Double-stranded RNA interference in the spider Cupiennius salei: the role of Distal-less is evolutionarily conserved in arthropod appendage formation

Chelicerates represent a basal arthropod group, which makes them an excellent system for the study of evolutionary processes in arthropods. To enable functional studies in chelicerates, we developed a double-stranded RNA-interference (RNAi) protocol for spiders while studying the function of the Distal-less gene. We isolated the Distal-less gene from the spider Cupiennius salei. Cs-Dll gene expression is first seen in cells of the prosomal segments before the outgrowth of the appendages. After the appendages have formed, Cs-Dll is expressed in the distal portion of the prosomal appendages, and in addition, in the labrum, in two pairs of opisthosmal (abdominal) limb buds, in the head region, and at the posterior-most end of the spider embryo. In embryos, in which Dll was silenced by RNAi, the distal part of the prosomal appendages was missing and the labrum was completely absent. Thus, Dll also plays a crucial role in labrum formation. However, the complete lack of labrum in RNAi embryos may point to a different nature of the labrum from the segmental appendages. Our data show that the expression of Dll in the appendages is conserved among arthropods, and furthermore that the role of Dll is evolutionarily conserved in the formation of segmental appendages in arthropods.     

Expression of dachshund in wild-type and Distal-less mutant Tribolium corroborates serial homologies in insect appendages

We isolated the homologue of the Drosophila gene dachshund (dac) from the beetle Tribolium castaneum. Tc'dac is expressed in all appendages except urogomphi and pleuropodia. Tc'dac is also active in the head lobes, in the ventral nervous system, in the primordia of the Malpighian tubules and in bilateral stripes corresponding to the presumptive dorsal midline. Expression of Tc'dac in the labrum lends support to the interpretation that the insect labrum is derived from a metameric appendage. The legs of Tribolium accommodate two Tc'dac domains, of which the more distal one corresponds to the single dac domain described for Drosophila leg discs. In contrast to Drosophila, where this domain is thought to intercalate between the homothorax (hth) and the Distal-less (Dll) domains, in Tribolium it arises from within the Dll domain. In embryos mutant for the Tc'Dll gene we find that the distal Tc'dac domain in the legs, as well as the expression in the labrum, are deleted while the proximal leg domain and the mandibular expression are unaffected. Based on Tc'dac expression in wild-type and mutant embryos, we demonstrate serial homology of the complete mandible with the coxa of the thoracic legs, which affirms the gnathobasic nature of the insect mandible.     

Early development of leg and wing primordia in the Drosophila embryo

The development of the leg and wing primordia in the Drosophila embryo has been traced using molecular markers. Distal-less and disconnected gene expression provide molecular labels for the leg primordia throughout embryonic development, disconnected expression in the developing leg primordia depends on Distal-less activity. The leg primordia arise as discrete clusters of cells that occupy well defined positions in the embryonic ectoderm. At later stages of embryogenesis the primordia become morphologically recognizable and are intimately associated with the development of the Keilin's organs. The presumptive leg disc and the Keilin's organ appear to derive from a common primordium. Similarly the Abnormal leg pattern gene provides a molecular label for the wing and haltere primordia. The dorsal thoracic primordia appear to be of independent origin from the legs.     

Functional analyses in the hemipteran Oncopeltus fasciatus reveal conserved and derived aspects of appendage patterning in insects

The conservation of expression of appendage patterning genes, particularly Distal-less, has been shown in a wide taxonomic sampling of animals. However, the functional significance of this expression has been tested in only a few organisms. Here we report functional analyses of orthologues of the genes Distal-less, dachshund, and homothorax in the appendages of the milkweed bug Oncopeltus fasciatus (Hemiptera). This hemimetabolous insect has typical legs but highly derived mouthparts. Distal-less, dachshund, and homothorax are conserved in their individual expression patterns and functions in the legs of Oncopeltus, but their functions in other appendages are in some cases divergent. We find that specification of antennal identity does not require wild-type Distal-less activity in Oncopeltus as it does in Drosophila. Additionally, the mouthparts of Oncopeltus show novel patterns of gene expression and function, relative to other insects. Expression of Distal-less in the maxillary stylets of Oncopeltus does not seem necessary for proper development of this appendage, while dachshund and homothorax are crucial for formation of the mandibular and maxillary stylets. These data are used to evaluate hypotheses for the evolution of hemipteran mouthparts and the evolution of developmental mechanisms in insect appendages in general.     

The expression of the proximodistal axis patterning genes Distal-less and dachshund in the appendages of Glomeris marginata (Myriapoda: Diplopoda) suggests a special role of these genes in patterning the head appendages

The genes Distal-less, dachshund, extradenticle, and homothorax have been shown in Drosophila to be among the earliest genes that define positional values along the proximal-distal (PD) axis of the developing legs. In order to study PD axis formation in the appendages of the pill millipede Glomeris marginata, we have isolated homologues of these four genes and have studied their expression patterns. In the trunk legs, there are several differences to Drosophila, but the patterns are nevertheless compatible with a conserved role in defining positional values along the PD axis. However, their role in the head appendages is apparently more complex. Distal-less in the mandible and maxilla is expressed in the forming sensory organs and, thus, does not seem to be involved in PD axis patterning. We could not identify in the mouthparts components that are homologous to the distal parts of the trunk legs and antennnae. Interestingly, there is also a transient premorphogenetic expression of Distal-less in the second antennal and second maxillary segment, although no appendages are eventually formed in these segments. The dachshund gene is apparently involved both in PD patterning as well as in sensory organ development in the antenna, maxilla, and mandible. Strong dachshund expression is specifically correlated with the tooth-like part of the mandible, a feature that is shared with other mandibulate arthropods. homothorax is expressed in the proximal and medial parts of the legs, while extradenticle RNA is only seen in the proximal region. This overlap of expression corresponds to the functional overlap between extradenticle and homothorax in Drosophila.     

Coexpression of the homeobox genes Distal-less and homothorax determines Drosophila antennal identity

The Distal-less gene is known for its role in proximodistal patterning of Drosophila limbs. However, Distal-less has a second critical function during Drosophila limb development, that of distinguishing the antenna from the leg. The antenna-specifying activity of Distal-less is genetically separable from the proximodistal patterning function in that certain Distal-less allelic combinations exhibit antenna-to-leg transformations without proximodistal truncations. Here, we show that Distal-less acts in parallel with homothorax, a previously identified antennal selector gene, to induce antennal differentiation. While mutations in either Distal-less or homothorax cause antenna-to-leg transformations, neither gene is required for the others expression, and both genes are required for antennal expression of spalt. Coexpression of Distal-less and homothorax activates ectopic spalt expression and can induce the formation of ectopic antennae at novel locations in the body, including the head, the legs, the wings and the genital disc derivatives. Ectopic expression of homothorax alone is insufficient to induce antennal differentiation from most limb fields, including that of the wing. Distal-less therefore is required for more than induction of a proximodistal axis upon which homothorax superimposes antennal identity. Based on their genetic and biochemical properties, we propose that Homothorax and Extradenticle may serve as antenna-specific cofactors for Distal-less.     

Distal-less expression in embryos of Limulus polyphemus (Chelicerata, Xiphosura) and Lepisma saccharina (Insecta, Zygentoma) suggests a role in the development of mechanoreceptors, chemoreceptors, and the CNS

The homeobox gene Distal-less (Dll) is well known for its participation in the development of arthropod limbs and their derivatives. Dll activity has been described for all groups of arthropods, but also for molluscs, echinoderms and vertebrates. Generally, Dll participates in the establishment of the proximo-distal-axis and differentiation along this axis. During our investigation of the expression pattern in the silverfish Lepisma saccharina and the horseshoe crab Limulus polyphemus, we found several expressions in late stages which cannot be explained with the "normal" limb-specific function. The antenna, cerci and terminal filament of the silverfish show a striped expression; single cells on the labrum, mandibles, maxillary palps and anal valves are also strongly stained by the Dll antibody. In addition to cell groups in the developing ganglia of the CNS, in the coxal endites and several nerve cells in femur and the trochanter of the prosomal limbs, the whole prosomal shield of Limulus polyphemus is surrounded by Dll-positive cell clusters. Furthermore, the lateral processes of the opisthosoma and the edges of the opisthosomal appendages are Dll positive. To get an indication of the cell fate of these regions, we examined hatched larvae and juvenile stages of both species with the SEM. We found a striking correlation of these Dll-positive areas and different sense organs, especially mechanoreceptors. Since many sense organs in arthropods are situated on the limbs, interpretation of the Dll expression in limbs is problematical. This has critical implications for comparative analysis of Dll expression patterns between arthropods and for the claim of homology between limb-like structures. Furthermore, we discuss the possibility of convergent appendage evolution in various bilaterian groups based on the improvement of spatial sensory resolution.     

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.     

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.     

The embryonic development of Stylops ovinae (Strepsiptera,Stylopidae) with emphasis on external morphology

External features of the embryonic development of Stylops ovinae (Strepsiptera) were examined. Eighteen distinct embryological stages are suggested. Many embryological traits are closely correlated to the parasitic life style of the first instar larvae or to vivipary. The high number of eggs, their small size, the characteristic egg membrane, and the lack of micropyles are derived groundplan features of Strepsiptera. The development with a semi-long germ embryo is shared with several other groups of Holometabola. The reduction of the labrum and antennae are autapomorphies of Strepsiptera. The cephalic ventral plate of the first instar larva of S. ovinae is formed by parts of the head capsule and the anlagen of the maxillae and labium. It is involved in the formation of the specific entognathous condition, and the entire character complex is autapomorphic for Stylopidae. The trochanter is recognizable in the anlagen of all three legs. Its fusion with the femur in the later stages is an autapomorphy of Stylopidia. The extreme spiralization and compression of the abdomen during blastokinesis is a derived feature, like the reduction of the anlagen of the anterior abdominal appendages. The caudal bristles on segment XI are possibly re-activated cerci. The same is likely in the case of segment XI.     

Evolutionary changes in sensory precursor formation in arthropods: embryonic development of leg sensilla in the spider Cupiennius salei

We describe here for the first time the development of mechanosensory organs in a chelicerate, the spider Cupiennius salei. It has been shown previously that the number of external sense organs increases with each moult. While stage 1 larvae do not have any external sensory structures, stage 2 larvae show a stereotyped pattern of touch sensitive ‘tactile hairs’ on their legs. We show that these mechanosensory organs develop during embryogenesis. In contrast to insects, groups of sensory precursors are recruited from the leg epithelium, rather than single sensory organ progenitors. The groups increase by proliferation, and neural cells delaminate from the cluster, which migrate away to occupy a position proximal to the accessory cells of the sense organ. In addition, we describe the development of putative internal sense organs, which do not differentiate until larval stage 2. We show by RNA interference that, similar to Drosophila, proneural genes are responsible for the formation and subtype identity of sensory organs. Furthermore, we demonstrate an additional function for proneural genes in the coordinated invagination and migration of neural cells during sensory organ formation in the spider.     

Morphological and behavioural development of halibut, Hippoglossus hippoglossus (L.) larvae

Live yolk-sac halibut, Hippoglossus hippoglossus (L.) larvae from rearing experiments at Austevoll Aquaculture Station, Norway, were examined from hatching to past first feeding for developmental morphology and behaviour. The findings include development of the respiratory and circulatory organs, eye pigmentation, mouth formation, organs of the digestive system and the process of yolk absorption, as well as swimming speed and activity levels.
A stomodeum is not present at hatching although drinking is possible through a pair of branchial pits which gradually develop into the operculum and gill basket. The mouth normally opens slowly, the gape being restricted by a transverse septum until bones are formed. The amount of time spent swimming varies from less than 15% of the observation period during the first 2 weeks after hatching to between 70 and 100% around the seventh week after hatching, when individual differences become more apparent. Larvae generally react with a burst of swimming when two come into contact. Speed and duration of swimming seems to be correlated with development of eye pigment, heart size and fin formation. The yolk-sac period is divided into four stages.     

The segmental origin of the appendages of the head and anterior body segments of a spiroboloid milliped, Narceus annularis

Study of a series of embryos showed that the spiroboloid leg arrangement (1 pair of legs on each of the first 5 segments) is derived from the typical leg arrangement (no legs on segment 1, 1 pair on segments 2 through 4, and 2 pairs on segment 5) by a shifting forward one segment of the first four pairs of legs. A careful re-examination of the literature, especially papers by Robinson ('07), Silvestri ('03, '49), Pflugfelder ('32), and Manton ('61), combined with observations of Narceus embryos led to the conclusion that (1) the anterior body segments are primatively single (2) the gnathochilarium is composed of only one pair of mouthparts, the diplopod head having but two gnathal segments, and (3) the intercalary segment is present in the Diplopoda.     

Functional morphology and development of tibial organs in the legs I,II and III of the bushcricketEphippiger ephippiger (Insecta,Ensifera)

Summary The anatomy of the complex tibial organs in the pro-, meso- and metathoracic legs of adults and larvae of the bushcricketEphippiger ephippiger is described comparatively. The subgenual organ and the intermediate organ are differentiated in the same way in legs I, II and III; the anatomy of the crista acustica and the tracheal morphology are significantly different. The final number of scolopidia in the tibial organ of each leg is present at the time of hatching. In the subgenual organ, the number of scolopidia is the same in all legs; in the intermediate organ, and especially in the crista acustica, the number of scolopidia decreases from leg I to legs II and III. In the first larval instar, the morphology of the tibia, the course of the trachea and the anatomy of accessory structures are developed in the same way in each leg. The specific differentiations forming the auditory receptor organ in leg I, such as the acoustic trachea, the tympana and tympanal cavities, develop step by step in subsequent instars. The auditory threshold recorded from the tympanal nerve in the prothoracic leg of adults is remarkably lower than in the meso- and metathoracic legs. Morphometrical analyses of structures that are suggested to play a role in stimulus transduction on scolopidia of the crista acustica reveal significant differences in the three legs.     

Contribution a l'étude de l'ontogenèse sensori-nerveuse du termiteCalotermes flavicollis Fab.

Summary The author studies the developpement of the neuro-sensorial organs on the legs ofCalotermes flavicollis Fab. He has found that the outline of the neuro-sensorial organs developing in the larvae of the first instar do not change their structure. After each molt the number of sensorial organs increases and in correlation with this the small sensorial nerves ramify and secondary nerfs branch off the primary nerfs. On the legs of the larvae of the forth and mainly on those of the fifth instar the number of sensorial organs increases considerably. These latter are of different size and sit according to the region of the leg more or less densely together. The same is observed on the nymphs and imagines.A regression of the number and size of the sensorial organs accompanies the development of the pseudergates.The anatomy of the neurosensorial system of the third pair of legs is alike that of the second pair. The first pair of legs shows on the front surface the anatomy found inthe hind surface of the second pair of legs and the hind surface of the first pair the one of the front surface of the second pair.

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Zusammenfassung Die Studie behandelt die Innervation der Beine vonCalotermes flavicollis Fab. Es wurde festgestellt, dass die gesamte sensoriel-nervöse Anlage schon bei den Larven des ersten Entwicklungsstadiums vorhanden ist und sich nicht mehr verändert, und dass nur mit jeder stattfindenden Häutung die Anzahl der sensoriellen Organe zunimmt und begleitet ist von einer reicher werdenden Verzweigung der Sinnesnerven. Gleichzeitig entstehen sekundäre Abzweigungen der Sinnesnerven, welche von den Hauptnerven ausgehen. Die Extremitäten der Larven des vierten und hauptsächlich des fünften Stadiums weisen eine sehr starke Zunahme der Sinnesorgane auf. Diese letzteren haben dann verschiedene Grösse und stehen je nach Region des Beines mehr oder weniger dicht beisammen. Dieselben Verhältnisse finden sich auch bei den Nymphen und Imagines.Eine zahlen- und grössenmässige Regression der Sinnesorgane wurde bei der Entstehung der Pseudergaten festgestellt. Die Anatomie der Sinnesorgane des dritten Beinpaares ist derjenigen des mittleren Beinpaares ähnlich. Die vordere Fläche des vordersten Beinpaares ist der hinteren Fläche des mittleren Paares ähnlich und die hintere Fläche derjenigen der Vorderfläche des mittleren Beinpaares.

     

Evolving role of Antennapedia protein in arthropod limb patterning

Evolutional changes in homeotic gene functions have contributed to segmental diversification of arthropodan limbs, but crucial molecular changes have not been identified to date. The first leg of the crustacean Daphnia lacks a prominent ventral branch found in the second to fourth legs. We show here that this phenotype correlates with the loss of Distal-less and concomitant expression of Antennapedia in the limb primordium. Unlike its Drosophila counterpart, Daphnia Antennapedia represses Distal-less in Drosophila assays, and the protein region conferring this activity was mapped to the N terminal region of the protein. The results imply that Dapnia Antennapedia specifies leg morphology by repressing Distal-less, and this activity was acquired through a change in protein structure after separation of crustaceans and insects.     

Cloning of a<Emphasis Type="Italic"> decapentaplegic</Emphasis> orthologue from the sawfly,<Emphasis Type="Italic"> Athalia rosae</Emphasis> (Hymenoptera), and its expression in the embryonic appendages

The cDNA of a decapentaplegic (dpp) orthologue from the sawfly, Athalia rosae (Hymenoptera), was cloned and characterized. The clone (Ar dpp) was 2,566 bp long and encoded 395 amino acids in a single open reading frame. Genomic Southern blotting showed that Ar dpp is a single copy gene. The deduced amino acid sequence can be aligned along its entire length with known insect DPPs. It shared common characteristics such as a signal sequence, a pro-domain region, and a ligand domain with seven cysteines at conserved locations. Ar dpp was expressed as a single 5.0-kb mRNA in embryos, larvae, pupae and adults. In situ hybridization showed that Ar dpp was expressed in the dorsal region proper in early embryonic stages and in the embryonic appendages of cephalic segments (labrum, antenna, mandible, maxilla, and labium), thoracic segments (thoracic legs), and all abdominal segments except the tenth segment (pleuropodia and proleg primordia). The present results indicate that Ar dpp expression reflects the primary determination of embryonic appendages.Edited by D. TautzThe sequence reported in this paper has been deposited in the DDBJ/EMBL/GenBank database with the accession number AB121072