Insertional mutagenesis screening identifies the <Emphasis Type="Italic">zinc finger homeodomain 2</Emphasis> (<Emphasis Type="Italic">zfh2</Emphasis>) gene as a novel factor required for embryonic leg development in <Emphasis Type="Italic">Tribolium castaneum</Emphasis>

The genetic control of leg development is well characterized in the fly Drosophila melanogaster. These control mechanisms, however, must differ to some degree between different insect species to account for the morphological diversity of thoracic legs in the insects. The legs of the flour beetle Tribolium castaneum differ from the Drosophila legs in their developmental mode as well as in their specific morphology especially at the larval stage. In order to identify genes involved in the morphogenesis of the Tribolium larval legs, we have analyzed EGFP enhancer trap lines of Tribolium. We have identified the zfh2 gene as a novel factor required for normal leg development in Tribolium. RNA interference with zfh2 function leads to two alternative classes of leg phenotype. The loss of a leg segment boundary and the generation of ectopic outgrowths in one class of phenotype suggest a role in leg segmentation and segment growth. The malformation of the pretarsal claw in the second class of phenotype suggests a role in distal development and the morphogenesis of the claw-shaped morphology of the pretarsus. This suggests that zfh2 is involved in the regulation of an unidentified target gene in a concentration-dependent manner. Our results demonstrate that enhancer trap screens in T. castaneum have the potential to identify novel gene functions regulating specific developmental processes.     

Notch-Mediated Segmentation and Growth Control of the Drosophila Leg

The possession of segmented appendages is a defining characteristic of the arthropods. By analyzing both loss-of-function and ectopic expression experiments, we show that the Notch signaling pathway plays a fundamental role in the segmentation and growth of the Drosophila leg. Local activation of Notch is necessary and sufficient to promote the formation of joints between segments. This segmentation process requires the participation of the Notch ligands, Serrate and Delta, as well as Fringe. These three proteins are each expressed in the developing leg and antennal imaginal discs in a segmentally repeated pattern that is regulated downstream of the action of Wingless and Decapentaplegic. Our studies further show that Notch activation is both necessary and sufficient to promote leg growth. We also identify target genes regulated both positively and negatively downstream of Notch signaling that are required for normal leg development. Together, these observations outline a regulatory hierarchy for the segmentation and growth of the leg. The Notch pathway is also deployed for segmentation during vertebrate somitogenesis, which raises the possibility of a common origin for the segmentation of these distinct tissues.     

Ancestral functions of Delta/Notch signaling in the formation of body and leg segments in the cricket Gryllus bimaculatus

Delta/Notch signaling controls a wide spectrum of developmental processes, including body and leg segmentation in arthropods. The various functions of Delta/Notch signaling vary among species. For instance, in Cupiennius spiders, Delta/Notch signaling is essential for body and leg segmentation, whereas in Drosophila fruit flies it is involved in leg segmentation but not body segmentation. Therefore, to gain further insight into the functional evolution of Delta/Notch signaling in arthropod body and leg segmentation, we analyzed the function of the Delta (Gb'Delta) and Notch (Gb'Notch) genes in the hemimetabolous, intermediate-germ cricket Gryllus bimaculatus. We found that Gb'Delta and Gb'Notch were expressed in developing legs, and that RNAi silencing of Gb'Notch resulted in a marked reduction in leg length with a loss of joints. Our results suggest that the role of Notch signaling in leg segmentation is conserved in hemimetabolous insects. Furthermore, we found that Gb'Delta was expressed transiently in the posterior growth zone of the germband and in segmental stripes earlier than the appearance of wingless segmental stripes, whereas Gb'Notch was uniformly expressed in early germbands. RNAi knockdown of Gb'Delta or Gb'Notch expression resulted in malformation in body segments and a loss of posterior segments, the latter probably due to a defect in posterior growth. Therefore, in the cricket, Delta/Notch signaling might be required for proper morphogenesis of body segments and posterior elongation, but not for specification of segment boundaries.     

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     

Gene expression patterns in an onychophoran reveal that regionalization predates limb segmentation in pan‐arthropods

SUMMARY In arthropods, such as Drosophila melanogaster, the leg gap genes homothorax (hth), extradenticle (exd), dachshund (dac), and Distal‐less (Dll) regionalize the legs in order to facilitate the subsequent segmentation of the legs. We have isolated homologs of all four leg gap genes from the onychophoran Euperipatoides kanangrensis and have studied their expression. We show that leg regionalization takes place in the legs of onychophorans even though they represent simple and nonsegmented appendages. This implies that leg regionalization evolved for a different function and was only later co‐opted for a role in leg segmentation. We also show that the leg gap gene patterns in onychophorans (especially of hth and exd) are similar to the patterns in crustaceans and insects, suggesting that this is the plesiomorphic state in arthropods. The reversed hth and exd patterns in chelicerates and myriapods are therefore an apomorphy for this group, the Myriochelata, lending support to the Myriochelata and Tetraconata clades in arthropod phylogeny.     

RNAi analysis of nubbin embryonic functions in a hemimetabolous insect,Oncopeltus fasciatus

SUMMARY Although the expression of the POU homeodomain gene nubbin (nub) has been examined in several arthropod species, its function has been studied only in Drosophila. Here, we provide the first insight into functional roles of this gene in a hemimetabolous insect species, Oncopeltus fasciatus. The analysis of its function using RNAi resulted in the altered morphology of antennae and labial tubes in the head, legs in the thorax, and, most notably, the growth of ectopic appendages originating from abdominal segments A2–A6. This change in the morphology of the abdomen can largely be attributed to the altered expression patterns of two hox genes, Ubx and abd‐A, in RNAinub embryos. First, abd‐A expression is completely abolished in A3–A6. Second, weak Ubx expression expands posteriorly to encompass novel domains in A2 and A3. Concomitant with these changes, limbs on A2 and A3 are small and less developed, whereas limbs on A4–A6 are large thoracic‐like legs. These results show that nub function is necessary for normal abd‐A expression and thus plays a critical role in suppressing leg formation on the abdomen. The loss of this regulation leads to upregulation of Distal‐less, and subsequent development of appendages. In Drosophila, however, abd‐A expression is unaffected in a nub‐depleted background, indicating that no such regulatory relationship exists between these two genes in the fruit fly. These differences reveal that variation exists in the genetic mechanisms that maintain an ancient insect feature, the limbless abdomen.     

The Sp8 zinc-finger transcription factor is involved in allometric growth of the limbs in the beetle Tribolium castaneum

Members of the Sp gene family are involved in a variety of developmental processes in both vertebrates and invertebrates. We identified the ortholog of the Drosophila Sp-1 gene in the red flour beetle Tribolium castaneum, termed T-Sp8 because of its close phylogenetic relationship to the vertebrate Sp8 genes. During early embryogenesis, T-Sp8 is seen in segmental stripes. During later stages, TSp8 is dynamically expressed in the limb buds of the Tribolium embryo. At the beginning of bud formation, TSp8 is uniformly expressed in all body appendages. As the limbs elongate, a ring pattern develops sequentially and the expression profile at the end of embryogenesis correlates with the final length of the appendage. In limbs that do not grow out like the labrum and the labium, T-Sp8 expression remains uniform, whereas a two-ring pattern develops in the longer antennae and the maxillae. In the legs that elongate even further, four rings of T-Sp8 expression can be seen at the end of leg development. The role of T-Sp8 for appendage development was tested using RNAi. Upon injection of double stranded T-Sp8 RNA, larvae develop with dwarfed appendages. Affected T-Sp8(RNAi) legs were tested for the presence of medial and distal positional values using the expression marker genes dachshund and Distal-less, respectively. The results show that a dwarfed TSp8(RNAi) leg consists of proximal, medial and distal parts and argues against T-Sp8 being a leg gap gene. Based on the differential expression pattern of T-Sp8 in the appendages of the head and the thorax and the RNAi phenotype, we hypothesise that T-Sp8 is involved in the regulation of limb-length in relation to body size - a process called allometric growth.     

Expression patterns of leg genes in the mouthparts of the spider<Emphasis Type="Italic"> Cupiennius salei</Emphasis> (Chelicerata: Arachnida)

The leg genes extradenticle, homothorax, dachshund, and Distal-less define three antagonistic developmental domains in the legs, but not in the antenna, of Drosophila. Here we report the expression patterns of these leg genes in the prosomal appendages of the spider Cupiennius salei. The prosoma of the spider bears six pairs of appendages: a pair of cheliceres, a pair of pedipalps, and four pairs of walking legs. Three types of appendages thus can be distinguished in the spider. We show here that in the pedipalp, the leg-like second prosomal appendage, the patterns are very similar to those in the legs themselves, indicating the presence of three antagonistic developmental domains in both appendage types. In contrast, in the chelicera, the fang-like first prosomal appendage, the patterns are different and there is no evidence for antagonistic domains. Together with data from Drosophila this suggests that leg-shaped morphology of arthropod appendages requires an underlying set of antagonistic developmental domains, whereas other morphologies (e.g. antenna, chelicera) may result from the loss of such antagonistic domains.Edited by M. Akam     

Loss of <Emphasis Type="Italic">Tc-arrow</Emphasis> and canonical Wnt signaling alters posterior morphology and pair-rule gene expression in the short-germ insect, <Emphasis Type="Italic">Tribolium castaneum</Emphasis>

Wnt signaling has been implicated in posterior patterning in short-germ insects, including the red flour beetle Tribolium castaneum (Bolognesi et al. Curr Biol 18:1624–1629, 2008b; Angelini and Kaufman Dev Biol 283:409–423, 2005; Miyawaki et al. Mech Dev 121:119–130, 2004). Specifically, depletion of Wnt ligands Tc-Wnt1 and Tc-WntD/8 produces Tribolium embryos lacking abdominal segments. Similar phenotypes are produced by depletion of Tc-porcupine (Tc-porc) or Tc-pangolin (Tc-pan), indicating that the signal is transmitted through the canonical Wnt pathway (Bolognesi et al. Curr Biol 18:1624–1629, 2008b). Here we show that RNAi for the receptor Tc-arrow produced similar truncated phenotypes, providing additional evidence supporting canonical signal transduction. Furthermore, since in Tribolium segments are defined sequentially by a pair-rule gene circuit that, when interrupted, produces truncated phenotypes (Choe et al. Proc Natl Acad Sci U S A 103:6560–6564, 2006), we investigated the relationship between loss of Wnt signaling and this pair-rule gene circuit. After depletion of the receptor Tc-arrow, expression of Tc-Wnt1 was noticeably absent from the growth zone, while Tc-WntD/8 was restricted to a single spot of expression in what remained of the posterior growth zone. The primary pair-rule genes Tc-runt (Tc-run) and Tc-even-skipped (Tc-eve) were expressed normally in the anterior segments, but were reduced to a single spot in the remnants of the posterior growth zone. Thus, expression of pair-rule genes and Tc-WntD/8 are similarly affected by depletion of Wnt signal and disruption of the posterior growth zone.     

Evolution of nubbin function in hemimetabolous and holometabolous insect appendages

Insects display a whole spectrum of morphological diversity, which is especially noticeable in the organization of their appendages. A recent study in a hemipteran, Oncopeltus fasciatus (milkweed bug), showed that nubbin (nub) affects antenna morphogenesis, labial patterning, the length of the femoral segment in legs, and the formation of a limbless abdomen. To further determine the role of this gene in the evolution of insect morphology, we analyzed its functions in two additional hemimetabolous species, Acheta domesticus (house cricket) and Periplaneta americana (cockroach), and re-examined its role in Drosophila melanogaster (fruit fly). While both Acheta and Periplaneta nub-RNAi first nymphs develop crooked antennae, no visible changes are observed in the morphologies of their mouthparts and abdomen. Instead, the main effect is seen in legs. The joint between the tibia and first tarsomere (Ta-1) is lost in Acheta, which in turn, causes a fusion of these two segments and creates a chimeric nub-RNAi tibia–tarsus that retains a tibial identity in its proximal half and acquires a Ta-1 identity in its distal half. Similarly, our re-analysis of nub function in Drosophila reveals that legs lack all true joints and the fly tibia also exhibits a fused tibia and tarsus. Finally, we observe a similar phenotype in Periplaneta except that it encompasses different joints (coxa–trochanter and femur–tibia), and in this species we also show that nub expression in the legs is regulated by Notch signaling, as had previously been reported in flies and spiders. Overall, we propose that nub acts downstream of Notch on the distal part of insect leg segments to promote their development and growth, which in turn is required for joint formation. Our data represent the first functional evidence defining a role for nub in leg segmentation and highlight the varying degrees of its involvement in this process across insects.     

Two cDNAs from the purple sea urchin,<Emphasis Type="Italic"> Strongylocentrotus purpuratus</Emphasis>, encoding mosaic proteins with domains found in factor H,factor I,and complement components C6 and C7

The vertebrate complement system is composed of about 30 serum and cell surface proteins that make up three activation pathways, a lytic pathway, and a set of proteins that regulate complement. Regulatory proteins are required for host protection against autologous complement attack and to control the amplification feedback loop of the alternative pathway. Purple sea urchin, Strongylocentrotus purpuratus, homologues of complement C3 (SpC3) and factor B (SpBf) have been identified, suggesting the presence of an alternative complement pathway. This implies that echinoderms require a complement regulatory system for the same reasons that it is required in higher vertebrates. Two cDNAs, Sp5 and Sp5013, have been characterized from coelomocytes and the deduced structures of the encoded mosaic proteins, SpCRL (S. purpuratus complement related protein, long form) and SpCRS (short form), have domains that are also found in regulatory proteins such as factor H and factor I and the terminal pathway components C6 and C7. These domains include multiple short consensus repeats, a fucolectin domain, Ser/Thr/Pro-rich regions, a Cys-rich region, and a factor I-membrane attack complex domain. The genes are constitutively expressed in all tissues of the sea urchin and are not induced in response to immune challenge. Multiple bands of varying intensity on both genome blots and RNA blots suggest that Sp5 and Sp5013 are members of a small gene family and that they might undergo alternative splicing. Based on the domains present in SpCRL and SpCRS, they might be either examples of complement regulatory proteins or members of the terminal pathway of complement.     

Differential expression of ubiquitin-conjugating enzyme E2r in the developing ovary and testis of penaeid shrimp <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>

In order to identify genes involved in oogenesis and spermatogenesis in penaeid shrimp Marsupenaeus japonicus, a modified annealing control primer (ACP) system was adapted to identify genes differentially expressed in ovary and testis at different developmental stages. By using 20 pairs of ACP primers, 8 differentially expressed genes were obtained. One of these genes is ubiquitin-conjugating enzyme E2r (UBE2r). Bioinformatics analyses show that this gene encodes a protein of 241 amino acids with a predicted molecular mass of 27.4 kDa. Real time PCR analyses demonstrated that the expression level changed significantly in the developing testis and ovary. In the stage 2 of testis, it reached its highest expression level, the lowest expression level present in the stage 1 of ovary. The significantly different expression levels in developing testis and ovary suggest that UBE2r has an important role in oogenesis and spermatogenesis. This article is the first report of UBE2r in crustaceans and also is the first report showing that UBE2r is differentially expressed at different stages of the developing ovary and testis in an animal.     

Kinematics of nonstationary locomotion in the centipede <Emphasis Type="Italic">Scolopendra cingulata</Emphasis>: A single instant change of speed

The spatiotemporal parameters of leg movement in Scolopendra instantly changing the locomotion speed from V ₁ to V ₂ ≠ V ₁ are investigated. It is shown that (i) the principle of “in trail” leg placement is kept upon a change of speed; (ii) the continuity of the metachronal coordination is not disturbed; but (iii) for some time after changing speed the set of ipsilateral legs comprises (a) head-proximal legs creating a new trackway of steps with pace ℓ₂, and (b) distal legs still using the old trackway with pace ℓ₁. The two groups differ in kinematic parameters. Group (a) works in the stationary mode corresponding to speed V ₂, while group (b) works in a transitory mode. Consecutively, with a metachronal wave propagating backwards along the body but immobile relative to the ground, more and more legs from group (b) switch to the new stationary mode.     

Molecular patterning mechanism underlying metamorphosis of the thoracic leg in Manduca sexta

The tobacco hornworm Manduca sexta, like many holometabolous insects, makes two versions of its thoracic legs. The simple legs of the larva are formed during embryogenesis, but then are transformed into the more complex adult legs at metamorphosis. To elucidate the molecular patterning mechanism underlying this biphasic development, we examined the expression patterns of five genes known to be involved in patterning the proximal-distal axis in insect legs. In the developing larval leg of Manduca, the early patterning genes Distal-less and Extradenticle are already expressed in patterns comparable to the adult legs of other insects. In contrast, Bric-a-brac and dachshund are expressed in patterns similar to transient patterns observed during early stages of leg development in Drosophila. During metamorphosis of the leg, the two genes finally develop mature expression patterns. Our results are consistent with the hypothesis that the larval leg morphology is produced by a transient arrest in the conserved adult leg patterning process in insects. In addition, we find that, during the adult leg development, some cells in the leg express the patterning genes de novo suggesting that the remodeling of the leg involves changes in the patterning gene regulation.