wing field into two cell populations and creates an important boundary required for further development. The maintenance of Ap expression is not dependent on the EGF-R pathway and it is at present not understood. Unexpectedly, however, the Vein/EGF-R pathway is involved in maintenance of the en expression pattern. Several alleles of vein and other genes encoding members of the EGF-R pathway, such as pointed, when homozygous cause duplications of A-P specific pattern elements in the posterior half of the wing [2••,9,13]. The duplications, although located in the posterior compartment, develop anterior traits, such as the bristles of the anterior margin,
suggesting that the cells have adopted anterior fates. Analysis of the mutant wing discs, revealed that en expression is partly lost, resulting in the establishment of an extra A-P organizing center, which induces the pattern duplication [2••,9]. These observations show that the EGF-R pathway is required to maintain the expression of En and hence the integrity of the posterior compartment. In summary the EGF-R pathway is required to initiate the Ap expression domain and to maintain that of En. Therefore the activity of the EGF-R pathway is crucial for the definition of the major cell populations in the wing field, the boundaries of which establish the organizing centers.
Formation of the hinge region The wing is subdivided along its proximal axis into the hinge and blade. Although a lot is known about the mechanisms that form and pattern the wing blade, relatively little was known about the processes involved in the development of the hinge. Recent work has begun to shed some light on these processes. Two transcription factors, encoded by the genes homothorax (hth) and teashirt (tsh), are required for proper development of the hinge [14••,15••]. Both factors are expressed in all cells of the wing disc during the second larval instar stage, before becoming restricted to the hinge region during the third larval instar [14••,15••]. The expression of hth is regulated by wg and tsh [14••,15••]. tsh and hth together seem to be required to suppress pouch formation in the proximal region of the wing area and lateral regions of the notum by inhibiting the activation of one of the enhancers
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the genetic network controlling initiation of appendage formation might be conserved throughout phyla.
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Molecules involved in subdivision of the hinge region. The picture shows the wg expression pattern of a late third instar wing disc. Wg is expressed in the intermediate and distal part of the hinge. The dorsal proximal hinge the hinge forms at the expression boundary of the Iro-C genes. It seems that interactions at this boundary are required for the formation of this part of the hinge and the boundary between notum and wing. Expression of the Iro-C genes is initiated with the help of the Vein/EGF-R pathway. Wg is required for the formation of the intermediate part of the hinge, which forms even in the absence of Ap function and therefore in the absence of further distalization of the primordium. For the formation of the distal hinge, a Vg-controlled signal is required emerging from the wing pouch. One important target of this unknown signal is probably wg, which is essential for the development of this region.
of the pouch-determining gene vg [14••,15••]. This enhancer is called the vg quadrant enhancer (vgQE) and activates vg-expression in the wing pouch [16] (see Figure 2). Ectopic expression of vg suppresses the expression of hth in the wing hinge and loss of vg in the wing pouch induces ectopic hth expression [14••,15••]. These mutual antagonistic relationships between hth and vg seem to initially help subdivide the wing field in the proximodistal direction into the hinge and blade regions. Interestingly, the vertebrate homologs of hth, Meis1 and Meis2, appear to have a similar role in establishing proximal fates in vertebrate limbs [17]. This supports the notion that
The hinge region is further subdivided into at least three parts: proximal, intermediate and distal (Figure 4). The molecules involved are largely unknown but recent work has identified some pieces of this puzzle. One report has revealed that the transcription factors encoded by the iroquois gene complex (iro-C) are required for the proper formation of the most proximal dorsal part of the hinge and definition of the border between notum (body wall) and wing [18••]. The results suggest that a signaling center is established at the expression boundary of the iro-C genes, which directs the development of the dorsal proximal hinge region [18••]. The iro-C genes are activated by the EGF-R pathway [3••] and could account for the requirement of the pathway in formation of the notum. Other experiments, however, suggest that the Vein/EGF-R pathway has an independent function in the formation of the notum prior to the expression of the iro-C genes [3••]. Several ectopic expression experiments indicate that wg activity is required nonautonomously for induction of the intermediate fate. This is based on ectopic expression studies of wg in the notum [7,19]. Recently, another study has revealed that formation of the distal hinge region depends on an unknown signal(s) emitted from the wing blade that is under the control of Vg [7,20••]. One important target of this signal is Wg, whose activity is essential for the development of this region [21]. Hence the distinct regions of the hinge appear to be established through interactions between different cell populations. The genes that have been identified either help to define these cell populations (Vg, Iro-C proteins) or are involved in communication between them (Wg).
Conclusions These observations provide an insight into poorly understood aspects of Drosophila wing development. They have started to clarify how the wing disc is subdivided into its major domains: notum and wing. They also indicate how the wing field becomes polarized in the D-V direction through initiation of ap expression by the EGF-pathway. The functional duality of the EGF-R pathway in wing development is an elegant principle for pattern formation. The question that still remains to be solved, however, is how the D-V polarity of the wing disc is initiated. The question is now shifted from how ap expression is defined to how Vein expression is defined. In vertebrates, two homologs of Ap, Lhx2 and Lmx-1, are involved in limb development in a similar fashion to Ap [22]. Given the high degree of conservation in the genetic network controlling the development of the Drosophila wing and vertebrate limb, it will be interesting to determine if the EGF-R pathway plays is involved in the regulation of these genes. It has been revealed that genes encoding members of the EGF-R pathway are expressed at the right time in the limb of the chick [23].
Wing disc development in the fly: the early stages Klein
Update After completion of this manuscript, a work by Kumar and Moses [29] was published describing that the EGF-R and Notch pathways act antagonistically as homeotic determinants of the eye antennal disc. They show that hyperactivation of the EGF-R pathway or downregulation of the Notch pathway leads the complete transformation of the eye into an antenna suggesting an antagonistic relationship of between the two pathways during the eye versus antenna decision in the eye antennal disc. As loss of Notch function in the wing disc leads to a wing to notum transformation (Couso and Martinez-Arias [30]), a similar antagonistic relationship between the Notch and EGF-R signalling pathways could exist in the wing disc. It will be very interesting to test the role of the Notch pathway and its relation to the EGF-R and Wg pathways during the wing versus notum decision in the wing imaginal disc.
Acknowledgements I would like to thank Robert Wilson and Alfonso Martinez-Arias for critical comments on the manuscript. The work of the author is supported by the Deutsche Forschungsgemeinschaft.
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