Divergent and conserved roles of extradenticle in body segmentation and appendage formation, respectively, in the cricket Gryllus bimaculatus

The cricket Gryllus bimaculatus is a typical hemimetabolous intermediate germ insect, in which the processes of segmentation and appendage formation differ from those in Drosophila, a holometabolous long germ insect. In order to compare their developmental mechanisms, we have focused on Gryllus orthologs of the Drosophila developmental regulatory genes and studied their functions. Here, we report a functional analysis of the Gryllus ortholog of extradenticle (Gb′exd) using embryonic and parental RNA interference (RNAi) techniques. We found the following: (1) RNAi suppression of Gb′exd results in the deletion or fusion of body segments. Especially the head was often very severely affected. This gap-like phenotype may be related to reduced expression of the gap genes hunchback and Krüppel in early RNAi germbands. (2) In the appendages, several segments (podomeres) were fused. (3) Head appendages including the antenna were transformed to a leg-like structure consisting of at least one proximal podomere as well as several tarsomeres. The defects in appendages are reminiscent of the phenotype caused by large exd clones in Drosophila antennal discs. These findings led us to the conclusion that (1) Gb′exd is required for segment patterning in the gnathal to abdominal region, acting in a gap gene-like manner in the anterior region. (2) Gb′exd plays important roles in formation of the appendages and the determination of their identities, acting as a regulatory switch that chooses between the fates of head appendages versus the appendage ground state. Although functions of Gb′exd in appendage patterning appear fundamentally conserved between Gryllus and Drosophila, its role in body segmentation may differ from that of Drosophila exd.     

A conserved genetic mechanism specifies deutocerebral appendage identity in insects and arachnids

The segmental architecture of the arthropod head is one of the most controversial topics in the evolutionary developmental biology of arthropods. The deutocerebral (second) segment of the head is putatively homologous across Arthropoda, as inferred from the segmental distribution of the tripartite brain and the absence of Hox gene expression of this anterior-most, appendage-bearing segment. While this homology statement implies a putative common mechanism for differentiation of deutocerebral appendages across arthropods, experimental data for deutocerebral appendage fate specification are limited to winged insects. Mandibulates (hexapods, crustaceans and myriapods) bear a characteristic pair of antennae on the deutocerebral segment, whereas chelicerates (e.g. spiders, scorpions, harvestmen) bear the eponymous chelicerae. In such hexapods as the fruit fly, Drosophila melanogaster, and the cricket, Gryllus bimaculatus, cephalic appendages are differentiated from the thoracic appendages (legs) by the activity of the appendage patterning gene homothorax (hth). Here we show that embryonic RNA interference against hth in the harvestman Phalangium opilio results in homeonotic chelicera-to-leg transformations, and also in some cases pedipalp-to-leg transformations. In more strongly affected embryos, adjacent appendages undergo fusion and/or truncation, and legs display proximal defects, suggesting conservation of additional functions of hth in patterning the antero-posterior and proximo-distal appendage axes. Expression signal of anterior Hox genes labial, proboscipedia and Deformed is diminished, but not absent, in hth RNAi embryos, consistent with results previously obtained with the insect G. bimaculatus. Our results substantiate a deep homology across arthropods of the mechanism whereby cephalic appendages are differentiated from locomotory appendages.     

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     

The roles of wingless and decapentaplegic in axis and appendage development in the red flour beetle, Tribolium castaneum

Axis patterning and appendage development have been well studied in Drosophila melanogaster, a species in which both limb and segment morphogenesis are derived. In Drosophila, positional information from genes important in anteroposterior and dorsoventral axis formation, including wingless (wg) and decapentaplegic (dpp), is required for allocating and patterning the appendage primordia. We used RNA interference to characterize the functions of wg and dpp in the red flour beetle, Tribolium castaneum, which retains more ancestral modes of limb and segment morphogenesis. We also characterized the expression of potential targets of the WG and DPP signaling pathways in these embryos. Tribolium embryos in which dpp had been downregulated had defects in the dorsalmost body wall, but did not appear to have been globally repatterned and had normal appendages. Downregulation of wg led to the loss of segment boundaries, gnathal and thoracic appendages, and lateral head lobes, and to changes in the expression of dpp, Distal-less, and Engrailed. The functions of wg varied along both the anteroposterior and dorsoventral axes of the embryo. Phylogenetic comparisons indicate that the role of WNT signaling in segment boundary formation is evolutionarily old, but that its role in appendage allocation originated in the common ancestor of holometabolous insects.     

The Tribolium ortholog of knirps and knirps-related is crucial for head segmentation but plays a minor role during abdominal patterning

Segment formation in the long germ insect Drosophila is dominated by overlapping gap gene domains in the syncytial blastoderm. In the short germ beetle Tribolium castaneum abdominal segments arise from a cellular growth zone, implying different patterning mechanisms. We describe here the single Tribolium ortholog of the Drosophila genes knirps and knirps-related (called Tc-knirps). Tc-knirps expression is conserved during head patterning and at later stages. However, posterior Tc-knirps expression in the ectoderm is limited to a stripe in A1, instead of a broad abdominal domain covering segment primordia A2-A5 as in Drosophila. Tc-knirps RNAi yields only mild defects in the abdomen, at a position posterior to the abdominal Tc-knirps domain. In addition, Tc-knirps RNAi larvae lack the antennal and mandibular segments. These defects are much more severe than the head defects caused by combined inactivation of Dm-knirps and Dm-knirps-related. Our findings support the notion that the role of gap gene homologs in abdominal segmentation differs fundamentally in long and short germ insects. Moreover, the pivotal role of Tc-knirps in the head suggests an ancestral role for knirps as head patterning gene. Based on this RNAi analysis, Tc-knirps functions neither in the head nor the abdomen as a canonical gap gene.     

The pleiohomeotic gene is required for maintaining expression of genes functioning in ventral appendage formation in Drosophila melanogaster

Polycomb group (PcG) proteins are negative regulators that maintain the expression of homeotic genes and affect cell proliferation. Pleiohomeotic (Pho) is a unique PcG member with a DNA-binding zinc finger motif and was proposed to recruit other PcG proteins to form a complex. The pho null mutants exhibited several mutant phenotypes such as the transformation of antennae to mesothoracic legs. We examined the effects of pho on the identification of ventral appendages and proximo-distal axis formation during postembryogenesis. In the antennal disc of the pho mutant, Antennapedia (Antp), which is a selector gene in determining leg identity, was ectopically expressed. The homothorax (hth), dachshund (dac) and Distal-less (Dll) genes involved in proximo-distal axis formation were also abnormally expressed in both the antennal and leg discs of the pho mutant. The engrailed (en) gene, which affects the formation of the anterior-posterior axis, was also misexpressed in the anterior compartment of antennal and leg discs. These mutant phenotypes were enhanced in the mutant background of Posterior sex combs (Psc) and pleiohomeotic-like (phol), which are another PcG genes. These results suggest that pho functions in maintaining expression of genes involved in the formation of ventral appendages and the proximo-distal axis.     

Abd-B suppresses lepidopteran proleg development in posterior abdomen

Pterygotes lack abdominal appendages except for pleuropods and prolegs. The larvae of some holometabolous insects develop prolegs, which are used for locomotion. We analyzed the role of the homeotic genes abd-A and Abd-B in lepidopteran proleg development using mutant analysis and embryonic RNAi in the silkworm Bombyx mori. The E^Mu mutant developed extra prolegs in its posterior abdomen and showed the misexpression of both genes, suggesting their involvement in proleg formation. The depletion of Abd-B by embryonic RNAi caused the development of extra prolegs on all segments posterior to A6, indicating the suppressive function of Abd-B. The abd-A RNAi animals failed to develop prolegs. These results indicate that abd-A and Abd-B are involved in proleg development in B. mori.     

Co-option of an anteroposterior head axis patterning system for proximodistal patterning of appendages in early bilaterian evolution

The enormous diversity of extant animal forms is a testament to the power of evolution, and much of this diversity has been achieved through the emergence of novel morphological traits. The origin of novel morphological traits is an extremely important issue in biology, and a frequent source of this novelty is co-option of pre-existing genetic systems for new purposes (Carroll et al., 2008). Appendages, such as limbs, fins and antennae, are structures common to many animal body plans which must have arisen at least once, and probably multiple times, in lineages which lacked appendages. We provide evidence that appendage proximodistal patterning genes are expressed in similar registers in the anterior embryonic neurectoderm of Drosophila melanogaster and Saccoglossus kowalevskii (a hemichordate). These results, in concert with existing expression data from a variety of other animals suggest that a pre-existing genetic system for anteroposterior head patterning was co-opted for patterning of the proximodistal axis of appendages of bilaterian animals.     

Functional analysis of the circadian clock gene period by RNA interference in nymphal crickets Gryllus bimaculatus

The circadian clock gene period (Gryllus bimaculatus period, Gb’per) plays a core role in circadian rhythm generation in adults of the cricket Gryllus bimaculatus. We examined the role of Gb’per in nymphal crickets that show a diurnal rhythm rather than the nocturnal rhythm of the adults. As in the adult optic lobes, Gb’per mRNA levels in the head of the third instar nymphs showed daily cycling in light-dark cycles with a peak at mid night, and the rhythm persisted in constant darkness. Injection of Gb’per double-stranded RNA (dsRNA) into the abdomen of third instar nymphs knocked-down the mRNA levels to 25% of that in control animals. Most Gb’per dsRNA injected nymphs lost their circadian locomotor activity rhythm, while those injected with DsRed2 dsRNA as a negative control clearly maintained the rhythm. These results suggest that nymphs and adults share a common endogenous clock mechanism involving the clock gene Gb’per.     

The <Emphasis Type="Italic">Tribolium spineless</Emphasis> ortholog specifies both larval and adult antennal identity

The morphology of insect antennae varies widely among species, but our understanding of antennal development comes almost solely from studies of one species—the fruit fly, Drosophila melanogaster. Moreover, this knowledge applies mostly to adult structures, since Drosophila lacks external larval appendages. In contrast to Drosophila, the red flour beetle, Tribolium castaneum, has both larval and adult antennae, which are very different from one another in morphology. Thus, Tribolium provides an ideal system to compare modes of antennal development both within and between species. Here, we report that the Tribolium ortholog of spineless (Tc-ss) is required in both the larval and adult antennae. Knockdown of Tc-ss by RNAi during either larval or imaginal development causes transformation of the distal portion of the antennae to legs. Thus, the function of ss is conserved between Drosophila and Tribolium with respect to adult antennal specification and also between Tribolium larval and adult antennal development. The similarity of the Tc-ss RNAi phenotype to that of a classically described Tribolium mutation, antennapedia (ap) (of no relationship to the Drosophila Hox gene of the same name), led us to characterize the original ap mutation and two newly identified ap alleles. Our mapping and phenotypic data suggest that Tc-ss is the best candidate for the ap locus. These results represent a first step in characterizing larval and adult antennal patterning in Tribolium, which should provide important insights into the evolution of insect antennal development.     

Larval leg integrity is maintained by Distal-less and is required for proper timing of metamorphosis in the flour beetle, Tribolium castaneum

The dramatic transformation from a larva to an adult must be accompanied by a coordinated activity of genes and hormones that enable an orchestrated transformation from larval to pupal/adult tissues. The maintenance of larval appendages and their subsequent transformation to appendages in holometabolous insects remains elusive at the developmental genetic level. Here the role of a key appendage patterning gene Distal-less (Dll) was examined in mid- to late-larval stages of the flour beetle, Tribolium castaneum. During late larval development, Dll was expressed in appendages in a similar manner as previously reported for the tobacco hornworm, Manduca sexta. Removal of this late Dll expression resulted in disruption of adult appendage patterning. Intriguingly, earlier removal resulted in dramatic loss of structural integrity and identity of larval appendages. A large amount of variability in appendage morphology was observed following Dll dsRNA injection, unlike larvae injected with dachshund dsRNA. These Dll dsRNA-injected larvae underwent numerous supernumerary molts, which could be terminated with injection of either JH methyltransferase or Methoprene-tolerant dsRNA. Apparently, the partial dedifferentiation of the appendages in these larvae acts to maintain high JH and, hence, prevents metamorphosis.     

Formation of the insect head involves lateral contribution of the intercalary segment, which depends on Tc-labial function

The insect head is composed of several segments. During embryonic development, the segments fuse to form a rigid head capsule where obvious segmental boundaries are lacking. Hence, the assignment of regions of the insect head to specific segments is hampered, especially with respect to dorsal (vertex) and lateral (gena) parts. We show that upon Tribolium labial (Tc-lab) knock down, the intercalary segment is deleted but not transformed. Furthermore, we find that the intercalary segment contributes to lateral parts of the head cuticle in Tribolium. Based on several additional mutant and RNAi phenotypes that interfere with gnathal segment development, we show that these segments do not contribute to the dorsal head capsule apart from the dorsal ridge. Opposing the classical view but in line with findings in the vinegar fly Drosophila melanogaster and the milkweed bug Oncopeltus fasciatus, we propose a “bend and zipper” model for insect head capsule formation.     

Functional analysis of Scr during embryonic and post-embryonic development in the cockroach, Periplaneta americana

The cockroach, Periplaneta americana represents a basal insect lineage that undergoes the ancestral hemimetabolous mode of development. Here, we examine the embryonic and post-embryonic functions of the hox gene Scr in Periplaneta as a way of better understanding the roles of this gene in the evolution of insect body plans. During embryogenesis, Scr function is strictly limited to the head with no role in the prothorax. This indicates that the ancestral embryonic function of Scr was likely restricted to the head, and that the posterior expansion of expression in the T1 legs may have preceded any apparent gain of function during evolution. In addition, Scr plays a pivotal role in the formation of the dorsal ridge, a structure that separates the head and thorax in all insects. This is evidenced by the presence of a supernumerary segment that occurs between the labial and T1 segments of RNAiScr first nymphs and is attributed to an alteration in engrailed (en) expression. The fact that similar Scr phenotypes are observed in Tribolium but not in Drosophila or Oncopeltus reveals the presence of lineage-specific variation in the genetic architecture that controls the formation of the dorsal ridge. In direct contrast to the embryonic roles, Scr has no function in the head region during post-embryogenesis in Periplaneta, and instead, strictly acts to provide identity to the T1 segment. Furthermore, the strongest Periplaneta RNAiScr phenotypes develop ectopic wing-like tissue that originates from the posterior region of the prothoracic segment. This finding provides a novel insight into the current debate on the morphological origin of insect wings.     

Over-expression of Ultrabithorax alters embryonic body plan and wing patterns in the butterfly Bicyclus anynana

In insects, forewings and hindwings usually have different shapes, sizes, and color patterns. A variety of RNAi experiments across insect species have shown that the hox gene Ultrabithorax (Ubx) is necessary to promote hindwing identity. However, it remains unclear whether Ubx is sufficient to confer hindwing fate to forewings across insects. Here, we address this question by over-expressing Ubx in the butterfly Bicyclus anynana using a heat-shock promoter. Ubx whole-body over-expression during embryonic and larvae development led to body plan changes in larvae but to mere quantitative changes to adult morphology, respectively. Embryonic heat-shocks led to fused segments, loss of thoracic and abdominal limbs, and transformation of head limbs to larger appendages. Larval heat-shocks led to reduced eyespot size in the expected homeotic direction, but neither additional eyespots nor wing shape changes were observed in forewings as expected of a homeotic transformation. Interestingly, Ubx was found to be expressed in a novel, non-characteristic domain – in the hindwing eyespot centers. Furthermore, ectopic expression of Ubx on the pupal wing activated the eyespot-associated genes spalt and Distal-less, known to be directly repressed by Ubx in the fly?s haltere and leg primordia, respectively, and led to the differentiation of black wing scales. These results suggest that Ubx has been co-opted into a novel eyespot gene regulatory network, and that it is capable of activating black pigmentation in butterflies.     

The specification of a highly derived arthropod appendage, the Drosophila labial palps, requires the joint action of selectors and signaling pathways

The remarkable diversity of form in arthropods reflects flexible genetic programs deploying many conserved genes. In the insect model Drosophila melanogaster, diversity of form can be observed between serially homologous appendages or when a single appendage is transformed by homeotic mutations, such as the adult labial mouthparts that can present alternative antennal, prothoracic, or maxillary identities. We have examined the roles of the Hox selector genes proboscipedia (pb) and Sex combs reduced (Scr), and the antennal selectors homothorax (hth) and spineless (ss) in labial specification, by tissue-directed mitotic recombination. Whereas loss of pb function transforms labium to prothoracic leg, loss of Scr, hth, or ss functions results in little or no change in labial specification. Results of analysis of single and multiple mutant combinations support a genetic hierarchy in which the homeotic pb gene possesses a primary role. It is surprising to note that while loss of ss activity alone had no detected effect, all mutant combinations lacking both pb and ss yielded the most severe phenotype observed: stunted, apparently tripartite legs that may correspond to a default state. The roles of the four selector genes are functionally linked to a cell nonautonomous mechanism involving the coupled activities of the decapentaplegic (dpp)/TGF-β and wingless (wg)/Wnt signaling pathways. Accordingly, several mutant combinations impaired in dpp signaling were seen to reorient labial-to-leg transformations toward antennal aristae. A crucial aspect of selector function in development and evolution may be in regulating diffusible signals, including those emitted by dpp and wg.     

Functional analyses in the milkweed bug Oncopeltus fasciatus (Hemiptera) support a role for Wnt signaling in body segmentation but not appendage development

Specification of the proximal-distal (PD) axis of insect appendages is best understood in Drosophila melanogaster, where conserved signaling molecules encoded by the genes decapentaplegic (dpp) and wingless (wg) play key roles. However, the development of appendages from imaginal discs as in Drosophila is a derived state, while more basal insects produce appendages from embryonic limb buds. Therefore, the universality of the Drosophila limb PD axis specification mechanism has been debated since dpp expression in more basal insect species differs dramatically from Drosophila. Here, we test the function of Wnt signaling in the development of the milkweed bug Oncopeltus fasciatus, a species with the basal state of appendage development from limb buds. RNA interference of wg and pangolin (pan) produce defects in the germband and eyes, but not in the appendages. Distal-less and dachshund, two genes regulated by Wg signaling in Drosophila and expressed in specific PD domains along the limbs of both species, are expressed normally in the limbs of pan-depleted Oncopeltus embryos. Despite these apparently paradoxical results, Armadillo protein, the transducer of Wnt signaling, does not accumulate properly in the nuclei of cells in the legs of pan-depleted embryos. In contrast, engrailed RNAi in Oncopeltus produces cuticular and appendage defects similar to Drosophila. Therefore, our data suggest that Wg signaling is functionally conserved in the development of the germband, while it is not essential in the specification of the limb PD axis in Oncopeltus and perhaps basal insects.