The role of wingless in the development of multibranched crustacean limbs

 Arthropods are the most diverse and speciose group of organisms on earth. A key feature in their successful radiation is the ease with which various appendages become readily adapted to new functions in novel environments. Arthropod limbs differ radically in form and function, from unbranched walking legs to multibranched swimming paddles. To uncover the developmental and genetic mechanisms underlying this diversification in form, we ask whether a three-signal model of limb growth based on Drosophila experiments is used in the development of arthropod limbs with variant shape. We cloned a Wnt-1 ortholog (Tlwnt-1) from Triops longicaudatus, a basal crustacean with a multibranched limb. We examined the mRNA in situ hybridization pattern during larval development to determine whether changes in wg expression are correlated with innovation in limb form. During larval growth and segmentation Tlwnt-1 is expressed in a segmentally reiterated pattern in the trunk. Unexpectedly, this pattern is restricted to the ventral portion of the epidermis. During early limb formation the single continuous stripe of Tlwnt-1 expression in each segment becomes ventrolaterally restricted into a series of shorter stripes. Some but not all of these shorter stripes correspond to what becomes the ventral side of a developing limb branch. We conclude that the Drosophila model of limb development cannot explain all types of arthropod proximodistal outgrowths, and that the multibranched limb of Triops develops from an early reorganization of the ventral body wall. In Triops, Tlwnt-1 plays a semiconservative role similar to that played by Drosophila wingless in segmentation and limb formation, and morphological innovation in limb form arises in part through an early modulation in the expression of the Tlwnt-1 gene. Received: 22 September 1998 / Accepted: 12 January 1999     

Control of Distal-less expression in the Drosophila appendages by functional 3' enhancers

The expression of the Hox gene Distal-less (Dll) directs the development of appendages in a wide variety of animals. In Drosophila, its expression is subjected to a complex developmental control. In the present work we have studied a 17 kb genomic region in the Dll locus which lies downstream of the coding sequence and found control elements of primary functional importance for the expression of Dll in the leg and in other tissues. Of particular interest is a control element, which we have called LP, which drives expression of Dll in the leg primordium from early embryonic development, and whose deletion causes severe truncation and malformation of the adult leg. This is the first Dll enhancer for which, in addition to the ability to drive expression of a reporter, a role can be demonstrated in the expression of the endogenous Dll gene and in the development of the leg. In addition, our results suggest that some enhancers, contrary to the widely accepted notion, may require a specific 5′ or 3′ position with respect to the transcribed region.     

Loss of <Emphasis Type="Italic">spineless</Emphasis> function transforms the <Emphasis Type="Italic">Tribolium</Emphasis> antenna into a thoracic leg with pretarsal,tibiotarsal, and femoral identity

The Drosophila spineless (ss) gene is regulated downstream of the appendage gene Distal-less (Dll) and is involved in leg and antenna development. Specifically, loss of ss leads to the homeotic transformation of the arista, the distalmost antennal segment, into tarsal identity, and the loss or fusion of distal leg segments. Here we show that the ss homolog from the red flour beetle Tribolium castaneum also homeotically transforms the beetle antenna into leg, but the extent of the transformation is significantly larger than in Drosophila, as the entire antenna (except for the basal antennifer) is transformed into pretarsal, tibiotarsal, and femoral identity; i.e., the transformation comprises the Dll positive area in both appendages. We interpret the antennal phenotype in Tribolium as evidence for a more exclusive role of ss in antennal determination downstream of Dll in the beetle. By contrast, the fact that, in Drosophila ss mutants, only a small portion of the Dll positive area in the antenna is homeotically transformed indicates that Dll uses additional targets to govern the development of the other antennal segments in the fly.     

Limb development in a primitive crustacean,Triops longicaudatus: subdivision of the early limb bud gives rise to multibranched limbs

 Recent advances in developmental genetics of Drosophila have uncovered some of the key molecules involved in the positioning and outgrowth of the leg primordia. Although expression patterns of these molecules have been analyzed in several arthropod species, broad comparisons of mechanisms of limb development among arthropods remain somewhat speculative since no detailed studies of limb development exist for crustaceans, the postulated sister group of insects. As a basis for such comparisons, we analysed limb development in a primitive branchiopod crustacean, Triops longicaudatus. Adults have a series of similar limbs with eight branches or lobes that project from the main shaft. Phalloidin staining of developing limbs buds shows the distal epithelial ridge of the early limb bud exhibits eight folds that extend in a dorsal ventral (D/V) arc across the body. These initial folds subsequently form the eight lobes of the adult limb. This study demonstrates that, in a primitive crustacean, branched limbs do not arise via sequential splitting. Current models of limb development based on Drosophila do not provide a mechanism for establishing eight branches along the D/V axis of a segment. Although the events that position limbs on a body segment appear to be conserved between insects and crustaceans, mechanisms of limb branching may not. Received: 28 February 1996/Accepted: 24 June 1996     

Functional stability of the aristaless gene in appendage tip formation during evolution

The appendages of an insect are subdivided into distinct segments or podomeres. Many genes responsible for the regionalization of the growing limb into subdomains have been isolated from Drosophila. So far, only one gene is known in the leg that is solely required for specifying the distal-most pattern element—the pretarsal claw. In Drosophila, the gene aristaless is expressed in the centre of the antennal and leg imaginal disc that represents the most distal position of appendages, and in a proximal region. When Drosophila aristaless function is impaired, antennae and legs develop without their distal-most structures—the arista and the claw. We describe here the analysis of aristaless in the beetle Tribolium—an insect that shows a different, more ancestral mode of appendage formation than Drosophila. In Tribolium, appendages grow out continuously during embryogenesis, and no imaginal discs are formed. Tribolium aristaless (Tc-al) expression starts midway during appendage elongation, and is seen in a distal and a proximal position of head and trunk appendages. At the end of embryogenesis, Tc-al is seen in four expression domains in the leg, in the dorsal epidermis, and ventrally in every segment in lateral groups of cells, presumably the histoblasts. Like in the Drosophila adult, Tc-al is required in the larva for the formation of the most distal structures of the leg and the antenna as revealed by RNAi experiments. We conclude that aristaless is evolutionarily robust, meaning that it has retained its expressional and functional characteristics, although a heterochronic change of the process of appendage elongation took place towards the evolution of the highly derived diptera.Edited by D. Tautz     

Cell migration within the embryonic limb primordium of Drosophila as revealed by a novel fluorescence method to visualize mRNA and protein

 We report a new technique using fluorescent probes to detect a mRNA and a protein simultaneously in the Drosophila embryo. For in situ hybridization, 3-hydroxy-N-2′-biphenyl-2-naphthalenecarboxamide phosphate ester (HNPP)/Fast Red TR was used as a fluorescent substrate for alkaline phosphatase. It was possible to compare protein and mRNA expression on a cell by cell basis with a laser scanning confocal microscope. We applied this technique to analyse the dynamics of Distal-less (Dll) enhancer activity in the thoracic limb primordium in the early Drosophila embryo. We stained embryos bearing the Dll early enhancer (Dll-304) fused to the Escherichia coli lacZ gene. LacZ mRNA was detectable in the ventral region of the limb primordium, and β-galactosidase protein in the dorsal region. In the middle, both mRNA and protein were detectable. These results suggest that the Dll enhancer is activated in the ventral region of the limb primordium and that Dll-positive cells migrate from a ventral position to a dorsal one within a single limb primordium. Received: 7 April 1997 / Accepted: 15 May 1997     

Clonal analysis of <Emphasis Type="Italic">Distal-less</Emphasis> and <Emphasis Type="Italic">engrailed</Emphasis> expression patterns during early morphogenesis of uniramous and biramous crustacean limbs

In order to investigate the correlation of cell lineage, gene expression, and morphogenesis of uniramous and biramous limbs we studied limb formation in the thorax and pleon of the amphipod Orchestia cavimana and the isopod Porcellio scaber. We took advantage of the fact that in amphipod and isopod crustaceans—both Malacostraca—uniramous limbs evolved independently in the thorax whereas ancestral biramous limbs are formed in the pleon (abdomen). The gene Distal-less is expressed in the early limb buds as in other arthropods. Accordingly, it is likely to be responsible for the development of the proximodistal axis of the appendages. Double staining of Distal-less and Engrailed proteins suggests that Distal-less in the pleon of the amphipod Orchestia might not be under the control of the Wingless protein. Additionally, we studied axis formation of the uniramous and biramous limbs. In both species investigated, biramous limbs originate exclusively by the subdivision of the original limb bud. Both distal elements continuously express Distal-less. There is flexibility in the suppression of the development of additional branches in the crustacean limb. In the amphipod O. cavimana, uniramous thoracopods are formed by downregulation of Distal-less in the area where, in biramous limbs, the exopodites would occur. In contrast, this region never expresses Distal-less in the uniramous thoracopods of the isopod P. scaber. Our results suggest that the gene expression pattern is independent of the cell division pattern. Gene expression domains and morphogenesis of limbs and segments, on the other hand, show a good correlation.Edited by D. Tautz     

Appendage patterning in the South American bird spider <Emphasis Type="Italic">Acanthoscurria geniculata</Emphasis> (Araneae: Mygalomorphae)

Pattern formation by the genes dachshund (dac), Distal-less (Dll), extradenticle (exd) and homothorax (hth) in spider appendages has been studied previously only in members of the higher spiders (Araneomorphae). In order to study the diversity and conservation of pattern formation in spiders as a whole, we studied homologs of these genes in embryos of the bird spider Acanthoscurria geniculata, which belongs to the Mygalomorphae, a more primitive spider group. We show that the patterns of dac and Dll are largely conserved in all spiders studied so far. We find a duplication of hth and exd genes as previously identified in the higher spider Cupiennius salei. These data suggest that pattern formation shows little diversity in all spiders, including the duplication of hth and exd that likely occurred before the split of Mygalomorphae and Araneomorphae. We also find that the legs and pedipalps bear endites of which only the pedipalpal endite expresses Dll and is retained in the adult. Similarly, the limb buds of the posterior spinnerets express Dll and become segmented appendages in the adult, whereas the anterior spinnerets lack Dll expression and are absent in postembryonic stages. In both cases, the expression of Dll or the lack of it indicates structures which will be retained as adult traits or rudimentary structures that degenerate, respectively. The presence of embryonic rudiments of leg endites in Acanthoscurria and the leg-like pattern formation in the posterior spinnerets are interpreted as primitive traits that have been lost in the Araneomorphae.     

Gene silencing in the spider mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>: dsRNA and siRNA parental silencing of the <Emphasis Type="Italic">Distal-less</Emphasis> gene

A major prerequisite to understanding the evolution of developmental programs includes an appreciation of gene function in a comparative context. RNA interference (RNAi) represents a powerful method for reverse genetics analysis of gene function. However, RNAi protocols exist for only a handful of arthropod species. To extend functional analysis in basal arthropods, we developed a RNAi protocol for the two-spotted spider mite Tetranychus urticae focusing on Distal-less (Dll), a conserved gene involved in appendage specification in metazoans. First, we describe limb morphogenesis in T. urticae using confocal and scanning electron microscopy. Second, we examine T. urticae Dll (Tu-Dll) mRNA expression patterns and correlate its expression with appendage development. We then show that fluorescently labeled double-stranded RNA (dsRNA) and short interfering RNA (siRNA) molecules injected into the abdomen of adult females are incorporated into the oviposited eggs, suggesting that dsRNA reagents can be systemically distributed in spider mites. Injection of longer dsRNA as well as siRNA induced canonical limb truncation phenotypes as well as the fusion of leg segments. Our data suggest that Dll plays a conserved role in appendage formation in arthropods and that such conserved genes can serve as reliable starting points for the development of functional protocols in nonmodel organisms.     

The involvement of <Emphasis Type="Italic">engrailed</Emphasis> and <Emphasis Type="Italic">wingless</Emphasis> during segmentation in the onychophoran <Emphasis Type="Italic">Euperipatoides kanangrensis</Emphasis> (Peripatopsidae: Onychophora) (Reid 1996)

As the putative sister group to the arthropods, onychophorans can provide insight into ancestral developmental mechanisms in the panarthropod clade. Here, we examine the expression during segmentation of orthologues of wingless (Wnt1) and engrailed, two genes that play a key role in defining segment boundaries in Drosophila and that appear to play a role in segmentation in many other arthropods. Both are expressed in segmentally reiterated stripes in all forming segments except the first (brain) segment, which only shows an engrailed stripe. Engrailed is expressed before segments are morphologically visible and is expressed in both mesoderm and ectoderm. Segmental wingless expression is not detectable until after mesodermal somites are clearly distinct. Early engrailed expression lies in and extends to both sides of the furrow that first demarcates segments in the ectoderm, but is largely restricted to the posterior part of somites. Wingless expression lies immediately anterior to engrailed expression, as it does in many arthropods, but there is no precise cellular boundary between the two expression domains analogous to the overt parasegment boundary seen in Drosophila. Engrailed stripes extend along the posterior part of each limb bud, including the antenna, while wingless is restricted to the distal tip of the limbs and the neurectoderm basal to the limbs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular evidence for the gnathobasic derivation of arthropod mandibles and for the appendicular origin of the labrum and other structures

 Mandibles are feeding appendages functioning as ”jaws” in the arthropod groups in which they occur. Which part of this appendage is involved in food manipulation (limb tip versus limb base), has been used to suggest phylogenetic relationships among some of the major taxa of arthropods (myriapods, crustaceans, and insects). As a way to independently verify the conclusions drawn from previous morphological analyses, we have studied the expression pattern of the gene Distal-less (Dll), which specifies the distal part of appendages. Our results show, in contrast to the traditional view, that both insect and crustacean adult mandibles are gnathobasic, handling food with the basal portion of the appendage. Furthermore, as is evident by the reduction in the number of Dll-expressing cells in the later developmental stages, adult diplopod jaws are also gnathobasic. Thus, jaws of all mandibulates (myriapods, crustaceans, and insects) seem to have a similar gnathobasic structure. We have also found that Dll is expressed in the labra of all arthropod taxa examined, suggesting that this structure is of appendicular derivation. Additionally, the spinnerets and book lungs of spiders, long considered on other grounds to be modified appendages, express Dll, confirming this interpretation. This study shows that, in addition to their use in phylogenetic and population genetic studies, molecular markers can be very useful for inferring the origins of a particular morphological feature. Received: 12 January 1998 / Accepted: 23 March 1998     

Limb ontogeny and trunk segmentation in Nebalia species (Crustacea, Malacostraca, Leptostraca)

The ’egg-larval’ development of two species of Nebalia has been examined with SEM. Various details concerning limb ontogeny and trunk segmentation are described. The most important of these are the following. The tripartite state of the peduncle of antenna 2 in the adult of Nebalia species is derived from the fusion of the third and fourth podomeres, present in late larvae. The proximal portion of the mandible in the adult of Nebalia brucei, carrying the ’coxal process’, is, based on the ontogenetic evidence, interpreted as the combined basis and coxa, and the bipartite palp is interpreted as the endopod. The early development of the thoracopods and the three anteriormost pleopods is identical. They all start as laterally directed, biramous limb buds. This suggests that tagmatisation of the trunk of the Leptostraca (and other Malacostraca) has been developed from an ancestor with an undivided trunk region with serially similar limbs. Certain early stages reveal an extra, ’eighth’, limbless pleon segment, as compared with the normal number of seven pleomeres of adult Leptostraca. The presence of a row of ventral, sternitic, triangular processes between the bases of the thoracopods, as they are found in certain stages of a species of Nebalia, is suggested as a possible ground pattern for the Malacostraca. Accepted: 1 February 2000     

Expression of arthropod distal limb-patterning genes in the onychophoran Euperipatoides kanangrensis

A current hypothesis states that the ancestral limb of arthropods is composed of only two segments. The proximal segment represents the main part of the modern leg, and the distal segment represents the tarsus and claw of the modern leg. If the distal part of the limb is an ancestral feature, one would expect conserved regulatory gene networks acting in distal limb development in all arthropods and possibly even their sister group, the onychophorans. We investigated the expression patterns of six genes known to function during insect distal limb development in the onychophoran Euperipatoides kanangrensis, i.e., clawless (cll), aristaless (al), spineless (ss), zinc finger homeodomain 2 (zfh2), rotund (rn), and Lim1. We find that all investigated genes are expressed in at least some of the onychophoran limbs. The expression patterns of most of these genes, however, display crucial differences to the known insect patterns. The results of this study question the hypothesis of conserved distal limb evolution in arthropods and highlight the need for further studies on arthropod limb development.     

Patterning of the branched head appendages in Schistocerca americana and Tribolium castaneum

Much of our understanding of arthropod limb development comes from studies on the leg imaginal disc of Drosophila melanogaster. The fly limb is a relatively simple unbranched (uniramous) structure extending out from the body wall. The molecular basis for this outgrowth involves the overlap of two signaling molecules, Decapentaplegic (Dpp) and Wingless (Wg), to create a single domain of distal outgrowth, clearly depicted by the expression of the Distal-less gene (Dll). The expression of wg and dpp during the development of other arthropod thoracic limbs indicates that these pathways might be conserved across arthropods for uniramous limb development. The appendages of crustaceans and the gnathal appendages of insects, however, exhibit a diverse array of morphologies, ranging from those with no distal elements, such as the mandible, to appendages with multiple distal elements. Examples of the latter group include branched appendages or those that possess multiple lobes; such complex morphologies are seen for many crustacean limbs as well as the maxillary and labial appendages of many insects. It is unclear how, if at all, the known patterning genes for making a uniramous limb might be deployed to generate these diverse appendage forms. Experiments in Drosophila have shown that by forcing ectopic overlaps of Wg and Dpp signaling it is possible to generate artificially branched legs. To test whether naturally branched appendages form in a similar manner, we detailed the expression patterns of wg, dpp, and Dll in the development of the branched gnathal appendages of the grasshopper, Schistocerca americana, and the flour beetle, Tribolium castaneum. We find that the branches of the gnathal appendages are not specified through the redeployment of the Wg-Dpp system for distal outgrowth, but our comparative studies do suggest a role for Dpp in forming furrows between tissues.     

Separable functions of wingless in distal and ventral patterning of the Tribolium leg

The gene wingless (wg) in Drosophila is an important factor in leg development. During embryonic development wg is involved in the allocation of the limb primordia. During imaginal disk development wg is involved in distal development and it has a separate role in ventral development. The expression pattern of wg is highly conserved in all arthropods (comprising data from insects, myriapods, crustaceans, and chelicerates), suggesting that its function in leg development is also conserved. However, recent work in other insects (e.g. the milkweed bug Oncopeltus fasciatus) argued against a role of wg in leg development. We have studied the role of wg in leg development of the flour beetle Tribolium castaneum. Using stage-specific staggered embryonic RNAi in wild-type and transgenic EGFP expressing enhancer trap lines we are able to demonstrate separable functions of Tribolium wg in distal and in ventral leg development. The distal role affects all podomeres distal to the coxa, whereas the ventral role is restricted to cells along the ventral midline of the legs. In addition, severe leg defects after injection into early embryonic stages are evidence that wg is also involved in proximal development and limb allocation in Tribolium. Our data suggest that the roles of wg in leg development are highly conserved in the holometabolous insects. Further studies will reveal the degree of conservation in other arthropod groups.     

From development to biodiversity—Tribolium castaneum, an insect model organism for short germband development

Insect embryogenesis is best understood in the fruit fly Drosophila. However, Drosophila embryogenesis shows evolutionary-derived features: anterior patterning is controlled by a highly derived Hox gene bicoid, the body segments form almost simultaneously and appendages develop from imaginal discs. In contrast, embryogenesis of the red flour beetle Tribolium castaneum displays typical features in anterior patterning, axis and limb formation shared with most insects, other arthropods as well as with vertebrates. Anterior patterning depends on the conserved homeobox gene orthodenticle, the main body axis elongates sequentially and limbs grow continuously starting from an appendage bud. Thus, by analysing developmental processes in the beetle at the molecular and cellular level, inferences can be made for similar processes in other arthropods. With the completion of sequencing the Tribolium genome, the door is now open for post-genomic studies such as RNA expression profiling, proteomics and functional genomics to identify beetle-specific gene circuits.