Segment polarity gene expression in a myriapod reveals conserved and diverged aspects of early head patterning in arthropods

Arthropods show two kinds of developmental mode. In the so-called long germ developmental mode (as exemplified by the fly Drosophila), all segments are formed almost simultaneously from a preexisting field of cells. In contrast, in the so-called short germ developmental mode (as exemplified by the vast majority of arthropods), only the anterior segments are patterned similarly as in Drosophila, and posterior segments are added in a single or double segmental periodicity from a posterior segment addition zone (SAZ). The addition of segments from the SAZ is controlled by dynamic waves of gene activity. Recent studies on a spider have revealed that a similar dynamic process, involving expression of the segment polarity gene (SPG) hedgehog (hh), is involved in the formation of the anterior head segments. The present study shows that in the myriapod Glomeris marginata the early expression of hh is also in a broad anterior domain, but this domain corresponds only to the ocular and antennal segment. It does not, like in spiders, represent expression in the posterior adjacent segment. In contrast, the anterior hh pattern is conserved in Glomeris and insects. All investigated myriapod SPGs and associated factors are expressed with delay in the premandibular (tritocerebral) segment. This delay is exclusively found in insects and myriapods, but not in chelicerates, crustaceans and onychophorans. Therefore, it may represent a synapomorphy uniting insects and myriapods (Atelocerata hypothesis), contradicting the leading opinion that suggests a sister relationship of crustaceans and insects (Pancrustacea hypothesis). In Glomeris embryos, the SPG engrailed is first expressed in the mandibular segment. This feature is conserved in representatives of all arthropod classes suggesting that the mandibular segment may have a special function in anterior patterning.     

Expression of pair rule gene orthologs in the blastoderm of a myriapod: evidence for pair rule-like mechanisms?

ABSTRACT: BACKGROUND: A hallmark of Drosophila segmentation is the stepwise subdivision of the body into smaller and smaller units, and finally into the segments. This is achieved by the function of the well-understood segmentation gene cascade. The first molecular sign of a segmented body appears with the action of the pair rule genes, which are expressed as transversal stripes in alternating segments. Drosophila development, however, is derived, and in most other arthropods only the anterior body is patterned (almost) simultaneously from a pre-existing field of cells; posterior segments are added sequentially from a posterior segment addition zone. A long-standing question is to what extent segmentation mechanisms known from Drosophila may be conserved in short-germ arthropods. Despite the derived developmental modes, it appears more likely that conserved mechanisms can be found in anterior patterning. RESULTS: Expression analysis of pair rule gene orthologs in the blastoderm of the pill millipede Glomeris marginata (Myriapoda: Diplopoda) suggests that these genes are generally involved in segmenting the anterior embryo. We find that the Glomeris pairberry-1 (pby-1) gene is expressed in a pair rule pattern that is also found in insects and a chelicerate, the mite Tetraynchus urticae. Other Glomeris pair rule gene orthologs are expressed in double segment wide domains in the blastoderm, which at subsequent stages split into two stripes in adjacent segments. CONCLUSIONS: The expression patterns of the millipede pair rule gene orthologs resemble pair rule patterning in Drosophila and other insects, and thus represent evidence for the presence of an ancestral pair rule-like mechanism in myriapods. We discuss the possibilities that blastoderm patterning may be conserved in long-germ and short-germ arthropods, and that a posterior double segmental mechanism may be present in short-germ arthropods.     

From development to biodiversity—Tribolium castaneum, an insect model organism for short germband development

Insect embryogenesis is best understood in the fruit fly Drosophila. However, Drosophila embryogenesis shows evolutionary-derived features: anterior patterning is controlled by a highly derived Hox gene bicoid, the body segments form almost simultaneously and appendages develop from imaginal discs. In contrast, embryogenesis of the red flour beetle Tribolium castaneum displays typical features in anterior patterning, axis and limb formation shared with most insects, other arthropods as well as with vertebrates. Anterior patterning depends on the conserved homeobox gene orthodenticle, the main body axis elongates sequentially and limbs grow continuously starting from an appendage bud. Thus, by analysing developmental processes in the beetle at the molecular and cellular level, inferences can be made for similar processes in other arthropods. With the completion of sequencing the Tribolium genome, the door is now open for post-genomic studies such as RNA expression profiling, proteomics and functional genomics to identify beetle-specific gene circuits.     

Expression of hunchback during trunk segmentation in the branchiopod crustacean Artemia franciscana

Comparative studies have shown that some aspects of segmentation are widely conserved among arthropods. Yet, it is still unclear whether the molecular prepatterns that are required for segmentation in Drosophila are likely to be similarly conserved in other arthropod groups. Homologues of the Drosophila gap genes, like hunchback, show regionally restricted expression patterns during the early phases of segmentation in diverse insects, but their expression patterns in other arthropod groups are not yet known. Here, we report the cloning of a hunchback orthologue from the crustacean Artemia franciscana and its expression during the formation of trunk segments. Artemia hunchback is expressed in a series of segmental stripes that correspond to individual thoracic/trunk, genital, and postgenital segments. However, this expression is not associated with the segmenting ectoderm but is restricted to mesodermal cells that associate with the ectoderm in a regular metameric pattern. All cells in the early segmental mesoderm appear to express hunchback. Later, mesodermal expression fades, and a complex expression pattern appears in the central nervous system (CNS), which is comparable to hunchback expression in the CNS of insects. No regionally restricted expression, reminiscent of gap gene expression, is observed during trunk segmentation. These patterns suggest that the expression patterns of hunchback in the mesoderm and in the CNS are likely to be ancient and conserved among crustaceans and insects. In contrast, we find no evidence for a conserved role of hunchback in axial patterning in the trunk ectoderm.     

Gene expression during postembryonic segmentation in the centipede <Emphasis Type="Italic">Lithobius peregrinus</Emphasis> (Chilopoda,Lithobiomorpha)

Postembryonic segmentation (anamorphosis) is widespread among arthropods, but only partially known as for its developmental mechanics and control. Studies on developmental genetics of segmentation in anamorphic arthropods are mostly limited to the germ band stage, during early phases of embryonic development. This work presents the first data on the postembryonic expression of a segmentation gene in a myriapod. Using real-time PCR, we analyzed engrailed expression patterns during the anamorphic stages of the centipede Lithobius peregrinus. A variation pattern in en RNA level during anamorphosis suggests that gene expression is precisely modulated during this period of development and that engrailed is mainly expressed in the posterior part of the body, in the newly differentiating segments of each stage. As anamorphosis is possibly the primitive segmentation mode in arthropods, the postembryonic en expression pattern documented here provides evidence for a conservation of en role in ontogeny, across the embryonic/postembryonic boundary, as well as in phylogeny, across the same boundary, but in the opposite direction, from primitive postembryonic expression to the more derived expression in clades with exclusively embryonic segmentation.     

Deciphering the onychophoran ‘segmentation gene cascade’: Gene expression reveals limited involvement of pair rule gene orthologs in segmentation,but a highly conserved segment polarity gene network

The hallmark of the arthropods is their segmented body, although origin of segmentation, however, is unresolved. In order to shed light on the origin of segmentation we investigated orthologs of pair rule genes (PRGs) and segment polarity genes (SPGs) in a member of the closest related sister-group to the arthropods, the onychophorans. Our gene expression data analysis suggests that most of the onychophoran PRGs do not play a role in segmentation. One possible exception is the even-skipped (eve) gene that is expressed in the posterior end of the onychophoran where new segments are likely patterned, and is also expressed in segmentation-gene typical transverse stripes in at least a number of newly formed segments. Other onychophoran PRGs such as runt (run), hairy/Hes (h/Hes) and odd-skipped (odd) do not appear to have a function in segmentation at all. Onychophoran PRGs that act low in the segmentation gene cascade in insects, however, are potentially involved in segment-patterning. Most obvious is that from the expression of the pairberry (pby) gene ortholog that is expressed in a typical SPG-pattern. Since this result suggested possible conservation of the SPG-network we further investigated SPGs (and associated factors) such as Notum in the onychophoran. We find that the expression patterns of SPGs in arthropods and the onychophoran are highly conserved, suggesting a conserved SPG-network in these two clades, and indeed also in an annelid. This may suggest that the common ancestor of lophotrochozoans and ecdysozoans was already segmented utilising the same SPG-network, or that the SPG-network was recruited independently in annelids and onychophorans/arthropods.     

Short and long germ segmentation: unanswered questions in the evolution of a developmental mode

The insect body plan is very well conserved, yet the developmental mechanisms of segmentation are surprisingly varied. Less evolutionarily derived insects undergo short germ segmentation where only the anterior segments are specified before gastrulation whereas the remaining posterior segments are formed during a later secondary growth phase. In contrast, derived long germ insects such as Drosophila specify their entire bodies essentially simultaneously. These fundamental embryological differences imply potentially divergent molecular patterning events. Numerous studies have focused on comparing the expression and function of the homologs of Drosophila segmentation genes between Drosophila and different short and long germ insects. Here we review these comparative data with special emphasis on understanding how short germ insects generate segments and how this ancestral mechanism may have been modified in derived long germ insects such as Drosophila. We break down the larger issue of short versus long germ segmentation into its component developmental problems and structure our discussion in order to highlight the unanswered questions in the evolution of insect segmentation.     

Divergent functions of orthodenticle, empty spiracles and buttonhead in early head patterning of the beetle Tribolium castaneum (Coleoptera)

The head gap genes orthodenticle (otd), empty spiracles (ems) and buttonhead (btd) are required for metamerization and segment specification in Drosophila. We asked whether the function of their orthologs is conserved in the red flour beetle Tribolium castaneum which in contrast to Drosophila develops its larval head in a way typical for insects. We find that depending on dsRNA injection time, two functions of Tc-orthodenticle1 (Tc-otd1) can be identified. The early regionalization function affects all segments formed during the blastoderm stage while the later head patterning function is similar to Drosophila. In contrast, both expression and function of Tc-empty spiracles (Tc-ems) are restricted to the posterior part of the ocular and the anterior part of the antennal segment and Tc-buttonhead (Tc-btd) is not required for head cuticle formation at all. We conclude that the gap gene like roles of ems and btd are not conserved while at least the head patterning function of otd appears to be similar in fly and beetle. Hence, the ancestral mode of insect head segmentation remains to be discovered. With this work, we establish Tribolium as a model system for arthropod head development that does not suffer from the Drosophila specific problems like head involution and strongly reduced head structures.     

Gene silencing in the spider mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>: dsRNA and siRNA parental silencing of the <Emphasis Type="Italic">Distal-less</Emphasis> gene

A major prerequisite to understanding the evolution of developmental programs includes an appreciation of gene function in a comparative context. RNA interference (RNAi) represents a powerful method for reverse genetics analysis of gene function. However, RNAi protocols exist for only a handful of arthropod species. To extend functional analysis in basal arthropods, we developed a RNAi protocol for the two-spotted spider mite Tetranychus urticae focusing on Distal-less (Dll), a conserved gene involved in appendage specification in metazoans. First, we describe limb morphogenesis in T. urticae using confocal and scanning electron microscopy. Second, we examine T. urticae Dll (Tu-Dll) mRNA expression patterns and correlate its expression with appendage development. We then show that fluorescently labeled double-stranded RNA (dsRNA) and short interfering RNA (siRNA) molecules injected into the abdomen of adult females are incorporated into the oviposited eggs, suggesting that dsRNA reagents can be systemically distributed in spider mites. Injection of longer dsRNA as well as siRNA induced canonical limb truncation phenotypes as well as the fusion of leg segments. Our data suggest that Dll plays a conserved role in appendage formation in arthropods and that such conserved genes can serve as reliable starting points for the development of functional protocols in nonmodel organisms.     

An analysis of segmentation dynamics throughout embryogenesis in the centipede <Emphasis Type="Italic">Strigamia maritima</Emphasis>

Background

Most segmented animals add segments sequentially as the animal grows. In vertebrates, segment patterning depends on oscillations of gene expression coordinated as travelling waves in the posterior, unsegmented mesoderm. Recently, waves of segmentation gene expression have been clearly documented in insects. However, it remains unclear whether cyclic gene activity is widespread across arthropods, and possibly ancestral among segmented animals. Previous studies have suggested that a segmentation oscillator may exist in Strigamia, an arthropod only distantly related to insects, but further evidence is needed to document this.

Results

Using the genes even skipped and Delta as representative of genes involved in segment patterning in insects and in vertebrates, respectively, we have carried out a detailed analysis of the spatio-temporal dynamics of gene expression throughout the process of segment patterning in Strigamia. We show that a segmentation clock is involved in segment formation: most segments are generated by cycles of dynamic gene activity that generate a pattern of double segment periodicity, which is only later resolved to the definitive single segment pattern. However, not all segments are generated by this process. The most posterior segments are added individually from a localized sub-terminal area of the embryo, without prior pair-rule patterning.

Conclusions

Our data suggest that dynamic patterning of gene expression may be widespread among the arthropods, but that a single network of segmentation genes can generate either oscillatory behavior at pair-rule periodicity or direct single segment patterning, at different stages of embryogenesis.

     

The ten Hox genes of the millipede Glomeris marginata

We have isolated the ten Hox genes from the pill millipede Glomeris marginata (Myriapoda:Diplopoda). All ten genes are expressed in characteristic Hox-gene-like expression patterns. The register of Hox gene expression borders is conserved and the expression profiles show that the anterior-most limb-bearing segment in arthropods (antennal/cheliceral segment) does not express any Hox gene, while the next segment (intercalary/second-antennal/premandibular/pedipalpal segment) does express Hox genes. The Hox expression patterns in this millipede thus support the conclusion that all arthropods possess a deuterocerebral segment. We find that there is an apparent posterior shift of Hox gene expression domains dorsally relative to their ventral patterns, indicating that the decoupling of dorsal and ventral segmentation is not restricted to the level of segment polarity genes but apparently includes the Hox genes. Although the mechanism for the decoupling of dorsal and ventral segmentation remains unsolved, the decoupling must be at a level higher in the hierarchy than that of the segment polarity and Hox genes. The expression patterns of Ultrabithorax and abdominal-A suggest a correlation between the function of these genes and the delayed outgrowth of posterior trunk appendages. This delay may be caused by an assumed repressor function of Ultrabithorax, which might partially repress the activation of the Distal-less gene. The Glomeris fushi tarazu gene is expressed in a Hox-like domain and in the developing central nervous system, but not in segmental stripes such as has been reported in another myriapod species, the centipede Lithobius. In contrast to the Lithobius fushi tarazu gene, there is no indication for a role in segment formation for the millipede fushi tarazu gene, suggesting that fushi tarazu first acquired its segmentation function in the lineage of the insects.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Notch/Delta signalling is not required for segment generation in the basally branching insect Gryllus bimaculatus

Arthropods and vertebrates display a segmental body organisation along all or part of the anterior-posterior axis. Whether this reflects a shared, ancestral developmental genetic mechanism for segmentation is uncertain. In vertebrates, segments are formed sequentially by a segmentation 'clock' of oscillating gene expression involving Notch pathway components. Recent studies in spiders and basal insects have suggested that segmentation in these arthropods also involves Notch-based signalling. These observations have been interpreted as evidence for a shared, ancestral gene network for insect, arthropod and bilaterian segmentation. However, because this pathway can play multiple roles in development, elucidating the specific requirements for Notch signalling is important for understanding the ancestry of segmentation. Here we show that Delta, a ligand of the Notch pathway, is not required for segment formation in the cricket Gryllus bimaculatus, which retains ancestral characteristics of arthropod embryogenesis. Segment patterning genes are expressed before Delta in abdominal segments, and Delta expression does not oscillate in the pre-segmental region or in formed segments. Instead, Delta is required for neuroectoderm and mesectoderm formation; embryos missing these tissues are developmentally delayed and show defects in segment morphology but normal segment number. Thus, what initially appear to be 'segmentation phenotypes' can in fact be due to developmental delays and cell specification errors. Our data do not support an essential or ancestral role of Notch signalling in segment generation across the arthropods, and show that the pleiotropy of the Notch pathway can confound speculation on possible segmentation mechanisms in the last common bilaterian ancestor.     

Tracking down the "head blob": comparative analysis of wingless expression in the developing insect procephalon reveals progressive reduction of embryonic visual system patterning in higher insects

The evolution of larval head morphology in holometabolous insects is characterized by reduction of antennal appendages and the visual system components. Little insight has been gained into molecular developmental changes underlying this morphological diversification. Here we compare the expression of the segment polarity gene wingless (wg) in the pregnathal head of fruit fly, flour beetle and grasshopper embryos. We provide evidence that wg activity contributes to segment border formation, and, subsequently, the separation of the visual system and protocerebrum anlagen in the anterior procephalon. In directly developing insects like grasshopper, seven expression domains are formed during this process. The activation of four of these, which correspond to polar expression pairs in the optic lobe anlagen and the protocerebral ectoderm, has shifted to postembryonic stages in flour beetle and Drosophila. The remaining three domains map to the protocerebral neuroectoderm, and form by disintegration of a large precursor domain in flour beetle and grasshopper. In Drosophila, the precursor domain remains intact, constituting the previously described “head blob”. These data document major changes in the expression of an early patterning gene correlated with the dramatic evolution of embryonic visual system development in the Holometabola.     

A molecular view of onychophoran segmentation

This paper summarizes our current knowledge on the expression and assumed function of Drosophila and (other) arthropod segmentation gene orthologs in Onychophora, a closely related outgroup to Arthropoda. This includes orthologs of the so-called Drosophila segmentation gene cascade including the Hox genes, as well as other genetic factors and pathways involved in non-drosophilid arthropods.Open questions about and around the topic are addressed, such as the definition of segments in onychophorans, the unclear regulation of conserved expression patterns downstream of non-conserved factors, and the potential role of mesodermal patterning in onychophoran segmentation.     

Bmdelta phenotype implies involvement of Notch signaling in body segmentation and appendage development of silkworm,Bombyx mori

The domesticated silkworm, Bombyx mori, belongs to the intermediate germband insects, in which the anterior segments are specified in the blastoderm, while the remaining posterior segments are sequentially generated from the cellularized growth zone. The pattern formation is distinct from Drosophila but somewhat resembles a vertebrate. Notch signaling is involved in the segmentation of vertebrates and spiders.Here, we studied the function of Notch signaling in silkworm embryogenesis via RNA interference (RNAi). Depletion of Bmdelta, the homolog of the Notch signaling ligand, led to severe defects in segment patterning, including a loss of posterior segments and irregular segment boundaries. The paired appendages on each segment were symmetrically fused along the ventral midline in Bmdelta RNAi embryos. An individual segment seemed to possess only one segmental appendage. Segmentation in prolegs could be observed.Our results show that Notch signaling is employed in not only appendage development but also body segmentation. Thus, conservation of Notch-mediated segmentation could also be extended to holometabolous insects. The involvement of Notch signaling seems to be the ancestral segmentation mechanism of arthropods.     

even-skipped has gap-like, pair-rule-like, and segmental functions in the cricket Gryllus bimaculatus, a basal, intermediate germ insect (Orthoptera)

Developmental mechanisms of segmentation appear to be varied among insects in spite of their conserved body plan. Although the expression patterns of the segment polarity genes in all insects examined imply well conserved function of this class of genes, expression patterns and function of the pair-rule genes tend to exhibit diversity. To gain further insights into the evolution of the segmentation process and the role of pair-rule genes, we have examined expression and function of an ortholog of the Drosophila pair-rule gene even-skipped (eve) in a phylogenetically basal insect, Gryllus bimaculatus (Orthoptera, intermediate germ cricket). We find that Gryllus eve (Gb'eve) is expressed as stripes in each of the prospective gnathal, thoracic, and abdominal segments and as a broad domain in the posterior growth zone. Dynamics of stripe formation vary among Gb'eve stripes, representing one of the three modes, the segmental, incomplete pair-rule, and complete pair-rule mode. Furthermore, we find that RNAi suppression of Gb'eve results in segmentation defects in both anterior and posterior regions of the embryo. Mild depletion of Gb'eve shows a pair-rule-like defect in anterior segments, while stronger depletion causes a gap-like defect showing deletion of anterior and posterior segments. These results suggest that Gb'eve acts as a pair-rule gene at least during anterior segmentation and also has segmental and gap-like functions. Additionally, Gb'eve may be involved in the regulation of hunchback and Krüppel expression. Comparisons with eve functions in other species suggest that the Gb'eve function may represent an intermediate state of the evolution of pair-rule patterning by eve in insects.     

The involvement of <Emphasis Type="Italic">engrailed</Emphasis> and <Emphasis Type="Italic">wingless</Emphasis> during segmentation in the onychophoran <Emphasis Type="Italic">Euperipatoides kanangrensis</Emphasis> (Peripatopsidae: Onychophora) (Reid 1996)

As the putative sister group to the arthropods, onychophorans can provide insight into ancestral developmental mechanisms in the panarthropod clade. Here, we examine the expression during segmentation of orthologues of wingless (Wnt1) and engrailed, two genes that play a key role in defining segment boundaries in Drosophila and that appear to play a role in segmentation in many other arthropods. Both are expressed in segmentally reiterated stripes in all forming segments except the first (brain) segment, which only shows an engrailed stripe. Engrailed is expressed before segments are morphologically visible and is expressed in both mesoderm and ectoderm. Segmental wingless expression is not detectable until after mesodermal somites are clearly distinct. Early engrailed expression lies in and extends to both sides of the furrow that first demarcates segments in the ectoderm, but is largely restricted to the posterior part of somites. Wingless expression lies immediately anterior to engrailed expression, as it does in many arthropods, but there is no precise cellular boundary between the two expression domains analogous to the overt parasegment boundary seen in Drosophila. Engrailed stripes extend along the posterior part of each limb bud, including the antenna, while wingless is restricted to the distal tip of the limbs and the neurectoderm basal to the limbs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

JAK-STAT signalling is required throughout telotrophic oogenesis and short-germ embryogenesis of the beetle Tribolium

In Drosophila, the JAK-STAT signalling pathway regulates a broad array of developmental functions including segmentation and oogenesis. Here we analysed the functions of Tribolium JAK-STAT signalling factors and of Suppressor Of Cytokine Signalling (SOCS) orthologues, which are known to function as negative regulators of JAK-STAT signalling, during telotrophic oogenesis and short-germ embryogenesis. The beetle Tribolium features telotrophic ovaries, which differ fundamentally from the polytrophic ovary of Drosophila. While we found the requirement for JAK-STAT signalling in specifying the interfollicular stalk to be principally conserved, we demonstrate that these genes also have early and presumably telotrophic specific functions. Moreover, we show that the SOCS genes crucially contribute to telotrophic Tribolium oogenesis, as their inactivation by RNAi results in compound follicles. During short-germ embryogenesis, JAK-STAT signalling is required in the maintenance of segment primordia, indicating that this signalling cascade acts in the framework of the segment-polarity network. In addition, we demonstrate that JAK-STAT signalling crucially contributes to early anterior patterning. We posit that this signalling cascade is involved in achieving accurate levels of expression of individual pair-rule and gap gene domains in early embryonic patterning.     

Segmental expression of Pax3/7 and Engrailed homologs in tardigrade development

How morphological diversity arises through evolution of gene sequence is a major question in biology. In Drosophila, the genetic basis for body patterning and morphological segmentation has been studied intensively. It is clear that some of the genes in the Drosophila segmentation program are functioning similarly in certain other taxa, although many questions remain about when these gene functions arose and which taxa use these genes similarly to establish diverse body plans. Tardigrades are an outgroup to arthropods in the Ecdysozoa and, as such, can provide insight into how gene functions have evolved among the arthropods and their close relatives. We developed immunostaining methods for tardigrade embryos, and we used cross-reactive antibodies to investigate the expression of homologs of the pair-rule gene paired (Pax3/7) and the segment polarity gene engrailed in the tardigrade Hypsibius dujardini. We find that in H. dujardini embryos, Pax3/7 protein localizes not in a pair-rule pattern but in a segmentally iterated pattern, after the segments are established, in regions of the embryo where neurons later arise. Engrailed protein localizes in the posterior ectoderm of each segment before ectodermal segmentation is apparent. Together with previous results from others, our data support the conclusions that the pair-rule function of Pax3/7 is specific to the arthropods, that some of the ancient functions of Pax3/7 and Engrailed in ancestral bilaterians may have been in neurogenesis, and that Engrailed may have a function in establishing morphological boundaries between segments that is conserved at least among the Panarthropoda. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.