The mechanism of Drosophila leg development along the proximodistal axis

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1 Develop. Growth Differ. (2004) 46, Review The mechanism of Drosophila leg development along the proximodistal axis Tetsuya Kojima Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo , Japan During development of higher organisms, most patterning events occur in growing tissues. Thus, unraveling the mechanism of how growing tissues are patterned into final morphologies has been an essential subject of developmental biology. Limb or appendage development in both vertebrates and invertebrates has attracted great attention from many researchers for a long time, because they involve almost all developmental processes required for tissue patterning, such as generation of the positional information by morphogen, subdivision of the tissue into distinct parts according to the positional information, localized cell growth and proliferation, and control of adhesivity, movement and shape changes of cells. The Drosophila leg development is a good model system, upon which a substantial amount of knowledge has been accumulated. In this review, the current understanding of the mechanism of Drosophila leg development is described. Key words: Arthropods, Drosophila, growth and development, leg, limb. Introduction Appendages of insects are largely classified into two categories: dorsal appendages including wings and halteres, and ventral appendages including antennae, feeding appendages, legs and genitalia. Dorsal appendages are unique to insects and are not segmented, while ventral appendages are common feature of all arthropods and consist of several segments connected by joints, as the name of the phylum Arthropoda (arthron = joint, pod = foot) implies. Although ventral appendages are very different in both appearance and function, they have been considered to diverge from a shared ancestral appendage according to historical anatomical analysis (Snodgrass 1935), through discovery of homeotic mutations that transform one appendage into another (Gehring 1966; Kaufman 1975; Estrada & Sanchez- Herrero 2001), and recent understanding of the molecular mechanisms involved in their development. For example, ectopic expression of a homeoticselector gene Antennapedia (Antp) causes antennato-leg transformation (Schneuwly et al. 1987), and recent studies of axis formation have shown that skojima@mail.ecc.u-tokyo.ac.jp Received 3 February 2004; accepted 8 February basically the same signaling mechanisms are operating during anteroposterior (AP), dorsoventral (DV) and proximodistal (PD) patterning in the developing leg and antennal primordia (Campbell & Tomlinson 1995; Brook & Cohen 1996; Lecuit & Cohen 1997). Among ventral appendages, legs are the least divergent in appearance within arthropods. Moreover, a recent report demonstrated that removing homeoticselector gene influence from the leg and antenna results in a production of legs with a malformed proximal portion and normally patterned tarsus and pretarsus, indicating that the developmental ground state possibly reflecting the ancestral arthropod appendage is leg-like in character (Casares & Mann 2001). In this review, I concentrate on describing the molecular mechanism of Drosophila leg development, which have been best studied among ventral appendages during past decade. Overview of the adult leg structure and its development Adult legs of Drosophila consist of several segments along the PD axis: from proximal to distal, the coxa, trochanter, femur, tibia, tarsus and pretarsus bearing two claws (Fig. 1A). The tarsus is further subdivided into five segments (tarsal segments 1 5). The neighboring leg segments are separated by the flexible

2 116 T. Kojima structures called joints, which allow them to move. Each segment has their own characteristic morphology and is distinguishable from other segments by its length and width along with the types, number and locations of sensory organs it has (Bryant 1978). Drosophila is a holometabolous insect and almost all adult tissues are derived from groups of imaginal cells that set aside from the larval tissues during embryogenesis (Cohen 1993; Fristrom & Fristrom 1993). They proliferate but remain undifferentiated in the larval body without contributing to the larval patterns. During pupal stage, nearly all larval structures disintegrate and are replaced by the structures of the adult fly formed from the imaginal cells (Fristrom & Fristrom 1993). Appendages such as legs, antennae and wings are differentiated by the imaginal discs, sac-like structures composed from the sheet of monolayered epithelial cells that invaginated during embryogenesis. Each imaginal disc is named after the appendage it forms: the leg disc, the eye-antennal disc, the wing disc and so on (Cohen 1993; Fristrom & Fristrom 1993). The leg disc consists of only about cells at its birth (Cohen 1993). The number of cells in the leg disc increases to over cells by proliferation during the second and third instar larval periods. By the beginning of third instar stage, two types of cells become recognizable: relatively large, squamous cells constituting the peripordial membrane that are thought to contribute to the adult body wall; and thick, columnar cells of the disc epithelium that differentiate into the leg (Fristrom & Fristrom 1993; Fig. 1(C). The disc epithelium is morphologically a flat sheet during first and second instar stages. With onset of the third Fig. 1 Adult leg and leg disc development. (A) Female second leg. The adult leg consists of 10 segments along the PD axis. Distal is towards right and dorsal is to the top. co, coxa; tr, trochanter; fe, femur; ti, tibia; ta1-ta5, tarsal segments 1 5; pr, pretarsus. (B) Leg disc at late third larval instar. Dorsal is to the top and anterior is to the left. Concentric folding is visible. (C E) Cross sections through midline along the DV axis. The leg disc is a sac of monolayered epithelial cells. By the early third instar (C), two types of cell groups, peripordial membrane (pm) and disc epithelium (de), are already visible. At this early stage, the folding of the disc epithelium has not yet occurred and the disc epithelium appears as a flat sheet. During the third instar stage, the disc epithelium is folded concentrically several times (D). At the late third instar, each segment is recognizable as regions separated by constrictions. During pupal stages, the disc epithelium telescopes out from the center and each segment is clearly visible (E). Regional relationships between the leg disc and the adult leg is schematically represented as colored figures in (D F).

3 Drosophila leg development 117 instar, it begins to be folded concentrically several times over so that it is no longer flat in appearance, although it remains monolayered (Fig. 1D). During the pupal stage, the disc epithelium telescopes out from its center and elongates to make a slender adult leg by cell rearrangement (Fig. 1E; Condic et al. 1991; Fristrom & Fristrom 1993). Consequently, the center of the leg disc corresponds to the distal tip of the adult leg, and progressively more peripheral portions form progressively more proximal leg segments. Therefore, each leg segment is specified as a concentric domain in the leg disc. The sensory organ development and joint formation also occur in pupal periods, and the adult leg formation is completed before eclosion (Fristrom & Fristrom 1993; Mirth & Akam 2002). Progressive subdivision of the leg disc along the proximodistal axis During larval stages, concentric subdomains in the leg disc along the PD axis are recognized as the region specific, circular expression domains of genes encoding transcription factors (hereafter referred to as PD genes). According to studies of their expression profiles and function during leg development, it has been suggested that the subdivision of the leg field into leg segments does not occur simultaneously but proceeds sequentially. At the time when the leg disc is first formed (see following), a homeobox gene, Distal-less (Dll), is expressed at the central (future distal) region and another homeobox gene, homothorax (hth), is expressed in the peripheral (future proximal) region surrounding the Dll domain (Cohen et al. 1989; Abu-Shaar & Mann 1998; Wu & Cohen 1999; Fig. 2A). Hth binds to a protein product of extradenticle (exd), a ubiquitously transcribed homeobox gene (Rauskolb et al. 1993), and brings it into a nucleus, where they act cooperatively (Rieckhof et al. 1997). Thus, Exd is localized in nuclei and functions only in the peripheral cells expressing hth. The peripheral region also expresses escargot (esg) and teashirt (tsh), both encoding zinc-finger-containing proteins (Fasano et al. 1991; Whiteley et al. 1992; Fuse et al. 1994; Goto & Hayashi 1997, 1999; Wu & Cohen 1999). It has been reported that all leg segments, except for the most basal segment such as the coxa, derive from cells that express Dll at some stages during leg development and are missing in Dll mutant legs Fig. 2 Sequential subdivision of the leg disc by expression of proximodistal (PD) genes. (A) The leg disc is progressively subdivided by Dll, dac, and hth/esg/ tsh/nexd. Distal is to the top and anterior is to the left. (B) Relationships between expression domains of PD genes at the late third instar stage and the adult leg segments. Because ss and rn are only expressed transiently at early stages, their expression domains are deduced from their mutant phenotypes and persistent lacz expression of rn-lacz. al expression in the distal tibia and femur is only seen in the ventral and dorsal regions, respectively. Distal is to the right.

4 118 T. Kojima (Cohen et al. 1989; Cohen et al. 1993; Gorfinkiel et al. 1997; Campbell & Tomlinson 1998). By contrast, mutant legs of hth, exd and tsh exhibit defects in the proximal segments. The coxa is fused to more distal segments and the fused proximal segment produces bristle types typical of distal leg segments (Gonzalez-Crespo & Morata 1995; Rauskolb et al. 1995; Gonzalez-Crespo & Morata 1996; Erkner et al. 1999; Wu & Cohen 1999; Azpiazu & Morata 2002). Forced expression of hth, exd or tsh in the Dll domain causes truncation of the distal leg structures (Gonzalez-Crespo & Morata 1996; Gonzalez-Crespo et al. 1998; Erkner et al. 1999; Azpiazu & Morata 2002). According to these observations, it has been thought that the leg disc is subdivided into two regions at the initial stage of its development. During second instar stage, expression of dachshund (dac), which encodes a nuclear factor (Mardon et al. 1994), starts in a region between the Dll and hth domains (Fig. 2A; Abu-Shaar & Mann 1998). By the early third instar stage, the fourth domain expressing both Dll and dac emerges between Dll and dac domains (Fig. 2A; Abu-Shaar & Mann 1998). As dac mutant legs lack intermediate region along the PD axis (Mardon et al. 1994), dac is also implicated in the subdivision of the leg field. During the third instar, the dac domain expands proximally and overlaps with the hth domain. A new Dll ring also appears in this overlapping region, forming the fifth domain expressing all of Dll, dac and hth (Fig. 2A; Diaz-Benjumea et al. 1994; Gorfinkiel et al. 1997; Abu-Shaar & Mann 1998; Wu & Cohen 1999). At the late third instar, the most peripheral (proximal), hth only domain corresponds to the coxa and proximal trochanter, the hth plus dac plus Dll domain to the distal trochanter and proximal edge of the femur, the dac only domain to the femur and proximal tibia, the Dll plus dac domain to the distal tibia and tarsal segment 1, and the most central (distal), Dll only domain to the distal leg segments from tarsal segment 2. Thus, the leg disc is progressively subdivided into five domains by the expression of Dll, dac and hth. Any one of these five domains, however, does not correspond to a single segment and should be further subdivided. This process is well documented for that in the Dll domain, which is further subdivided into five tarsal segments and the pretarsus. At present, the following genes have been found to act as PD genes subdividing the Dll domain: spineless (ss), encoding a homologue of the dioxin receptor (Duncan et al. 1998); aristaless (al) and BarHI/BarH2 (collectively referred to as Bar), encoding homeodomain proteins (Kojima et al. 1991; Higashijima et al. 1992; Campbell et al. 1993; Schneitz et al. 1993); apterous (ap) and Lim1, encoding LIM-homeodomain proteins (Cohen et al. 1992; Lilly et al. 1999; Pueyo et al. 2000; Tsuji et al. 2000), bric a brac 1/bric a brac 2 (collectively referred to as bab), encoding BTB domain containing transcription factors (Godt et al. 1993; Couderc et al. 2002); and rotund (rn), encoding a zinc-finger protein (St Pierre et al. 2002). Relationships between the expression domains of these PD genes at the late third instar and the adult leg segments are shown in Figure 2(B). The subdivision of the tarsus and pretarsus regions also occurs in a stepwise fashion. During the late second to early third instar stages, ss is transiently expressed in the future tarsus region. Because tarsal Fig. 3 Morphogens in the leg disc development. (A) Schematic representation of regional relationship between expression domains of four morphogens. Distal is to the top and anterior is to the left. (B) Regulatory relationships between morphogens and PD genes. Arrows and T-bars indicate positive and negative regulation, respectively, and thickness of arrows indicates activities of the morphogens. Hh secreted from the posterior compartment induces dpp and wg expression dorsally and ventrally, respectively, and combined action of Dpp and Wg determines expression domains of Dll, dac, hth/tsh and production of EGFR ligands, which in turn determines expression domains of PD genes, such as al, Lim1, Bar, rn and bab.

5 Drosophila leg development 119 segments 2 4 are missing in ss mutant legs, these segments appear to emerge from the ss domain. (Duncan et al. 1998). rn is also expressed transiently in the tarsus region at early to mid third instar stages (St Pierre et al. 2002). Because persistent lacz expression of rn-lacz reporter is detected in the distal edge of tarsal segment 1 to the tarsal segment 4 at late third instar (Couso & Bishop 1998), and rn mutant legs show fusion of tarsal segments 1 and 5 by deleting tarsal segments 2 4, the rn domain at early third instar is thought to correspond to the tarsal segments 2 4. (St Pierre et al. 2002). bab expression pattern is temporally and spatially very similar to that of rn-lacz. The levels of bab expression at late third instar, however, is high in tarsal segments 3 and 4 while low in the tarsal segment 2 to the distal edge of tarsal segment 1. Weak bab expression also extends into the tarsal segment 5 (Godt et al. 1993; Couderc et al. 2002; de Celis Ibeas & Bray 2003). In bab null mutants, tarsal segments 2 5 are frequently fused into a single segment, indicating requirement of the bab function in specifying this region (Couderc et al. 2002). At the early third instar, Bar expression starts in a domain corresponding to future tarsal segments 3 5. By late third instar, Bar expression pattern changes: strong expression in the tarsal segment 5, moderate expression in the tarsal segment 4, and expression in the tarsal segment 3 disappears. Tarsal segments 3, 4 and 5 are each specified according to Bar expression levels in each segment at later stages, and loss of the late Bar expression leads to a fusion of tarsal segments 3 5. In contrast, loss of Bar activity at early stages results in a fusion of tarsal segments including tarsal segment 2, indicating that the distal tarsus region that differentiates into tarsal segments 3 5 is first separated from the proximal region corresponding to tarsal segments 1 2 by the early expression of Bar, and later subdivided into tarsal segments 3, 4 and 5 by the later expression of Bar (Kojima et al. 2000). In tarsal segment 4, ap is also expressed from the mid third instar stage and required for the proper development of this segment (Kojima et al. 2000; Pueyo et al. 2000). al and Lim1 expression occur in the pretarsus region from early third instar stage and together specify this segment (Campbell & Tomlinson 1998; Kojima et al. 2000; Pueyo et al. 2000; Tsuji et al. 2000). Because each leg segment can already be recognized morphologically just before the pupal stage, subdivision of the leg disc into each leg segment must have occur by this stage. Indeed, the pretarsus and tarsal segments 4 and 5 are prefigured, respectively, by the expression of al and Lim1, ap and Bar (the late weak expression), and Bar (the late strong expression). However, no such genes for other leg segments have been found. There may be unknown genes whose expression corresponds to any single segments other than tarsal segments 4, 5 and the pretarsus. Alternatively, these segments are determined by the combination of genes expressed in several segment widths. Nevertheless, there might be many PD genes remaining to be identified to explain all segments by the expression of genes encoding transcription factors. Morphogens in leg development In general, expression domains of region specific transcription factors that subdivide a developmental field are defined according to the positional information provided by the activity of secreted protein called morphogen (reviewed in Lawrence & Struhl 1996). In leg development, at least four kinds of secreted proteins such as Hedgehog (Hh), TGF- family member Decapentaplegic (Dpp), Wnt family member Wingless (Wg) and Ligand(s) for epidermal growth factor receptor (EGFR) have been found to act as morphogens (for review of these signaling pathways, see Barolo & Posakony 2002 and references therein). Cells in the leg disc are divided into two populations by separate cell lineages, the anterior (A) and posterior (P) compartments (Steiner 1976; Cohen 1993). hh is specifically expressed in the P compartment and Hh protein is secreted in a short distance into the A compartment (Lee et al. 1992; Tabata et al. 1992; Tashiro et al. 1993; Tabata & Kornberg 1994). In response to Hh, dpp and wg are induced, respectively, in dorsal and ventral cells near the boundary between A and P compartments (Fig. 3; Basler & Struhl 1994). Antagonistic action of Dpp and Wg provides positional information along the DV axis (Struhl & Basler 1993; Brook & Cohen 1996; Jiang & Struhl 1996; Penton & Hoffmann 1996), while they serve as morphogens for the PD information by acting cooperatively (Diaz-Benjumea et al. 1994; Lecuit & Cohen 1997). During first and second instar stages, higher activity of Dpp plus Wg signaling is required for induction of Dll expression, while dac is induced in cells receiving lower activity of Dpp plus Wg signaling (Fig. 3B; Lecuit & Cohen 1997). The expression of hth and tsh are repressed by Dpp plus Wg singaling, so that their expression is restricted to the most proximal portion (Fig. 3B; Abu-shaar & Mann 1998; Wu & Cohen 1999). At the early third instar stage, expression of vein (vn) and rhomboid (rho) starts at the most central

6 120 T. Kojima point of the leg disc (Fig. 3A; Campbell 2002; Galindo et al. 2002). vn encodes a neuregulin-like ligand of EGFR and rho encodes a membrane protein required for processing and activation of Spitz (Spi), a TGF- family member of EGFR ligand (Schnepp et al. 1996; Schweitzer & Shilo 1997; Bang & Kintner 2000). It has been reported that the expression of genes subdividing the tarsus and the pretarsus, such as al, Bar, bab and rn, is controlled by a gradient of EGFR activity from a distal to proximal direction (Fig. 3B; Campbell 2002; Galindo et al. 2002). While removal of Dpp or Wg signaling activities before the third instar stage causes truncation of the distal leg segments as well as patterning defects along the DV axis, those removed after the early third instar stage, when expression of the tarsus and pretarsus genes has occurred, produce legs with DV patterning defects but with normal organization along the PD axis (Galindo et al. 2002). Moreover, mosaic analyses for thickveins (tkv) or arrow (arr), which, respectively, encode receptors for Dpp and Wg (Nellen et al. 1994; Penton et al. 1994; Wehrli et al. 2000), showed that al expression is normal in the mutant clones if the most central portion producing EGFR ligands is not included in the clones (Campbell 2002). In addition, the initial vn expression depends on Dpp plus Wg activity (Galindo et al. 2002). From these observations, it has been suggested that the subdivision of the tarsus and pretarsus regions is only controlled by EGFR signaling and independent of Dpp and Wg activities, and that the progressive subdivision along the PD axis is accomplished by switching morphogens (Fig. 3B). Regulatory interactions between genes subdividing the leg field along the proximodistal axis As described in the preceding, the expression domains of PD genes are determined according to morphogen activity. However, morphogens are insufficient for proper PD gene expression patterns, and regulatory interactions between PD genes are required for accurately establishing their expression domains. There is mutually exclusive regulatory interaction between hth and dac, between tsh and dac, and between Dll and dac, helping creation of sharp boundaries between domains of these three genes at the late second instar (Fig. 4B; Abu-shaar & Mann 1998; Erkner et al. 1999; Wu & Cohen 1999; Dong et al. 2001). In the hth domain, hth activates and attenuates esg and tsh expression, respectively, while tsh positively regulates hth expression (Fig. 4B; Abushaar & Mann 1998; Goto & Hayashi 1999; Wu & Cohen 1999, 2000). During the third instar stage, bab and rn expression is positively regulated by Dll but repressed by dac, so that bab and rn expression is limited to the Dll only domain (Fig. 4C; Chu et al. 2002; Galindo et al. 2002). bab expression is also positively regulated by ss (Fig. 4C; Chu et al. 2002). Dll is also required for activating al, Bar and ss expression (Fig. 4C; Campbell & Tomlinson 1998; Duncan et al. 1998; Kojima et al. 2000). When Bar expression emerges, the dac domain is abutted distally by the Bar domain and its distal limit is determined by dac repression by Bar (Fig. 4C; Kojima et al. 2000). Bar is also required for ap expression in tarsal segment 4 and the late strong Bar expression in tarsal segment 5 (Fig. 4D; Kojima et al. 2000). It has also been demonstrated that proximal limits of al and Lim1 domains and distal limit of the Bar domain are determined by a mutual regulatory interactions between al, Lim1 and Bar, creating the sharp boundary between tarsal segment 5 and pretarsus (Fig. 4D; Kojima et al. 2000; Pueyo et al. 2000; Tsuji et al. 2000). Considering these observations, it seems that during leg development, expression domains of PD genes are only roughly defined by the morphogen signaling and strictly determined by regulatory interactions between them. The strict determination of PD gene expression domains may be important for subsequent patterning. One example for this is seen at the boundary between al/lim1 and Bar domains, which corresponds to the tarsal segment 5/pretarsus boundary. The expression of Fasciclin II (Fas II), a cell adhesion molecule (Grenningloh et al. 1991), is positively regulated by al/ Lim1. Bar non-autonomously activates Fas II expression in pretarsus, while it autonomously represses Fas II in tarsal segment 5 (Kojima et al. 2000; Tsuji et al. 2000). As a result, Fas II is expressed only in a row of the pretarsus cells abutting the Bar domain. These cells are rectangular in their apical cell shapes at the late third instar stage, while other cells around them have round apical cell shapes (Kojima et al. 2000). This characteristic shape of Fas II expressing cells is regulated by the localized Fas II expression (T. K., unpubl. data). Thus, the strict determination of the expression domains of al, Lim1 and Bar is essential for controlling the apical cell shape of cells at the boundary between their expression domains. Recently, de Celis and Bray (2003) reported an intriguing observation about regulation of bab, Bar and dac during development of the tarsus region. brother of odd with entrails limited (bowl) and oddskipped (odd), both encoding zinc-finger proteins

7 Drosophila leg development 121 Fig. 4 Regulatory relationships between PD genes. (A) Arrows and T-bars indicate positive and negative regulation, respectively. In the embryonic leg disc, Dll impedes nuclear localization of Exd (nexd). esg is also repressed by Dll. During the second instar stage, mutually exclusive interaction between hth/tsh, dac and Dll is involved in the strict determination of their expression domains. Around the onset of third instar stage, al, Bar, rn, ss and bab expression appears under the control of Dll and/or dac. From this stage onward, the Dll and dac domains overlap each other. The distal limit of the dac domain is determined by the early Bar expression, while activation and repression of rn/bab by Dll and dac, respectively, determine the proximal limit of the rn/ bab domains. By the late third instar, overlap between al and Bar domains is resolved by the regulatory interaction between al, Lim1 and Bar, creating the sharp boundary between tarsal segment 5 and pretarsus. Strong expression of Bar in the tarsal segment 5 and ap expression in tarsal segment 4 require early Bar expression. (B) The model of de Celis and Bray (2003) for specification of the tarsal segments. bowl and odd is expressed at the tibia/tarsal segment 1 and tarsal segment 5/pretarsus boundaries as well as at the boundaries between proximal segments. During the growth phase of the tarsus region, cells formerly expressing bowl/odd are added to the tarsus region, creating gradient of Bowl/Odd proteins at both edges of the tarsus region. According to this Bowl/Odd gradient, the wave of bab expression with its peak at the middle of the tarsus region are created. Near the edges of the tarsus region where bab expression levels are lowest, dac and strong Bar expression occurs and tarsal segments 1 and 5 are specified.

8 122 T. Kojima (Coulter et al. 1990; Wang & Coulter 1996), are expressed at the distal end of the tibia and the proximal end of the pretarsus at the late third instar. At the early third instar, their expression directly flanks the bab domain. (Fig. 4E; de Celis Ibeas & Bray 2003; Hao et al. 2003). Despite its absence in the tarsus region, bowl and odd activity is cell-autonomously required for downregulation of bab and upregulation of dac and Bar in the proximal and distal tarsus region, respectively, and bowl or odd mutant legs exhibit fusion of the tarsus (de Celis Ibeas & Bray 2003; Hao et al. 2003). To explain this observation, de Celis and Bray proposed the following model. During the growth period of the tarsus region, the part of progeny of the bowl/odd expressing cells are progressively added to the proximal and distal parts of the tarsus region by ceasing bowl/odd expression. This creates local gradients of Bowl/Odd proteins, leading cells closer to the edges experiencing the control by Bowl/Odd for longer time than those closer to the center. Since bab expression appears to be under negative control of Bowl/Odd, this results in the wave of bab expression levels along the tarsus region with the strongest expression in tarsal segments 3 and 4 and weaker expression near the proximal and distal edges of the tarsus region. Consequently, dac expression in tarsal segment 1 and the strong Bar expression in tarsal segment 5 occur due to release from possible repression by bab (de Celis Ibeas & Bray 2003). This model is very intriguing since it can explain how subdivision of the growing tissue is accomplished. Furthermore, this model gives insight into how the extent of tarsal segmentation can be evolved and diverged (see following). As described in the preceding, subdivision of the tarsus region is also controlled by EGFR signaling, and an understanding of the relationship between the function of bowl/odd and that of EGFR signaling is very important. At present, however, it remains elusive. Allocation of the limb primordia and formation of the leg disc during embryogenesis The development of the leg disc starts during the embryonic stage. Dpp, Wg and EGFR signaling also play essential roles during this stage, although their way of functioning is quite different to those of the larval stages. At mid-embryonic stage, when hth expression and nuclear Exd is observed in almost all cells of the thoracic segments (Aspland & White 1997; Gonzalez-Crespo et al. 1998), small groups of cells in the ventrolateral region in the thoracic segments begin to express Dll (Fig. 5A; Cohen 1990; Gonzalez- Crespo et al. 1998). At this stage, wg is expressed in strips along the AP boundaries of the embryonic ectoderm, dpp is expressed as dorsal strips and EGFR ligand(s) are produced at the ventral midline (Cohen et al. 1993; Golembo et al. 1996, 1999; Goto & Hayashi 1997). The initial expression of Dll is induced by Wg, while it is repressed dorsally and ventrally by Dpp and EGFR activities, respectively (Cohen 1990; Goto & Hayashi 1997). Soon after the induction of Dll, dpp expression and the production of EGFR ligand(s) occur at the dorsal and mid-ventral Fig. 5 Allocation of the primordia for leg and wing discs. (A) By combination of activation by Wg and repression by Dpp and EGFR ligands, btd/sp1 is expressed in a small group of cells at ventrolateral region of the thorax in the embryo, and induces Dll expression. (B) Shortly after Dll induction, expression or production domains of dpp, wg and EGFR ligands are changed (left) and subdivides the Dll expression domain into three regions according to their activities (middle). Dorsal-most cells form the wing disc, and ventral-most and medial cells form the leg disc (right). Ventral-most cells continuing Dll but not hth/esg expression constitute the distal domain of the leg disc, while medial cells losing Dll expression constitute the proximal region.

9 Drosophila leg development 123 cells expressing Dll, respectively (Fig. 5B; Kubota et al. 2000). EGFR signaling activity antagonizes Dpp signaling activity and, according to the balance of the two signalings and cross talk between them, three populations of cells are determined in the primary Dll domain (Fig. 5B): dorsally located cells destined to form the wing and haltere discs; ventral cells to form the distal leg; and cells in the middle region to form the proximal leg (Cohen et al. 1993; Kubota et al. Fig. 6 Function of Notch pathway in the leg segmentation. (A) Two joints at the tarsal segments 2/3 and tarsal segments 3/4 boundaries. Characteristic structures called ball and socket are seen (arrowheads). Distal is to the right. (B) Notch is activated in the distal-most cells of each segment by Dl and Ser, which is expressed just proximally to the site of Notch activation (upper figure), and regulates the joint formation and growth/survival of cells in the segments. fng is expressed in the large region including the Dl/Ser expression domain and modulates Notch activity to prevent joint formation at the proximal side of the Dl/ Ser domain. Downstream of Notch pathway, odd family genes and AP-2 are involved in the joint formation. 2000). At this stage, wg expression retracts ventrally and its expression within Dll domain is restricted to ventral cells (Fig. 5B; Cohen et al. 1993; Kubota et al. 2003). Wg inhibits the formation of the wing and haltere discs, while it promotes the leg disc formation (Kubota et al. 2003). Thus, the primary Dll domain contains precursor cells of both dorsal (wing or haltere) and ventral (leg) appendages and is called limb primordia. By late embryonic stage, the precursor cells of the wing and haltere discs migrate dorsally, stop Dll expression, and start expression of genes required for the formation of them, such as vestigial (vg), snail (sna) and esg (Boulay et al. 1987; Cohen et al. 1993; Williams et al. 1991; Fuse et al. 1996; Kim et al. 1996; Goto & Hayashi 1997). The distal leg cells remain to express Dll but lose hth expression and nuclear Exd. Conversely, the proximal leg cells staying with expressing hth and having nuclear Exd cease Dll expression and surround Dll expressing cells (Gonzalez-Crespo et al. 1998; Kubota et al. 2003). esg expression also appears in the proximal cells (Goto & Hayashi 1997). This is the first stage, when the leg disc with the PD subdivision can be recognized by gene expression. During these processes, forced expression of Dll represses esg expression and prevents nuclear localization of Exd (Gonzalez-Crespo et al. 1998; Kubota et al. 2003), suggesting that Dll negatively regulates expression of the proximal genes at this early stage of leg disc formation (Fig. 4A). wg mutant embryos lose Dll expression (Cohen 1990; Cohen et al. 1993) and did not form all of the wing, haltere and leg discs in the transplantation experiment, in which fragments of the mutants are cultured in wild-type flies (Simcox et al. 1989). However, Dll mutant embryos can produce the wing and the proximal leg structures in a similar transplantation experiment (Cohen et al. 1993), indicating that although Dll is expressed downstream of wg in the limb primordia, it is only essential for the development of the distal leg segments. Recently, buttonhead (btd) and the Drosophila homologue of human Sp1, which encode zinc-finger transcription factors having partially redundant functions with each other (Wimmer et al. 1993, 1996; Schock et al. 1999), have been shown to be expressed in the limb primordia earlier than Dll (Fig. 5A; Estella et al. 2003). The expression of btd and Sp1 at this early stage is controlled by Dpp and Wg signalings in a manner similar to Dll. They are expressed in all cells of the embryonic leg disc, including the Dll and esg domains, and in embryos lacking both of btd and Sp1, Dll and esg expression for the embryonic leg disc is lost, suggesting that they are essential to the formation of both proximal

10 124 T. Kojima and distal portion of the leg disc (Estella et al. 2003). Moreover, btd and Sp1 can promote ectopic leg development when misexpressed in the wing and haltere discs (Estella et al. 2003). From these observations, it has been proposed that btd and Sp1 may be the most upstream genes for the leg disc formation. In embryos simultaneously lacking btd and Sp1, however, the wing and haltere discs seem to be formed, as esg expression in their primordia appears normal (Estella et al. 2003). There must be one or more genes that act(s) upstream of, or redundantly with, btd and Sp1 in the specification of the limb primordia. The formation of joints between leg segments Each leg segment is connected to the next by a specialized, flexible structure called a joint (Fig. 6A). The differentiation of joints is a complex process involving changes in the cell shape, in the adhesion between cells and in the distribution of filamentous actin and extracellular matrix proteins (Fristrom & Fristrom 1993; Mirth & Akam 2002). Although the morphogenesis of joints occurs during pupal stages, the territory of each segment later forming a joint seems to be already specified by the end of the third instar, as revealed by the expression of several marker genes for the joint (Mirth & Akam 2002). Notch pathway has been shown to be fundamental for the differentiation of the joint (Fig. 6B; de Celis et al. 1998; Bishop et al. 1999; Rauskolb & Irvine 1999). Two ligands of Notch, Delta (Dl) and Serrate (Ser), are expressed as a ring in each segment. Notch is activated in the most distal cells of each segment, which are distally adjacent to the Notch ligands expression domain, and specifies them as joint forming cells. Loss of function of Notch and Notch ligands leads to deletion of joint structures and fusion of leg segments. Notch signaling is also sufficient to promote joint formation, because ectopic joint structures are induced when Notch or its ligands are activated ectopically (de Celis et al. 1998; Bishop et al. 1999; Rauskolb & Irvine 1999). In contrast to the most distal cells, however, Notch is not activated in cells expressing the Notch ligands and cells proximally adjacent to them. In the proximal part of each segment including the Notch ligands expression domain, fringe (fng) is expressed and required for the proper joint formation. fng encodes a modulator of Notch activity (Irvine & Wieschaus 1994; Panin et al. 1997), and thus proposed to be responsible for the asymmetric activation of Notch along the PD axis in each segment (de Celis et al. 1998; Bishop et al. 1999; Rauskolb & Irvine 1999). Notch activity also seems to be regulated by genes involved in the planer cell polarity, because in several mutants for them, ectopic joints and Notch activation are observed at the proximal side of the Notch ligands domain (Held et al. 1986; Bishop et al. 1999). Interestingly, loss of the Notch pathway activity leads to reduced leg growth, and ectopic activation of it results in outgrowths of leg tissues, in addition to the defects in the joint formation (de Celis et al. 1998; Bishop et al. 1999; Rauskolb & Irvine 1999). Clonal analysis has indicated that Notch activity is nonautonomously required in joint forming cells for the proper leg growth (de Celis et al. 1998). It is thus suggested that Notch activity also controls growth of leg segments by regulating some kind of secreted factors promoting cell growth and/or preventing cell survival. Because every segment is connected to the next by the joint, formation of the joint is expected to occur by using regulatory input from PD genes. Indeed, functional studies for PD genes have shown that defects or ectopic formation of joints are associated with leg malformations produced by altering their activities (cf. Kojima et al. 2000). In addition, expression of Notch ligands and fng has been shown to be regulated by PD genes (Rauskolb 2001; St Pierre et al. 2002). For example, hth and dac, respectively, act as activators of Ser expression in the coxa and in the femur, while those in the tarsus region repressed and activated, respectively, by Dll and rn (Rauskolb 2001; St Pierre et al. 2002). Because Ser is only expressed in subsets of cells expressing hth, dac or rn, combinatorial action of them with other genes may be required to restrict Ser expression to limited cells in each segment. Interestingly, two distinct enhancers that independently regulate Ser expression in the femur and in the tarsus have been identified (Rauskolb 2001). Considering all of these observations, it seems that each ring of Notch ligands and fng expression may be individually regulated by different combinations of PD genes using different enhancers, as in the case of the regulation of pair-rule gene expression during embryonic segmentation (Small & Levine 1991; Pick 1998). Downstream of the Notch pathway, several genes encoding transcription factors are known to mediate the Notch function in leg development. One of which is Drosophila homologue of mammalian AP-2, which is activated in joint forming cells of all segments by the Notch pathway (Kerber et al. 2001; Monge et al. 2001). Although loss of AP-2 activity results in severely shortened legs with defects in the joint formation, its ectopic expression induces ectopic joint structures only in the presence of Notch activity, indicating that AP-2 is an essential mediator of Notch activity but only functions with another Notch target gene(s) (Kerber

11 Drosophila leg development 125 et al. 2001; Monge et al. 2001). In addition, ectopic expression of AP-2 does not produce leg outgrowths (Kerber et al. 2001; Monge et al. 2001), unlike in the case of the Notch pathway genes (de Celis et al. 1998; Bishop et al. 1999; Rauskolb & Irvine 1999). AP-2 is nonautonomously required for the survival of interjoint cells (Kerber et al. 2001; Monge et al. 2001). Thus, it has been proposed that AP-2 mediates subsets of the Notch pathway functions in leg development (Fig. 6B): it is involved in the specification of the joint forming cells and the regulation of the survival factor(s) but not in the regulation of the growth promoting factor(s). Other genes downstream of the Notch pathway in leg development are odd-skipped (odd) family genes, odd, bowl, sister of odd and bowl (sob), and drumstick (drm). All of them encode transcription factors showing similarity between their zinc-finger domains (Coulter et al. 1990; Hart et al. 1996; Wang & Coulter 1996; Green et al. 2002) and are co-expressed in joint forming cells by Notch activity, except those in the tarsus region (Rauskolb & Irvine 1999; de Celis & Bray 2003; Hao et al. 2003). Although none of their single mutants have apparent defects in the formation of joints in which they are expressed, possibly due to functional redundancy between them (de Celis & Bray 2003; Hao et al. 2003), ectopic expression studies have shown that they are capable of inducing ectopic joint structures as well as change in cell shape and actin distribution (Hao et al. 2003), which are also induced by ectopic activation of Notch (de Celis et al. 1998; Bishop et al. 1999; Rauskolb & Irvine 1999). However, leg outgrowths are not produced by ectopic expression of them (Hao et al. 2003). Therefore, the odd family genes are thought to be mediators of Notch activity in the joint formation outside the tarsus region but not in the leg growth regulation (Fig. 6B). As shown in the preceding, bowl and odd are also implicated in the process of proper subdivision of tarsal segments (de Celis & Bray 2003). Although sob and drm mutants show no abnormality in tarsal segments, the observation that ectopic sob expression frequently produces shortened legs with propagated tarsal segment 1 identities (Hao et al. 2003), which is similar to those observed in ectopic expression of bowl (de Celis & Bray 2003), might indicate the involvement of sob and drm in the proper subdivision of the tarsus region. Evolution of the arthropod leg development Based on comparative morphology, Snodgrass proposed that the appendage of an arthropod ancestor consisted of two segments, which he called the coxopodite and telopodite (Snodgrass 1935). The coxopodite is a basal segment that is the simple outgrowth from the body wall, and the telopodite is a distal segment. Studies on the mechanism of Drosophila leg development and on the expression patterns of PD genes in various arthropod species have brought wide acceptance to this view. As described in the preceding, Drosophila legs are initially subdivided into two domains by the expression and function of Dll in the distal region and of hth/exd in the proximal region. In addition, Dll expression in the distal region and hth and/or exd expression in the proximal region are conserved in legs of various arthropod species so far examined (Panganiban et al. 1994, 1995; Popadic & Panganiban 1998; Abzhanov & Kaufman 2000; Jockusch et al. 2000; Mittmann & Scholtz 2001; Schoppmeier & Damen 2001; Suzuki & Palopoli 2001; Inoue et al. 2002; Williams et al. 2002; Prpic & Tautz 2003; Prpic et al. 2003). Moreover, Dll activity has been shown to be essential for the distal leg formation in a spider and a beetle (Beermann et al. 2001; Schoppmeier & Damen 2001). These studies indicate that the subdivision of the leg into two parts by Dll and hth/exd is a fundamental property of arthropod appendages, and that Dll and hth/exd specify the telopodite and the coxopodite, respectively. Interestingly, most living arthropods have legs consisting of more than two segments and dac expression in the intermediate region of the leg is also conserved even in a spider (Chelicerata) and a millipede (Myriapoda) as well as in other insects (Prpic et al. 2001, 2003; Inoue et al. 2002; Prpic & Tautz 2003). It may thus follow that the subdivision of the leg by Dll, dac and hth/exd may have already occurred before diversification of arthropods into chelicerates, myriapods, crustaceans and insects. Between arthropod legs, the extent of segmentation in the tarsus is most highly variable (Snodgrass 1935). Even within the insect group, the number of tarsal segments varies from one to five although the number in other segments is completely conserved. In addition, tarsal segments do not have musculature to move them, unlike other segments that do (Snodgrass 1935). From these anatomical observations, it has been considered that multiple tarsal segments have been derived from the non-segmented tarsus of the ancestral leg. The expression pattern of odd family genes in the leg disc, in which they are expressed as a ring in each segment but tarsal segments (de Celis & Bray 2003; Hao et al. 2003), may reflect this idea. This high variability or rapid evolution of the tarsal segmentation pattern could be explained by considering the model of de Celis and Bray for the regulation of the tarsus PD genes by bowl and odd

12 126 T. Kojima at early stages of the tarsus development (see preceding; de Celis Ibeas & Bray 2003). Simple changes in the duration or the rate of proliferation during the growth phase of the tarsus, in the sensitivity of bab to the repressive activities of bowl and odd, or in the stability of their protein products could result in the variation of the tarsal segmentation. Further investigation of bowl, odd and bab functions in the tarsus development as well as those of other PD genes and signaling pathways in leg development of Drosophila and other arthropods should reveal how patterning and growth of the tissue are coordinated, and should help us to unravel the mechanism underlying the diversification of arthropod leg structures. Similarity between Drosophila leg development and vertebrate limbs Dll homologues have been found in a wide variety of species across the animal kingdom, even outside of the arthropod, and have been shown to mark the distal portion of all kinds of outgrowths from the body wall (Panganiban et al. 1997). In vertebrate limb development, Dll homologues are expressed in the apical ectodermal ridge of the limb bud, which regulates the patterned outgrowth of the limb, and mutants of Dll homologues have severe malformations of the distal limb (Panganiban & Rubenstein 2002; Robledo et al. 2002). Moreover, homologues of hth and exd, meis and Pbx1, respectively, have shown a striking functional similarity during limb development (Gonzalez- Crespo et al. 1998; Mercader et al. 1999). Subcellular localization of Pbx1 is regulated by meis activity, and meis and Pxb1 are only functional in the proximal but not in the distal portion of the limb. Ectopic expression of meis in the distal region impedes limb development (Gonzalez-Crespo et al. 1998; Mercader et al. 1999). Thus, the functions of Dll and hth/exd are ancient and may be shared by all animal species having outgrowths from the body wall, such as limbs or appendages. Acknowledgements I would like to thank Prof K. Saigo, University of Tokyo, for his support and encouragement throughout this study. I also thank K. Yasunaga for his helpful comments on the manuscript. T. K. is supported by grants from the Ministry of Education, Science and Culture of Japan. References Abu-Shaar, M. & Mann, R. S Generation of multiple antagonistic domains along the proximodistal axis during Drosophila leg development. Development 125, Abzhanov, A. & Kaufman, T. C Homologs of Drosophila appendage genes in the patterning of arthropod limbs. Dev. Biol. 227, Aspland, S. E. & White, R. A. 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