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.     

Heterochrony during skeletal development of Pseudis platensis (anura,hylidae) and the early offset of skeleton development and growth

The aquatic frog Pseudis platensis has a giant tadpole, long developmental time, and dissociated metamorphic events that include later offset of larval somatic morphologies. Moreover, when the tadpole metamorphoses, the young frog is nearly the size of an adult, suggesting that this species has low rates of postmetamorphic growth. Herein, we study the development of the skeleton during larval development up to the end of metamorphosis, which is denoted by the complete lost of the tail in P. platensis. Our study revealed heterochronic differences in skeletal development compared with that of most anurans; these involve the complete differentiation of skull bones and the extensive ossification of the postcranial skeleton before completion of metamorphosis. The skull of metamorphosing P. platensis has an ossified sphenethmoid and a fully formed plectral apparatus, thus differing with regard to the pattern observed in most anurans in which both developmental events take place during the postmetamorphic life. Despite the fact that the iliosacral articulation and the urostyle are present at the end of metamorphosis as in most anurans, ossification/calcification of carpus, tarsus, and limb epihyses during metamorphosis of P. platensis suggests that the postcranial skeleton lacks postmetamorphic growth. This study also includes a discussion of the pattern of development of the plectral apparatus, which allows us to propose a new hypothesis regarding pars externa plectri homology. J. Morphol., 2009. © 2008 Wiley‐Liss, Inc.     

The development of the sensory neuron pattern in the antennal disc of wild-type and mutant (lz3, ssa) Drosophila melanogaster

The development of the sensory neuron pattern in the antennal disc of Drosophila melanogaster was studied with a neuron-specific monoclonal antibody (22C10). In the wild type, the earliest neurons become visible 3 h after pupariation, much later than in other imaginal discs. They lie in the center of the disc and correspond to the neurons of the adult aristal sensillum. Their axons join the larval antennal nerve and seem to establish the first connection towards the brain. Later on, three clusters of neurons appear in the periphery of the disc. Two of them most likely give rise to the Johnston's organ in the second antennal segment. Neurons of the olfactory third antennal segment are formed only after eversion of the antennal disc (clusters t1-t3). The adult pattern of antennal neurons is established at about 27% of metamorphosis. In the mutant lozenge3 (lz3), which lacks basiconic antennal sensilla, cluster t3 fails to develop. This indicates that, in the wild type, a homogeneous group of basiconic sensilla is formed by cluster t3. The possible role of the lozenge gene in sensillar determination is discussed. The homeotic mutant spineless-aristapedia (ssa) transforms the arista into a leg-like tarsus. Unlike leg discs, neurons are missing in the larval antennal disc of ssa. However, the first neurons differentiate earlier than in normal antennal discs. Despite these changes, the pattern of afferents in the ectopic tarsus appears leg specific, whereas in the non-transformed antennal segments a normal antennal pattern is formed. This suggests that neither larval leg neurons nor early aristal neurons are essential for the outgrowth of subsequent afferents.     

A bacterial polysaccharide biosynthesis-related gene inversely regulates larval settlement and metamorphosis of Mytilus coruscus

Abstract

Larval settlement and metamorphosis is essential for the development of marine invertebrates. Although polysaccharides are involved in larval settlement and metamorphosis of Mytilus coruscus, the molecular basis of polysaccharides underlying this progression remains largely unknown. Here, the roles of the polysaccharide biosynthesis-related gene 01912 of Pseudoalteromonas marina ECSMB14103 in the regulation of larval settlement and metamorphosis were examined by gene-knockout technique. Compared with biofilms (BFs) of the wild-type P. marina, Δ01912 BFs with a higher colanic acid (CA) content showed a higher inducing activity on larval settlement and metamorphosis. Deletion of the 01912 gene caused an increase in c-di-GMP levels, accompanied by a decrease in the motility, an increase in cell aggregation, and overproduction of CA. Thus, the bacterial polysaccharide biosynthesis-related gene 01912 may regulate mussel settlement by producing CA via the coordination of c-di-GMP. This work provides a deeper insight into the molecular mechanism of polysaccharides in modulating mussel settlement.     

Regulation of gene expression in the distal region of the Drosophila leg by the Hox11 homolog, C15

The distal region of the Drosophila leg, the tarsus, is divided into five segments (ta I-V) and terminates in the pretarsus, which is characterized by a pair of claws. Several homeobox genes are expressed in distinct regions of the tarsus, including aristaless (al) and lim1 in the pretarsus, Bar (B) in ta IV and V, and apterous (ap) in ta IV. This pattern is governed by regulatory interactions between these genes; for example, Al and B are mutually antagonistic resulting in exclusion of B expression from the pretarsus. Although Al is necessary, it is not sufficient to repress B, indicating another factor is required. Here, this factor is identified as the product of the C15 gene, which is another homeodomain protein, a homolog of the human Hox11 oncogene. C15 is expressed in the same cells as al and, together, C15 and Al appear to directly repress B. C15/Al also act indirectly to repress ap in ta V, i.e., in surrounding cells. To do this, C15/Al autonomously repress expression of the gene encoding the Notch ligand Delta (Dl) in the pretarsus, restricting Dl to ta V and creating a Dl+/Dl- border at the interface between ta V and the pretarsus. This results in upregulation of Notch signaling, which induces expression of the bowl gene, the product of which represses ap.     

Global gene expression profiling of Homarus americanus (Crustacea) larval stages during development and metamorphosis

Homarus americanus has a life history that is similar to other arthropods, including a pelagic larval phase and a benthic adult phase. The larval phase is divided into three morphologically distinct stages, followed by metamorphosis to the post-larval phase. H. americanus larval development has been studied previously, although the molecular mechanisms that regulate the consequent changes are not fully elucidated. This study is the first to use an oligonucleotide microarray to investigate global gene expression during H. americanus larval development. Stage-specific gene expression profiles of larvae and postlarvae from two-year classes were assessed. We found the expression levels of 1851 genes to be significantly different among larval stages. Functional annotations indicated that various differentially expressed genes were involved with immune function, energy regulation, and development. Ten target genes of interest were selected for expression verification using RT-qPCR. Two Phosphoenolpyruvatecarboxykinases, Argonaute 2, Ecdysone-inducible protein 75, and Procollagen-lysine 2-oxoglutarate 5-dioxygenase 3, had significantly different expression (p?相似文献   

Expression patterns of aristaless in developing appendages of Gryllus bimaculatus (cricket)

We report the isolation and expression patterns of aristaless (al), a paired-type homeobox gene, of Gryllus bimaculatus (Gb), a hemimetabola model insect. Gryllus al (Gbal) is expressed in the most distal region of developing labrum, antenna, mandible, maxilla, labium, leg, cercus, and hindgut. Gbal is also expressed in the proximal region, corresponding to the presumptive coxopodite, of the developing antenna, mandible, maxilla, labium, and leg, but not in the developing labrum, cercus, and hindgut. During development of the leg, expression of Gbal changes dynamically with the progress in leg segmentation: Gbal is expressed in order in the presumptive pretarsus, coxa, femur, tibia and tarsus before appearance of morphological segmentation.     

Ecdysteroid control of ionic current development in Manduca sexta motoneurons

The steroid hormone 20-hydroxyecdysone (20-HE) regulates several processes during insect metamorphosis. We studied the effects of 20-HE on the development of voltage-sensitive ionic currents of thoracic leg motoneurons of Manduca sexta. The larval leg motoneurons persist throughout metamorphosis but undergo substantial morphological reorganization, which is under the control of 20-HE and accompanied by changes in Ca²⁺ and K⁺ current densities. To determine whether 20-HE controls the changes in Ca²⁺ and K⁺ current levels during postembryonic development, identified thoracic leg motoneurons isolated from late larval and early pupal stages were taken into primary cell culture. Whole-cell Ca²⁺ and K⁺ currents were measured after 1–4 days of steroid hormone incubation. In the presence of 20-HE, peak Ca²⁺ currents of pupal leg motoneurons increased from day 1 to day 4 in vitro. Thus, at culture day 4 the pupal Ca²⁺ current levels were larger in 20-HE–treated than in untreated cells. By contrast, 20-HE did not affect the Ca²⁺ current amplitudes of larval leg motoneurons. Whole-cell K⁺ currents, measured at 4 days in pupal motoneurons, consisted of a fast-activating transient current and a sustained, slowly inactivating current. 20-HE did not affect the amplitude of the transient or sustained currents after 4 days in vitro. Thus, a direct steroid hormone effect may control the proper maturation of voltage-sensitive Ca²⁺ currents in leg motoneurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 211–223, 1998     

Larval development and post-settlement metamorphosis of the barnacle Balanus albicostatus Pilsbry and the serpulid polychaete Pomatoleios kraussii Baird: Impact of a commonly used antifouling biocide,Irgarol 1051

Abstract

The impact of a commonly-used antifouling algicide, Irgarol 1051, on the larval development and post-settlement metamorphosis of the barnacle, Balanus albicostatus Pilsbry (Crustacea: Cirripedia), and the larval metamorphosis of a serpulid polycheate, Pomatoleios kraussii Baird, was evaluated. In the case of B. albicostatus, larval mortality increased with an increase in the concentration of Irgarol 1051, and there was a shift in the larval stage targeted from advanced instars to early instars. Nauplii that survived to the cyprid instar stage when reared in the presence of Irgarol 1051 showed prolonged instar and total naupliar duration when compared to the controls. The post-settlement metamorphosis of cyprids significantly varied with Irgarol concentration and also with biofilm age. One and 2-d-old untreated biofilms showed higher metamorphosis when compared to 5-d-old biofilms. However, when the biofilms that promoted cyprid metamorphosis were treated with Irgarol 1051 at low concentrations, metamorphosis rates decreased. Cyprids were prevented from metamorphosing completely by biofilms treated at the highest concentration of Irgarol 1051. Inhibition of metamorphosis was also observed in the case of competent polychaete larvae when exposed to Irgarol 1051 compared to those exposed to metamorphosis inducers such as 3-iso-butyl-1-methylxanthine (IBMX) and natural biofilms. Identification of the pathway(s) that caused the promotory biofilms to become toxic when exposed to Irgarol 1051 is discussed.     

BmMD-2A responds to 20-hydroxyecdysone and regulates Bombyx mori silkworm innate immunity in larva-to-pupa metamorphosis

20E-hydroxyecdysone (20E) plays important roles in larval molting and metamorphosis in insects and is also involved in the insect innate immune response. Insect metamorphosis is a highly successful strategy for environmental adaptation and is the most vulnerable stage during which the insect is susceptible to various pathogens. 20E regulates a series of antimicrobial peptides (AMPs) through the immunodeficiency (IMD) pathway activation in Drosophila; nevertheless, whether other immune pathways are involved in 20E-regulated insect immunity is unknown. Our previous studies showed that BmMD-2A is a member of the MD-2-related lipid recognition (ML) family of proteins that are involved in the Bombyx mori innate immunity Toll signaling pathway. In this study, we further demonstrate that BmMD-2A is also positively regulated by 20E, and the BmMD-2A neutralization experiment suggested that 20E activates some downstream immune effect factors, the AMP genes against Escherichia coli and Staphylococcus aureus, through the regulation of BmMD-2A in larval metamorphosis, implying that B. mori may use the Toll-ML signaling pathway to maintain innate immune balance in the larval-pupal metamorphosis stage, which is a different innate immunity pathway regulated by 20E compared to the IMD pathway in Drosophila.     

EGFR signaling is required for re-establishing the proximodistal axis during distal leg regeneration in the cricket Gryllus bimaculatus nymph

Nymphs of hemimetabolous insects, such as cockroaches and crickets, possess functional legs with a remarkable capacity for epimorphic regeneration. In this study, we have focused on the role of epidermal growth factor receptor (EGFR) signaling in regeneration of a nymphal leg in the cricket Gryllus bimaculatus. We performed loss-of-function analyses with a Gryllus Egfr homolog (Gb'Egfr) and nymphal RNA interference (RNAi). After injection of double-stranded RNA for Gb'Egfr in the body cavity of the third instar cricket nymph, amputation of the leg at the distal tibia resulted in defects of normal distal regeneration. The regenerated leg lacked the distal tarsus and pretarsus. This result indicates that EGFR signaling is required for distal leg patterning in regeneration during the nymphal stage of the cricket. Furthermore, we demonstrated that EGFR signaling acts downstream of the canonical Wnt/Wg signaling and regulates appendage proximodistal (PD) patterning genes aristaless and dachshund during regeneration. Our results suggest that EGFR signaling influences positional information along the PD axis in distal leg patterning of insects, regardless of the leg formation 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.     

<Emphasis Type="Italic">Rab32</Emphasis> and the remodeling of the imaginal midgut in <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Midgut remodeling is a complex physiological process in holometabolous insects. During midgut remodeling, the larval midgut is decomposed by apoptosis or autophagy during metamorphosis, and the degraded larval midgut is partially absorbed as nutrients by the imaginal midgut for its formation. The molecular mechanism involved in this process is not clear. Here, we found that a Rab protein, which we have named HaRab32, is related to the organogenesis of insect imaginal midgut. Results show that HaRab32 is up-regulated in epidermis and midgut during metamorphosis. Its expression could be up-regulated by 20E. Immunohistochemistry shows Rab32 is distributed in the epithelium of the imaginal midgut during metamorphosis. Knockdown of HaRab32 by RNA interference disturbs the formation of the imaginal midgut. These data imply HaRab32 plays important roles in midgut remodeling by participating in the imaginal midgut formation.     

Development of the adult leg epidermis in <Emphasis Type="Italic">Manduca sexta</Emphasis>: contribution of different larval cell populations

During metamorphosis of the tobacco hornworm Manduca sexta, the simple thoracic legs of the larva are remodeled into the more complex adult legs. Most of the adult leg epidermis derives from the adult primordia, small sets of epidermal cells located in specific regions of the larval leg, which proliferate rapidly in the final larval instar. In contrast, the contribution of the epidermal cells outside the primordia is unknown. In this study we have determined their contribution to the adult leg by labeling them with 5-bromodeoxyuridine (BUdR) and following their fate. Although the labeled cells diminished drastically in number, small groups of these cells persisted into the midpupal stage suggesting that they do contribute to the adult leg epidermis. We also found that during the wandering stage the adult primordia went through active proliferation and very little cell death, while the cells outside the primordia went through extensive cell death accounting for the decrease in their number. Our results indicate that two distinct cell populations exist outside the adult primordia. Most cells belong to the first population, which is larval-specific and disappears through apoptosis early in metamorphosis. The second population consists of polymorphic cells that contribute to the larval, pupal and adult leg epidermis.Edited by D. Tautz     

Thoracic leg motoneurons in the isolated CNS of adult Manduca produce patterned activity in response to pilocarpine,which is distinct from that produced in larvae

In the hawkmoth, Manduca sexta, thoracic leg motoneurons survive the degeneration of the larval leg muscles to innervate new muscles of the adult legs. The same motoneurons, therefore, participate in the very different modes of terrestrial locomotion that are used by larvae (crawling) and adults (walking). Consequently, changes in locomotor behavior may reflect changes in both the CNS and periphery. The present study was undertaken to determine whether motor patterns produced by the isolated CNS of adult Manduca, in the absence of sensory feedback, would resemble adult specific patterns of coordination. Pilocarpine, which evokes a fictive crawling motor pattern from the isolated larval CNS, also evoked robust patterned activity from leg motoneurons in the isolated adult CNS. As in the larva, levator and depressor motoneurons innervating the same leg were active in antiphase. Unlike fictive crawling, however, bursts of activity in levator or depressor motoneurons of one leg alternated with bursts in the homologous motoneurons innervating the opposite leg of the same segment and the leg on the same side in the adjacent segment. The most common mode of intersegmental activity generated by the isolated adult CNS resembled an alternating tripod gait, which is displayed, albeit infrequently, during walking in intact adult Manduca. A detailed analysis revealed specific differences between the patterned motor activity that is evoked from the isolated adult CNS and activity patterns observed during walking in intact animals, perhaps indicating an important role for sensory feedback. Nevertheless, the basic similarity to adult walking and clear distinctions from the larval fictive crawling pattern suggest that changes within the CNS contribute to alterations in locomotor activity during metamorphosis. Electronic Publication     

Formation and specification of distal leg segments in Drosophila by dual Bar homeobox genes, BarH1 and BarH2

Here, we show that BarH1 and BarH2, a pair of Bar homeobox genes, play essential roles in the formation and specification of the distal leg segments of Drosophila. In early third instar, juxtaposition of Bar-positive and Bar-negative tissues causes central folding that may separate future tarsal segments 2 from 3, while juxtaposition of tissues differentially expressing Bar homeobox genes at later stages gives rise to segmental boundaries of distal tarsi including the tarsus/pretarsus boundary. Tarsus/pretarsus boundary formation requires at least two different Bar functions, early antagonistic interactions with a pretarsus-specific homeobox gene, aristaless, and the subsequent induction of Fas II expression in pretarsus cells abutting tarsal segment 5. Bar homeobox genes are also required for specification of distal tarsi. Bar expression requires Distal-less but not dachshund, while early circular dachshund expression is delimited interiorly by BarH1 and BarH2.