The pleiohomeotic functions as a negative regulator of <Emphasis Type="Italic">Drosophila even-skipped</Emphasis> gene during embryogenesis

Polycomb group (PcG) proteins maintain the spatial expression patterns of genes that are involved in cell-fate specification along the anterior-posterior (A/P) axis. This repression requires cis-acting silencers, which are called PcG response elements (PREs). One of the PcG proteins, Pleiohomeotic (Pho), which has a zinc finger DNA binding protein, plays a critical role in recruiting other PcG proteins to bind to PREs. In this study, we characterized the effects of a pho mutation on embryonic segmentation. pho maternal mutant embryos showed various segmental defects including pair-rule gene mutant patterns. Our results indicated that engrailed and even-skipped genes were misexpressed in pho mutant embryos, which caused embryonic segment defects.     

The zinc finger homeodomain-2 gene of Drosophila controls Notch targets and regulates apoptosis in the tarsal segments

The development of the Drosophila leg is a good model to study processes of pattern formation, cell death and segmentation. Such processes require the coordinate activity of different genes and signaling pathways that progressively subdivide the leg territory into smaller domains. One of the main pathways needed for leg development is the Notch pathway, required for determining the proximo-distal axis of the leg and for the formation of the joints that separate different leg segments. The mechanisms required to coordinate such events are largely unknown. We describe here that the zinc finger homeodomain-2 (zfh-2) gene is highly expressed in cells that will form the leg joints and needed to establish a correct size and pattern in the distal leg. There is an early requirement of zfh-2 to establish the correct proximo-distal axis, but zfh-2 is also needed at late third instar to form the joint between the fourth and fifth tarsal segments. The expression of zfh-2 requires Notch activity but zfh-2 is necessary, in turn, to activate Notch targets such as Enhancer of split and big brain. zfh-2 is controlled by the Drosophila activator protein 2 gene and regulates the late expression of tarsal-less. In the absence of zfh-2 many cells ectopically express the pro-apoptotic gene head involution defective, activate caspase-3 and are positive for acridine orange, indicating they undergo apoptosis. Our results demonstrate the key role of zfh-2 in the control of cell death and Notch signaling during leg development.     

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.     

Fate map of the distal portion of Drosophila proboscis as inferred from the expression and mutations of basic patterning genes

The late-third-instar labial disc is comprised of two disc-proper cell layers, one representing mainly the ventral half of the anterior compartment (L-layer) and the other, the dorsal half of the anterior compartment and most, if not all, of the posterior compartment (M-layer). In the L-layer, Distal-less represses homothorax whereas no Distal-less-dependent homothorax repression occurs in the M-layer where Distal-less is coexpressed with homothorax. In wild-type labial discs, clawless, one of the two homeobox genes expressed in distal cells receiving maximum (Decapentaplegic+Wingless) signaling activity in leg and antennal discs, is specifically repressed by proboscipedia. A fate map, inferred from data on basic patterning gene expression in larval and pupal stages and mutant phenotypes, indicates the inner surface of the labial palpus, which includes the pseudotracheal region, to be a derivative of the distal portion of the M-layer expressing wingless, patched, Distal-less and homothorax. The outer surface of the labial palpus with more than 30 taste bristles derives from an L-layer area consisting of dorsal portions of the anterior and posterior compartments, each expressing Distal-less. Our analysis also indicates that, in adults and pupae, the anterior-posterior boundary, dividing roughly equally the outer surface of the distiproboscis, runs along the outer circumference of the inner surface of distiproboscis.     

An antennal-specific role for bowl in repressing supernumerary appendage development in Drosophila

In Drosophila, antennae and legs are serially homologous appendages, and yet they develop into organs of very different structure and function. This implies that different genetic mechanisms operate onto a common developmental ground state to produce antennae and legs. Still few such mechanisms have been uncovered. During leg development, bowl, a member of the odd-skipped gene family, has been shown to participate in the formation of the leg segmental joints. Here we report that, in the antennal disc, bowl has a dramatically different role: bowl is expressed in the ventral antennal disc to prevent inappropriate expression of wg early during development. The removal of bowl function leads to the activation of wg in the dpp-expressing domain. This ectopic expression of wg, together with dpp, results in a new proximo-distal axis that promotes non-autonomous antennal duplications. The role of bowl in suppressing a supernumerary PD axis is maintained even when the antennal disc is homeotically transformed into a leg-like appendage. Therefore, bowl is part of a genetic program that suppresses the formation of supernumerary appendages specifically in the fly's head.     

Notch signaling relieves the joint-suppressive activity of Defective proventriculus in the Drosophila leg

Segmentation plays crucial roles during morphogenesis. Drosophila legs are divided into segments along the proximal-distal axis by flexible structures called joints. Notch signaling is necessary and sufficient to promote leg growth and joint formation, and is activated in distal cells of each segment in everting prepupal leg discs. The homeobox gene defective proventriculus (dve) is expressed in regions both proximal and distal to the intersegmental folds at 4 h after puparium formation (APF). Dve-expressing region partly overlaps with the Notch-activated region, and they become a complementary pattern at 6 h APF. Interestingly, dve mutant legs resulted in extra joint formation at the center of each tarsal segment, and the forced expression of dve caused a jointless phenotype. We present evidence that Dve suppresses the potential joint-forming activity, and that Notch signaling represses Dve expression to form joints.     

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     

Evolutionary plasticity of collier function in head development of diverse arthropods

The insect intercalary segment represents a small and appendage-less head segment that is homologous to the second antennal segment of Crustacea and the pedipalpal segment in Chelicerata, which are generally referred to as “tritocerebral segment.” In Drosophila, the gene collier (col) has an important role for the formation of the intercalary segment. Here we show that in the beetle Tribolium castaneum col is required for the activation of the segment polarity genes hedgehog (hh), engrailed (en) and wingless (wg) in the intercalary segment, and is a regulatory target of the intercalary segment specific Hox gene labial (lab). Loss of Tc col function leads to increased cell death in the intercalary segment. In the milkweed bug Oncopeltus fasciatus, the loss of col function has a more severe effect in lacking the intercalary segment and also affecting the adjacent mandibular and antennal segments. By contrast, col is not expressed early in the second antennal segment in the crustacean Parhyale hawaiensis or in the pedipalpal segment of the spider Achaearanea tepidariorum. This suggests that the early expression of col in a stripe and its role in tritocerebral segment development is insect-specific and might correlate with the appendage-less morphology of the intercalary segment.     

The Drosophila Polycomb group gene Sex combs extra encodes the ortholog of mammalian Ring1 proteins

In Drosophila, the Polycomb group (PcG) of genes is required for the maintenance of homeotic gene repression during development. Here, we have characterized the Drosophila ortholog of the products of the mammalian Ring1/Ring1A and Rnf2/Ring1B genes. We show that Drosophila Ring corresponds to the Sex combs extra (Sce), a previously described PcG gene. We find that Ring/Sce is expressed and required throughout development and that the extreme Pc embryonic phenotype due to the lack of maternal and zygotic Sce can be rescued by ectopic expression of Ring/Sce. This phenotypic rescue is also obtained by ectopic expression of the murine Ring1/Ring1A, suggesting a functional conservation of the proteins during evolution. In addition, we find that Ring/Sce binds to about 100 sites on polytene chromosomes, 70% of which overlap those of other PcG products such as Polycomb, Posterior sex combs and Polyhomeotic, and 30% of which are unique. We also show that Ring/Sce interacts directly with PcG proteins, as it occurs in mammals.     

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.     

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.     

Ectodermal Wnt signaling regulates abdominal myogenesis during ventral body wall development

Defects of the ventral body wall are prevalent birth anomalies marked by deficiencies in body wall closure, hypoplasia of the abdominal musculature and multiple malformations across a gamut of organs. However, the mechanisms underlying ventral body wall defects remain elusive. Here, we investigated the role of Wnt signaling in ventral body wall development by inactivating Wls or β-catenin in murine abdominal ectoderm. The loss of Wls in the ventral epithelium, which blocks the secretion of Wnt proteins, resulted in dysgenesis of ventral musculature and genito-urinary tract during embryonic development. Molecular analyses revealed that the dermis and myogenic differentiation in the underlying mesenchymal progenitor cells was perturbed by the loss of ectodermal Wls. The activity of the Wnt-Pitx2 axis was impaired in the ventral mesenchyme of the mutant body wall, which partially accounted for the defects in ventral musculature formation. In contrast, epithelial depletion of β-catenin or Wnt5a did not resemble the body wall defects in the ectodermal Wls mutant. These findings indicate that ectodermal Wnt signaling instructs the underlying mesodermal specification and abdominal musculature formation during ventral body wall development, adding evidence to the theory that ectoderm-mesenchyme signaling is a potential unifying mechanism for the origin of ventral body wall defects.     

Effects of Polycomb Group Mutations on the Expression of Ultrabithorax in the Drosophila Visceral Mesoderm

The Polycomb group (PcG) genes encode repressors of many developmental regulatory genes including homeotic genes and are known to act by modifying chromatin structure through complex formation. We describe how Ultrabithorax (Ubx) expression is affected by the PcG mutants in the visceral mesoderm. Mutant embryos of the genes extra sex combs (esc), Polycomb (Pc), additional sex combs (Asx) and pleiohomeotic (pho) were examined. In each mutation, Ubx was ectopically expressed outside of their normal domains along the anterior-posterior axis in the visceral mesoderm, which is consistent with the effect of PcG proteins repressing the homeotic genes in other tissues. All of these four PcG mutations exhibit complete or partial lack of midgut constriction. However, two thirds of esc mutant embryos did not show Ubx expression in parasegment 7 (PS7). Even in the embryos showing ectopic Ubx expression, the level of Ubx expression in the PcG mutations was weaker than that in normal embryos. We suggest that in PcG mutations the ectopic Ubx expression is caused by lack of PcG repressor proteins, while the weaker or lack of Ubx expression is due to the repression of Ubx by Abd-B protein which is ectopically expressed in PcG mutations as well.     

Antenna and all gnathal appendages are similarly transformed by homothorax knock-down in the cricket Gryllus bimaculatus

Our understanding of the developmental mechanisms underlying the vast diversity of arthropod appendages largely rests on the peculiar case of the dipteran Drosophila melanogaster. In this insect, homothorax (hth) and extradenticle (exd) together play a pivotal role in appendage patterning and identity. We investigated the role of the hth homologue in the cricket Gryllus bimaculatus by parental RNA interference. This species has a more generalized morphology than Oncopeltus fasciatus, the one other insect besides Drosophila where homothorax function has been investigated. The Gryllus head appendages represent the morphologically primitive state including insect-typical mandibles, maxillae and labium, structures highly modified or missing in Oncopeltus and Drosophila. We depleted Gb’hth function through parental RNAi to investigate its requirement for proper regulation of other appendage genes (Gb’wingless, Gb’dachshund, Gb’aristaless and Gb’Distalless) and analyzed the terminal phenotype of Gryllus nymphs. Gb’hth RNAi nymphs display homeotic and segmentation defects similar to hth mutants or loss-of-function clones in Drosophila. Intriguingly, however, we find that in Gb’hth RNAi nymphs not only the antennae but also all gnathal appendages are homeotically transformed, such that all head appendages differentiate distally as legs and proximally as antennae. Hence, Gb’hth is not specifically required for antennal fate, but fulfills a similar role in the specification of all head appendages. This suggests that the role of hth in the insect antenna is not fundamentally different from its function as cofactor of segment-specific homeotic genes in more posterior segments.     

Lipodystrophies: Disorders of adipose tissue biology

The adipocytes synthesize and store triglycerides as lipid droplets surrounded by various proteins and phospholipids at its surface. Recently, the molecular basis of some of the genetic syndromes of lipodystrophies has been elucidated and some of these genetic loci have been found to contribute to lipid droplet formation in adipocytes. The two main types of genetic lipodystrophies are congenital generalized lipodystrophy (CGL) and familial partial lipodystrophy (FPL). So far, three CGL loci: 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2), Berardinelli–Seip Congenital Lipodystrophy 2 (BSCL2) and caveolin 1 (CAV1) and four FPL loci: lamin A/C (LMNA), peroxisome proliferator-activated receptor γ (PPARG), v-AKT murine thymoma oncogene homolog 2 (AKT2) and zinc metalloprotease (ZMPSTE24), have been identified. AGPAT2 plays a critical role in the synthesis of glycerophospholipids and triglycerides required for lipid droplet formation. Another protein, seipin (encoded by BSCL2 gene), has been found to induce lipid droplet fusion. CAV1 is an integral component of caveolae and might contribute towards lipid droplet formation. PPARγ and AKT2 play important role in adipogenesis and lipid synthesis. In this review, we discuss and speculate about the contribution of various lipodystrophy genes and their products in the lipid droplet formation.     

Larval legs of mulberry silkworm Bombyx mori are prototypes for the adult legs

Morphological diversity of leg appendages is one of the hallmarks of developmental evolution. Limbs in insects may develop either from their embryonic prototypes or from imaginal discs harbored inside the larva. Bombyx mori (B. mori), a Lepidopteran insect, develops adult wings from larval wing imaginal discs. However, it has been debated whether the adult legs of B. mori arise from imaginal discs or from the larval legs. Here we addressed how the larval legs relate to their adult counterparts. We present the morphological landmarks during early leg development. We used expression of developmental genes like Distalless and extradenticle to mark leg primordia. Finally, we employed classical excision approach to develop a fate map of the adult leg. Excision and ablation of thoracic legs along proximo-distal axis at various times during larval development resulted in the loss of corresponding adult leg segments. Our data suggest that B. mori legs develop from larval appendages rather than leg imaginal discs.     

Brista: a gene involved in the specification and differentiation of distal cephalic and thoracic structures in Drosophila melanogaster

Summary A gene Brista has been identified in chromosome 2R, in the region 60D11-E4, in which mutations cause homoeotic transformation of distal antennal structures to distal leg derivatives, and in which certain alleles also lead to upsets in the formation of distal elements of the legs. This gene is haploinsufficient for the homoeotic phenotype. Several putative null and two hypomorphic alleles have been recovered. The effects of exposure to the non-permissive temperature of a temperature-sensitive allele are cummulative and depend upon the length of the exposure during the period of antennal cell proliferation. It is suggested that this gene contributes to the stability of the state of determination in distal domain of the antennal and leg discs, and its relationship to other genes with similar mutant phenotype is discussed.