Insects bear a stereotyped set of limbs, or ventral body appendages. In the highly derived dipteran Drosophila melanogaster, the homeodomain transcription factor encoded by the Distal-less (Dll) gene plays a major role in establishing distal limb structures. We have isolated the Dll orthologue (TcDll) from the beetle Tribolium castaneum, which, unlike Drosophila, develops well-formed limbs during embryogenesis. TcDll is initially expressed at the sites of limb primordia formation in the young embryo and subsequently in the distal region of developing legs, antennae and mouthparts except the mandibles. Mutations in the Short antennae (Sa) gene of Tribolium delete distal limb structures, closely resembling the Dll phenotype in Drosophila. TcDll expression is severely reduced or absent in strong Sa alleles. Genetic mapping and molecular analysis of Sa alleles also support the conclusion that TcDll corresponds to the Sa gene. Our data indicate functional conservation of the Dll gene in evolutionarily distant insect species. Implications for evolutionary changes in limb development are discussed.
Arthropod limbs show a tremendous range in form and number, which is thought to have facilitated adaptation to various ecological niches (Siewing, 1969). Despite their diversity, arthropod limbs share a basic organisation consisting of a proximo-distal sequence of characteristic segments. In the leg, for example, the most proximal coxa is followed by the trochanter, the femur, the tibia and the tarsus, which bears the claw at the distal tip. This stereotyped organisation suggests that the underlying regulatory mechanisms of limb development have been conserved in evolution.
Limb development has been studied most thoroughly in Drosophila both genetically and molecularly (Cohen et al., 1993; Lecuit and Cohen, 1997). The limbs of Drosophila are formed from imaginal discs. These specialised groups of epithelial cells that develop inside the larva originate from small groups of precursor cells that invaginate from the epidermis of the nearly mature embryo (Bate and Martinez-Arias, 1991). Fate-mapping studies of the leg imaginal disc have shown that the segment primordia are arranged in concentric rings, such that the centre and the periphery of the disc give rise to the distal tip of the leg and to the thoracic body wall, respectively. During pupal metamorphosis, the leg segments are telescoped out in a distal-to-proximal sequence (Condic et al., 1991; Fristrom and Fristrom, 1975; Schubiger, 1968).
The Distal-less (Dll) gene plays a key role in the formation of the proximal-distal axis (Cohen and Jürgens, 1989a; Cohen and Jürgens, 1989b; Cohen, 1990; Diaz-Benjumea et al., 1994). Embryos carrying amorphic alleles of the Dll gene are lethal and lack the Keilin organs, the rudimentary anlagen of the larval legs, and sense organs specific for the mouthparts (Cohen and Jürgens, 1989a). Flies with hypomorphic Dll alleles survive to adulthood and display distinct limb defects (Cohen et al., 1989b; Sunkel and Whittle, 1987). The phenotypes of hypomorphic Dll alleles can be ordered in an allelic series according to their leg defects, with distal structures being more sensitive to a reduction in Dll activity than are proximal structures. Clonal analysis and the transplantation of Dll mutant leg discs has shown that the most proximal parts of the leg develop independently of Dll activity (Cohen et al., 1993; Cohen and Jürgens, 1989a).
Dll encodes a protein with a homeodomain (Cohen et al., 1989; Vachon et al., 1992), a motif typical for DNA-binding transcription factors. Dll RNA is expressed, in the Drosophila embryo, at the sites of the sense organs corresponding to the rudimentary anlagen of larval limbs and in the precursor cells of imaginal limbs. Post-embryonically, Dll expression is observed in the centre of the leg imaginal disc, in the antennal and maxillary primordia of the eye-antennal disc as well as in the labial disc and the labral primordium (B. Cohen, PhD Thesis, University of Munich, Germany, 1994). Through the combined activity of the secreted signalling molecules Hedgehog (Hh), Wingless (Wg) and Decapentaplegic (Dpp), which provide the positional information of the anterior-posterior and dorsal-ventral axes, Dll expression is confined to specific cells in the embryo (Cohen et al., 1993; Diaz-Benjumea et al., 1994). The segmental expression of Dll and thus the formation of the limbs is regulated by homeotic genes. In the abdomen of the embryo, the Dll gene is repressed by proteins of the Bithorax complex, which bind specifically to certain enhancer elements in the large Dll regulatory region (Vachon et al., 1992). Another maxilla-specific enhancer element mediates control of Dll expression by the ANT-C gene Deformed (O’Hara et al., 1993).
The isolation of Dll homologous sequences from arthropods and vertebrates suggests that Dll homologous genes may have been conserved in evolution. This is supported by the finding that a polyclonal antibody directed against the Dll protein of the butterfly Precis coenia crossreacts with presumably homologous proteins across the animal kingdom (Panganiban et al., 1997). Whether or not the role of Dll in limb development is also functionally conserved is unknown. One way to address this question is provided by mutants of the beetle Tribolium castaneum that appear to display Dll-related phenotypes. Limb development in Drosophila and Tribolium differs in some important aspects. The larval limbs of Drosophila are suppressed and are represented only by specific distal sense organs (Keilin, 1911), because the limb anlagen themselves are involuted during embryogenesis. By contrast, the extended germband of Tribolium displays segmentally repeated limb buds that subsequently develop into well-formed larval limbs. The Tribolium larva possesses three pairs of segmented legs as well as external gnathal appendages and antennae, which facilitates the recognition of mutants with limb defects. Mutations affecting leg and/or antenna development were first mentioned by Sokoloff (Dawson and Sokoloff, 1964; Krause et al., 1962; Sokoloff, 1964a) and later described by Kuld (I. Kuld, Diploma Thesis. University of Freiburg, Germany, 1976). Tribolium embryos homozygous for the mutants Short antennae (Sa) or Fused tarsi and antennae (Fta) die shortly after hatching. These mutants lack distal elements of the antennae, mouthparts and legs entirely. The only limb element retained in these embryos is the most proximal part of the limb corresponding to the body wall. This phenotype is strikingly reminiscent of the phenotype of amorphic Dll mutants in Drosophila.
We have cloned the Dll homologue of Tribolium castaneum using a PCR-based approach. TcDll expression was observed in all limb primordia of the embryo from the labrum to the thoracic legs. The expression pattern as revealed by the crossreacting anti-Dll antibody from Precis was indistinguishable from the pattern of TcDll RNA accumulation. Alleles of the Sa gene showed expression in only some limb primordia or lacked TcDll expression completely. In addition, TcDll was genetically mapped to the Sa locus. One of the Sa alleles, Sa-Fta, was shown to be associated with a chromosomal rearrangement. Our results indicate that Dll function is conserved between Drosophila and Tribolium.
MATERIALS AND METHODS
Wild-type Tribolium castaneum stocks GA-1 and Tiw-1, and mutants of the Short antenna locus (Sa-8, Sa-BQ, Sa-Fta, sa) were obtained from the stock collection in Manhattan, Kansas (S. Haas and R. W. B.). The new alleles Sa-8 and Sa-BQ were induced by gamma-rays (R. W. B., unpublished). Tribolium beetles were kept on whole wheat flour with 5% dry yeast at 30°C (Berghammer et al., 1999).
Morphological analysis, antibody staining and in situ hybridisation
Larval cuticles were prepared according to Meer (Meer, 1977). The antibody staining followed standard Drosophila protocols (Macdonald and Struhl, 1986). Commercial antibodies were obtained from Dianova. The anti-Dll polyclonal antibody was provided by G. Panganiban (Panganiban et al., 1995). In situ hybridisations were carried out as described previously (Tautz and Pfeifle, 1989). The riboprobes used were prepared according to the Boehringer Digoxigenin Labeling and Detection Kit. Images were processed using Adobe Photoshop 3.0 and Aldus Freehand 7.0 software.
The cDNA library, prepared from young Tribolium castaneum embryos, was a gift from R. Schröder. The genomic libraries were gifts from S. Brown (Brown et al., 1990), and from K. Handel and S. Roth (Tübingen; unpublished).
The interspecies PCR for obtaining the TcDll homeobox fragment was carried out on genomic Tc DNA using the following set of degenerate nested primers: Dll-1, 5′-ATGCGGAAGCCC(G)C(C/G)-GAACC(G)ATA(C/T)TA-3′ (aa: MRKPRTIY); Dll-2, 5′-GCG-(C)GCG(C)AGT(C)TCG(C)GCT(T)CT(G)T(C)TCG(C/A/T)GG-3′ (aa: PEREALAA); Dll-3, 5′-CTGG(C)GTCTGG(C/A)GTC(G)AA-(G)T(A/G/C)CC-3′ (aa: GLTQTQ). The annealing temperature was 45°C, the number of cycles 30. The first reaction with Dll-1 and Dll-3 resulted in a 134 bp fragment. 1 μl of this reaction was used as template for the second reaction with Dll-1 and Dll-2, resulting in a 107 bp fragment. The 107 bp fragment of the TcDll homeobox was verified by sequencing and by whole-mount in situ hybridisation.
For DNA preparations from single larvae, 0.2-0.5 ml Proteinase K buffer (0.2 M Tris pH 8.0, 0.1 M EDTA, 1% Sarcosyl, fresh 100 μg/ml Proteinase K in 1:100 dilution) was added and the larvae ground on ice. After one hour incubation at 48°C, 5 μl 100 mM PMSF was added and further incubated for 10 minutes. The DNA was precipitated and resuspended in TE with RNAse. DNA from one larva is sufficient for 20 PCR reactions.
For mapping the Dll gene relative to the Sa locus, a polymorphism between the ecotypes GA-1 and Tiw-1 in intron 4 was used to generate a CAPS (cleaved amplified polymorphic sequence) marker. The nucleotide polymorphism results in a restriction enzyme polymorphism for AluI that can easily be recognised on agarose gels. This CAPS marker was named CAPS Int4E-Alu, corresponding to its position in intron 4 of the TcDll gene, upstream of exon E (see Fig. 8). The PCR reaction with the primers EF-1 (5′ACCAAATTGA-AGAACAAC-3′) and EXE-1 (5′-TCAGTATTAAACAGCTGGCC-3′) results in a 162 bp fragment. In the Tiw-1 ecotype, digestion with AluI results in three fragments of 102bp, 46bp and 14bp. In GA-1 the 102 bp fragment is cut into two fragments of 49bp and 53bp. Mutant lines were outcrossed with that corresponding ecotype showing the different polymorphism and after intercrossing the F1, homozygous mutant single larvae of the F2 generation were tested for recombination using the CAPS Int4E-Alu marker.
Mutant alleles of the Tribolium Short antennae gene show limb defects that resemble the Drosophila Dll phenotype
Tribolium mutants displaying antennal and leg defects were originally described by Krause et al. (Krause et al., 1962) and by Sokoloff (Sokoloff, 1964a; Sokoloff, 1964b). Two of those mutants, the dominant mutant Fused tarsi and antennae (Fta) and the recessive mutant short antennae (sa), and two newly identified dominant mutants with comparable phenotypes, Sa-BQ and Sa-8, were available for the present study. As will be detailed below, all four mutants failed to complement one another and thus represent alleles of a single gene that we name Short antennae (Sa). The Fta mutant was renamed Sa-Fta to indicate its allelic status. In the following we will first describe the antennal and leg defects of mutant adult beetles before we analyse the embryonic phenotypes of homozygous dominant alleles.
The distal segments of antennae and legs are defective in Sa mutant beetles
The antenna of wild-type Tribolium castaneum consists of 11 segments. Two basal segments, pedicel and scape, bear the flagellum. Its six proximal segments are followed by three distal segments that are broadened to form the club (Fig. 1A). The base of the antenna corresponds to the coxa of the leg (Cohen et al., 1993; Cohen et al., 1989a). In Sa mutants, the proximal and distal segments of the flagellum were fused to various extents whereas the base of the antenna was not affected (Fig. 1A). Homozygous sa mutants showed the weakest defect, only the proximal segments of the flagellum being fused to give three segments. In heterozygous Sa-BQ mutants, all proximal segments were fused into one single segment. In both mutants, the three distal segments of the club were unaffected. By contrast, heterozygous Sa-8 and Sa-Fta mutants preferentially exhibited fusions of distal segments. In Sa-8, the three club segments formed a single knob that was still distinct, and the proximal segments of the flagellum were partially fused. The strongest antennal defect was displayed by Sa-Fta mutants. All segments distal to the base were fused into a club-shaped structure. To determine how reduced Sa gene function relates to the formation of distal segments, we analysed trans-heterozygous combinations of the recessive sa allele with the three dominant alleles (Fig. 1A). Their antennal defects were stronger than those of the heterozygous Sa alleles alone, indicating that the residual activity of the Sa gene was further reduced. The entire flagellum was reduced to a short, club-shaped structure attached to the base of reduced tibia that was occasionally distorted (in 10% of the cases). The adjacent tarsal segments were fused with the tibia, whereas the three distal segments of the tarsus remain separate. In trans-heterozygous combinations with the recessive sa allele, these leg defects were enhanced, resulting in a distorted femur or fusion of femur and tibia, respectively (Fig. 1B). In addition, the trans-heterozygous combination of the two the antenna. In summary, there is a quantitative requirement for Sa gene function in the formation of flagellar segments that correspond to the distal part of the antenna.
Two dominant mutants, Sa-Fta and Sa-BQ, also showed leg defects that were confined to distal segments (Fig. 1B). By contrast, the legs of both the dominant Sa-8 allele and the recessive sa allele were indistinguishable from wild-type legs. In heterozygous Sa-Fta mutants, all tarsal segments were fused with the distal end of the tibia. Heterozygous Sa-BQ mutants displayed a severely alleles, Sa-8 and sa, that did not display leg defects on their own, showed a shortened and distorted tibia similar to the leg phenotype of heterozygous Sa-BQ beetles. Thus, the formation of distal leg segments is sensitive to the level of residual Sa gene activity. This quantitative effect is reminiscent of distal leg defects reported for trans-heterozygous combinations of Dll alleles in Drosophila, as summarised in Fig. 1C (Cohen et al., 1989b).
Embryos homozygous for dominant Sa alleles lack the limbs of the head and thorax
Given the similarity of the adult defects of Sa mutants to the Drosophila Dll phenotype (Cohen et al., 1989b), we inspected earlier developmental stages of Sa mutants. Homozygous sa embryos and larvae were indistinguishable from wild type.
Among the progeny of dominant Sa-alleles, approximately 25% of the larvae died soon after hatching (before the second moult). These larvae showed severe defects of the legs and head appendages (Fig. 2). Wild-type larval legs consist of five distinct segments: coxa, trochanter, femur, tibia and a pointed tarsus with a claw at its tip (Fig. 2A). By contrast, the leg stumps of mutant larvae were represented only by the most proximal segment, the coxa (Fig. 2B). The head appendages of the wild-type larva consist of an unpaired labrum, a pair of antennae, a pair of mandibles, a pair of maxillae with maxillary palps and an unpaired labium with labial palps (Fig. 2C). As illustrated in Fig. 2D, mutant larvae lacked all head appendages that are regarded as limbs. The labial and maxillary palps were missing, whereas the most proximal part of both labium and maxilla was present. In addition, the antennae and the labrum were absent. By contrast, the mandibles were not affected (Fig. 2D). These specific defects of homozygous Sa embryos precisely match the lethal phenotype of amorphic Dll alleles in Drosophila (Cohen et al., 1989a; Cohen et al., 1989b). Homozygous Sa mutants showed one additional defect that has no counterpart in Drosophila: the posterior structures named urogomphi, which may correspond to limbs on a posterior abdominal segment, were missing (arrows in Fig. 2A,B).
The recessive embryonic phenotype that results in larval lethality is a common feature of the dominant Sa mutants. We performed complementation tests between the dominant mutants Sa-BQ, Sa-8 and Sa-Fta, using the lethal phenotype as the criterion. All possible combinations gave the same lethal phenotypes among the F1 progeny. Thus, all three dominant mutants and the recessive sa mutant represent the same complementation group, the Sa gene.
Altered or no expression of Dll-related protein in homozygous Sa mutant embryos
The phenotypic similarity between Sa mutants in Tribolium and Dll mutants in Drosophila raised the possibility that the Sa gene might encode the orthologue of the Dll protein. To explore this possibility, we used the crossreacting anti-Dll antibody (Panganiban et al., 1995) to study the expression of Dll-related protein in the three strong Sa mutants. Tribolium wild-type embryos accumulated Dll-related protein in the distal regions of the legs and all head appendages except the mandibles (Fig. 3A). Additional expression domains of Dll-related protein were observed: the inner lobe of the branched maxillae (Fig. 3A, asterisk), the pleuropodia on abdominal segment 1 and small groups of cells in the (peripheral) nervous system. At later stages of embryogenesis, Dll-related protein was also expressed in a proximal ring-shaped domain in the legs (Fig. 3A).
Expression of the Dll-related protein was altered in homozygous Sa mutant embryos in an allele-specific manner. For the X-ray-induced allele Sa-8, no expression was observed at any stage of embryogenesis (data not shown). Sa-Fta mutant embryos gave only a faint, if any, signal at late developmental stages (Fig. 3B). Sa-BQ mutant embryos lacked all limb-specific staining except for the inner lobe of the maxillae that, together with small groups of cells in the peripheral nervous system, exhibited strong Dll-related protein expression (Fig. 3C,D). Based on these findings, the Sa gene is a good candidate for the Tribolium Dll orthologue, TcDll.
Tribolium cDNA clones encoding the Dll orthologue
The Dll gene encodes a homeodomain transcription factor that is highly conserved in evolution (Akimenko et al., 1994; Cohen et al., 1989; Panganiban et al., 1994; Panganiban et al., 1995; Price et al., 1991; Simeone et al., 1994; Vachon et al., 1992). A comparison of the Drosophila Dll gene with the mouse Dll gene revealed a conserved intron in the homeobox (Price et al., 1991). We therefore used a set of degenerate nested primers to PCR amplify the 5′ part of the homeobox. The resulting 107 bp PCR fragment was used to screen a Tribolium cDNA library.
Four cDNA clones were isolated (data not shown). Although three clones were truncated at the 5′ end, the longest clone was 1.9 kb in length and included an open reading frame corresponding to a polypeptide of 314 aa, which is approximately the size of the Drosophila Dll protein. Moreover, the predicted protein showed 100% identity in the homeodomain and other significant sequence similarities with Dll from both Drosophila and Precis (data not shown). To determine whether the Tribolium genome contains more than one TcDll gene we used the longest cDNA clone as well as the 107 bp PCR fragment for low stringency Southern blot analysis. Both experiments yielded no additional bands (data not shown), indicating that the TcDll gene is unique and probably represents the true orthologue. The DNA sequence of the TcDll gene and the deduced amino acid sequence of the protein are shown in Fig. 4 (GenBank accession number AF317551).
Expression pattern of TcDll in wild-type and Sa mutant embryos
We used in situ hybridisation to study the expression pattern of TcDll at various stages of embryogenesis (Fig. 5). The earliest expression was confined to two small domains in the procephalic region of the young germband (Fig. 5A,B). During germband elongation, sites of strong TcDll expression appeared in the procephalic region and the antennal segment (Fig. 5C). Slightly later, TcDll expression was initiated in the labral, maxillary, labial and the two anterior thoracic segments (Fig. 5D). Before the embryo was fully segmented, the third thoracic segment also started to express TcDll (Fig. 5E). In addition, TcDll expression was observed in the pleuropodia of the first abdominal segment (Fig. 5F,G). When the maxillae started to branch, the newly formed inner maxillary lobe also showed TcDll expression at the tip (Fig. 5H). By contrast, the mandibular segment did not express TcDll at any time during embryogenesis (Fig. 5F, arrows). It should be noted that TcDll expression had commenced before the limb primordia started to grow out. During outgrowth, TcDll expression was exclusively found in the most distal part of limb primordia (Fig. 5F-H). At later stages of embryogenesis, an additional proximal ring of TcDll expression appeared in the developing legs (Fig. 5I).
The RNA expression pattern of TcDll in different Sa mutant embryos closely resembled the protein expression revealed with the cross-reacting anti-Dll antibody (Fig. 6; compare with Fig. 3). No TcDll expression was observed in Sa-8 mutant embryos (Fig. 6D-F). Sa-Fta mutants exhibited TcDll expression in all limb primordia during early developmental stages (Fig. 6G). However, the inner lobe of the maxillae showed no expression at any time. At later stages, strong TcDll expression was found only in the antennae and the labrum but eventually the antennal expression also disappeared (Fig. 6H,I). Sa-BQ mutants showed a similar time course (Fig. 6K-M). TcDll was initially expressed in all limb primordia, whereas in later stages, expression was confined to the antennae and the inner part of the maxillae. Thus, the Sa mutants displayed allele-specific alterations of TcDll expression.
Genomic organisation of the TcDll gene and molecular characterisation of Sa alleles
The analysis of genomic Lambda clones revealed that the TcDll gene consists of 5 exons (Fig. 4, 7A). The TcDll gene spans a region of more than 21kb. However, its absolute size remains undefined since no genomic clones were isolated that bridge the highly conserved intron in the homeodomain (Fig. 7A). The results presented above strongly suggested that the Sa gene is the Tribolium counterpart of the Dll locus in Drosophila. To provide additional evidence, we used two different strategies to molecularly link the TcDll gene with the genetically identified Sa gene. First, we mapped the cloned TcDll gene relative to the Sa locus. Second, we tested the four Sa alleles for molecular lesions in the TcDll gene.
We have previously mapped the TcDll locus to position 7(43) on the RAPD linkage map (Beeman and Brown, 1999), but the Sa locus was not positioned on that map. Mapping of the TcDll gene relative to the Sa locus was done by cosegregation analysis. A sequence polymorphism between different Tribolium ecotypes in the intron 5′ to exon E was used as a molecular CAPS marker for the TcDll gene that enabled ecotype-specific alleles to be recognised as distinct bands (Fig. 7A). Single homozygous mutant larvae of a segregating F2 population from a cross between Sa mutants and the appropriate Tribolium ecotype were PCR genotyped. If the TcDll gene mapped close to the Sa locus or was identical to the Sa gene, one would expect few or no recombinants, respectively. No recombinants were found among 30 larvae tested. Thus, at a resolution of approximately 2 cM, TcDll was not separated from the Sa locus.
To search for mutations in the TcDll gene of Sa mutants, all exons were sequenced in the Sa-8, Sa-Fta, Sa-BQ and sa alleles. Except for a few silent polymorphisms (data not shown), no deviations from the corresponding wild-type sequence were found. In addition, the two bona fide X-ray alleles, Sa-8 and Sa-Fta, and the Sa-BQ allele were also tested for chromosomal rearrangements by comparing restriction maps of the genomic TcDll region between heterozygous mutants and their wild-type siblings. No gross alterations were observed in Sa-8 and Sa-BQ (data not shown). By contrast, several probes from the 3′ region of the TcDll gene revealed new bands in heterozygous Sa-Fta mutants, indicating a chromosomal rearrangement in this region (Fig. 7B,C). Although we were unable to delineate the chromosomal rearrangement as a simple inversion or deletion, we confirmed the result with different restriction digests that yielded polymorphic bands.
Our results establish a role for TcDll in limb development. The TcDll protein is encoded by a single-copy gene and contains a homeodomain that is identical to those in Drosophila Dll and in the Precis Dll homologue. Furthermore, the initial expression patterns in embryos of these three insect species are remarkably similar, if not identical, to each other. Most importantly, both genetic mapping and molecular analysis of the Sa-Fta allele indicate that TcDll is encoded by the Sa gene, which is represented by four mutant alleles with striking phenotypic similarities to the allelic series of Dll phenotypes in Drosophila. By these criteria, Sa /TcDll is the Tribolium orthologue of the Drosophila Dll gene.
Evolutionary conservation of Dll function in limb development
Previous studies of Dll expression in various arthropod species suggested that Dll is a conserved key regulator in limb development (Cohen et al., 1989; Panganiban et al., 1994; Panganiban et al., 1995; Vachon et al., 1992). However, functional requirement of Dll expression could not be assessed because there was no genetic or other means to interfere specifically with Dll action. We have now identified in Tribolium, an insect species distantly related to Drosophila, mutations in a single gene, Sa/TcDll, that affect limb development essentially the same way as do Dll mutations, both qualitatively and quantitatively. Complete lack of Sa gene function, as evidenced by the lack of TcDll mRNA and protein accumulation, specifically abolishes the formation of all limb structures in both head and thorax as does complete lack of Dll gene function in Drosophila (Cohen and Jürgens, 1989a; Cohen and Jürgens, 1989b). Moreover, the development of adult antennae and legs is sensitive to reduced Sa activity, as shown in heterozygotes for dominant alleles and several trans-heterozygous allele combinations, which parallels the dosage-dependent effects of Dll activity in Drosophila (Cohen and Jürgens, 1989a; Cohen and Jürgens, 1989b). In addition, the expression analysis of two strong alleles, Sa-Fta and Sa-BQ, suggests that some aspects of Dll gene regulation are conserved between Tribolium and Drosophila. In Sa-Fta embryos, TcDll mRNA does not accumulate stably in most limb primordia but persists longer in the antennae. In Sa-BQ embryos, both RNA and protein accumulate in the inner lobe of the maxillae as well as in segmentally repeated cell groups of the peripheral nervous system. These observations suggest the existence of distinct enhancer elements in the as yet undefined cis-regulatory regions of the Sa/TcDll gene. Similarly, the expression patterns of Drosophila Dll alleles, Dll-J and Dll-IB, define elements that drive expression in corresponding regions of the mutant embryos (O’Hara et al., 1993). In summary, the available evidence supports the notion that Dll function in limb development is evolutionarily conserved.
Delayed Dll expression in Drosophila – a heterochronic change in limb development?
Across the insects, with the exception of the Diptera, limb primordia are established during embryogenesis, giving rise to well-formed larval limbs within which the founder cells of the imaginal limbs proliferate (e.g. Pieris; for a discussion, see Cohen et al., 1989b). By contrast, the dipteran larvae are limbless. In lower dipterans, the imaginal limb primordia of both head and thorax originate in the embryo in close association with distal sense organs. In higher dipterans such as Drosophila, this association is not obvious for the primordium of the eye-antennal disc and the distal sense organs of the antenna and the maxilla (Jürgens et al., 1986). These evolutionary changes, culminating in head involution, resulted in a derived condition that is characterised by divergent pathways of larval and imaginal limb development. However, the initial expression of Dll at specific distal positions within the segments of head and thorax closely resembles the TcDll expression pattern before limb primordia are morphologically discernible. Subsequently, larval limb development is suppressed in Drosophila. The small imaginal primordia are involuted towards the end of embryogenesis, they grow into large disc-shaped epithelia within the larva and are transformed into imaginal limbs by evagination during the pupal stage. Thus, a pupal leg in Drosophila appears to correspond to an embryonic leg in Tribolium. This view is supported by the equivalent expression of Dll in the distal region corresponding to the tarsus and the distal tibia, and in a proximal ring corresponding to the proximal part of the femur (Fig. 8; Diaz-Benjumea et al., 1994). Similar patterns of Dll expression were observed in the embryos of diverse insect species, the butterfly Precis, the grasshopper Schistocerca and the cricket Gryllus, using the crossreacting antibody (Jockusch et al., 2000; Panganiban et al., 1994; Niwa et al., 1997). Although it is not known when during disc development the mature pattern of Dll expression is established in Drosophila this is likely to occur after the imaginal disc has reached a certain cell number. Thus, the Dll expression pattern appears to reflect a heterochronic change in limb development.
Conserved Dll function in support of the Snodgrass hypothesis
Limbs are segmentally repeated ventral body appendages that are subject to segment-specific developmental modifications, including changes in shape and function as well as complete loss. A large body of evidence, mainly from Drosophila, indicates that Dll integrates the developmental signals that initiate and modify limb development (Cohen et al., 1989; Diaz-Benjumea et al., 1994; Lecuit and Cohen, 1997). Among the positive regulators of Dll expression are the segment-polarity genes such as wingless and engrailed, which provide the anterior-posterior coordinate, and genes like dpp, which provide the dorsoventral coordinate (Basler et al., 1994). Superimposed is a negative control of Dll expression that is exerted by the homeotic genes. In Drosophila, a negative cis-regulatory element of Dll has been shown to bind BXC gene products (Vachon et al., 1992). Similarly, larval prolegs in abdominal segments 2-6 of caterpillars are formed by cell groups that express Dll but not the abd-A gene (Warren et al.,1994; Weatherbee et al., 1999). This regulatory network appears to be largely conserved between Drosophila and Tribolium. Not only are the initial patterns of Dll expression very similar but also the requirement for Dll action, as evidenced by the Sa and Dll mutant phenotypes, is essentially the same. This parallel is best illustrated by the mandible whose limb status has been controversial (Manton, 1964; Popadic et al., 1998) In both Drosophila and Tribolium, the mandible does not express Dll nor does it require Dll function for normal development (Cohen et al., 1989; Cohen and Jürgens, 1989a; Cohen and Jürgens, 1989b). These results support the notion that the mandible is not a limb but corresponds to a basal structure derived from the body wall.
Snodgrass proposed a hypothetical ground state of the arthropod leg (Snodgrass, 1935). The leg was divided into the proximal part consisting of the coxa, a simple outpocketing of the body wall called the coxopodite, and into the distal telopodite representing the limb proper. The idea that the coxopodite and the telopodite are distinct is supported by the finding that genes regulating the proximal-distal axis in Drosophila define a sharp boundary at the coxa-trochanter junction. The proximal region corresponding to the coxa is characterised by the expression of Homothorax (hth) which facilitates the nuclear localisation of Extradenticle (Exd) (Rieckhoff et al., 1997). In the distal region, Wingless (Wg) and Decapentaplegic (Dpp) act through their targets Dll and dachshund (dac) to restrict hth expression and thereby nuclear localisation of exd. Thus, the domain of the ‘coxopodite genes’ hth and exd and that of the ‘telopodite genes’ of the Wg/Dpp pathway are mutually exclusive (Abu-Shaar and Mann, 1998; Gonzáles-Crespo and Morata, 1996; Gonzalez-Crespo et al., 1998). Comparable analyses have not been possible in other insect species. Our analysis of the lack-of-function phenotype of the Sa/TcDll gene, however, provides additional support for Snodgrass’ original idea. As in Drosophila, lack of Dll function abolishes the limb proper, or telopodite, but leaves the coxa and body wall unaffected, thus demonstrating that a basic subdivision of the leg into proximal and distal regions also holds true in another, evolutionarily distant insect species. Considering the potential of Tribolium as another genetic model for the study of insect development (Berghammer et al., 1999; Maderspacher et al., 1998; Sulston and Anderson, 1996), functional analysis of the regulatory network of Dll action in limb development may soon help to address mechanisms underlying the evolution of limb development beyond the level of comparative gene expression patterns.
We thank R. Schröder, S. Brown, S. Haas, K. Handel, G. Panganiban and M. Klingler for generously providing material for this study; and S. Roth and members of his lab for technical advice. We particularly thank S. M. Cohen for his interest and intellectual input during the course of this project. This work was supported by a Leibniz award from the DFG to G. J. and a HFSPO grant to D. G. J. and D. T.