The segmentation cascade in the centipede Strigamia maritima: involvement of the Notch pathway and pair-rule gene homologues

The centipede Strigamia maritima forms all of its segments during embryogenesis. Trunk segments form sequentially from an apparently undifferentiated disk of cells at the posterior of the germ band. We have previously described periodic patterns of gene expression in this posterior disc that precede overt differentiation of segments, and suggested that a segmentation oscillator may be operating in the posterior disc. We now show that genes of the Notch signalling pathway, including the ligand Delta, and homologues of the Drosophila pair-rule genes even-skipped and hairy, show periodic expression in the posterior disc, consistent with their involvement in, or regulation by, such an oscillator. These genes are expressed in a pattern of apparently expanding concentric rings around the proctodeum, which become stripes at the base of the germ band where segments are emerging. In this transition zone, these primary stripes define a double segment periodicity: segmental stripes of engrailed expression, which mark the posterior of each segment, arise at two different phases of the primary pattern. Delta and even-skipped are also activated in secondary stripes that intercalate between primary stripes in this region, further defining the single segment repeat. These data, together with observations that Notch mediated signalling is required for segment pattern formation in other arthropods, suggest that the ancestral arthropod segmentation cascade may have involved a segmentation oscillator that utilised Notch signalling.     

Generating patterns from fields of cells: Examples from Drosophila segmentation

In Drosophila, a cascade of maternal, gap, pair-rule and segment polarity genes subdivides the antero/posterior axis of the embryo into repeating segmental stripes. This review summarizes what happens next, i.e. how an intrasegmental pattern is generated and controls the differentiation of specific cell types in the epidermis. Within each segment, cells secreting the signalling molecules Wingless (the homologue of vertebrate Wnt-1) and Hedgehog are found in narrow stripes on both sides of the parasegmental boundary. The Wingless and Hedgehog organizing activities help to establish two more stripes per segment that localize ligands for the Epidermal Growth Factor and the Notch signalling pathways, respectively. These four signals then act at short range and in concert to control epidermal differentiation at the single cell level across the segment. This example from Drosophila provides a paradigm for how organizers generate precise patterns, and ultimately different cell types, in a naïve field of cells.     

Later stages of regeneration in the Polychaete, Nephtys

The sequence of events in posterior regeneration of the polychaete, Nephtys, has been examined in histological preparations from the fifth day to the end of the third week after amputation, that is from the time when wound healing is complete until several new segments are differentiated. The pygidium forms and begins to differentiate prior to segmentation. The first indication of each new segment is the appearance of a large pair of segmental blood vessels which arise from the vascular complex in the gut wall. Associated with these are fibroblasts, the anlagen of the new septum. Epidermal derivatives develop subsequently, appearing first ventrolaterally, close to the regenerating nerve cord. The mitotic rate appears to be highest prior to the period of maximum segment formation. Visible cell differentation follows, and subsequent growth of segments is primarily by cell enlargement. It appears likely that the blood-vascular system associated with the gut and the regenerating nerve cord, both of which are disproportionately large in the regenerate, are important for the initiation of new segments.     

Compartments in the abdomen of Drosophila and the role of the engrailed locus

A clonal analysis has shown that the dorsal surface of the first abdominal segment of Drosophila melanogaster is subdivided into anterior and posterior compartments. Cells of the posterior compartment grow up to but not beyond the anterior-posterior compartment border within the first abdominal segment and the intersegmental border that defines the boundary between the first and second abdominal segments. Growing within these boundaries, a narrow band of tissue clonally isolated from the adjoining tissue is formed. When these posterior cells are deficient for the engrailed locus, however, neither the compartment nor the segment border is maintained. The implications, that compartmentalization is essential for segmentation, and that all insect segments are subdivided by anterior and posterior compartments, are discussed.     

Regeneration of the segment boundary in Oncopeltus

The segment boundary of Oncopeltus is a compartment border. It is also an element in the pattern of the abdomen, being marked by a groove in the surface of the cuticle and an abrupt change in the pigmentation of the cells. If the segment boundary is either burnt or extirpated, the surviving cells of the two neighbouring segments migrate into the wounded area and form a new segment boundary where they confront each other. Grafting experiments with genetically marked cells demonstrate that a boundary is regenerated wherever cells from remote locations in the anteroposterior axis of any segment are apposed; thus anterior and posterior cells from the same segment form an ectopic boundary when brought together, while cells from equivalent positions in two segments heal together without forming a boundary. We consider the segment boundary to be an element in a pattern which reiterates down the longitudinal axis of the body—whenever cells from different positions in this pattern are brought together intercalation occurs. The intercalation can either be within a segment (no boundary forms) or between segments (a boundary forms). The route of intercalation appears to be the shortest available, so that when the apposed cells are more than half a segment length apart a new boundary forms.     

Morphology and ontogeny of the eodiscoid trilobite Sinodiscus changyangensis from the lower Cambrian of South China

Abstract: A large number of complete specimens together with numerous disarticulated sclerites of the eodiscinid trilobite Sinodiscus changyangensis Zhang in Zhou et al., 1977 have been collected from the lower Cambrian Shuijingtuo Formation in Changyang, Hubei Province, South China. An ontogenetic series is established based on the immature and mature exoskeletons including the previously unknown protaspides and meraspides, in particular. No further substages can be differentiated in the protaspid specimens herein. Changes that took place during the meraspid period include the addition of postcephalic segments and prominent pygidial larval notches in early meraspid development which became progressively less distinct and disappeared in degree 2. Two holaspid stages are recognized based on the addition of a new pygidial segment, indicating that the start of the holaspid phase preceded the onset of the epimorphic phase and accordingly, its developmental mode is attributed to the protarthrous pattern. The trunk segmentation schedule of S. changyangensis is discussed, which is similar to other primitive eodiscoid trilobites, that is, as the boundary between the thorax and pygidium migrated posteriorly, there is no change in the number of the trunk segments. The processes of liberation of the thoracic segment and segment insertion into the pygidium are separated from one another, and the two different mechanisms, somitogenesis and tagmosis, progress independently during the ontogenetic development of the postcephalic region of these primitive eodiscinids.     

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.     

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.     

Segment boundary formation in Drosophila embryos

In Drosophila embryos, segment boundaries form at the posterior edge of each stripe of engrailed expression. We have used an HRP-CD2 transgene to follow by transmission electron microscopy the cell shape changes that accompany boundary formation. The first change is a loosening of cell contact at the apical side of cells on either side of the incipient boundary. Then, the engrailed-expressing cells flanking the boundary undergo apical constriction, move inwards and adopt a bottle morphology. Eventually, grooves regress, first on the ventral side, then laterally. We noted that groove formation and regression are contemporaneous with germ band retraction and shortening, respectively, suggesting that these rearrangements could also contribute to groove morphology. The cellular changes accompanying groove formation require that Hedgehog signalling be activated, and, as a result, a target of Ci expressed, at the posterior of each boundary (obvious targets like stripe and rhomboid appear not to be involved). In addition, Engrailed must be expressed at the anterior side of each boundary, even if Hedgehog signalling is artificially maintained. Thus, there are distinct genetic requirements on either side of the boundary. In addition, Wingless signalling at the anterior of the domains of engrailed (and hedgehog) expression represses groove formation and thus ensures that segment boundaries form only at the posterior.     

Regeneration of the nervous and muscular system after caudal amputation in the polychaete <Emphasis Type="Italic">Alitta virens</Emphasis> (Annelida: Nereididae)

Regenerating segments in polychaetes offer a vivid example of epimorphic recovery of the lost organs and tissues. It is also a promising object for studying positional information and the mechanisms maintaining the body integrity. With the aim to develop a convenient standardized model, we described the dynamics of recovery of the major anatomical structures and created a staging system for the caudal regeneration in Alitta virens. In average the normal organization of the posterior body end is restored within 10 days after amputation (dpa). The whole regenerative process was divided into 5 stages: (1) wound healing (0–1 dpa), (2) blastema formation (1–2 dpa), (3) patterning and growth of the blastema (2–3 dpa), (4) differentiation of the first regenerated segment (3–5 dpa), (5) formation and differentiation of the subsequent 5–6 segments (5–10 dpa). The regeneration is carried out mainly by epimorphosis, although the elements of intercalary growth as well as the morphallactic transformation of the stump have been noted. Terminal structures of the pygidium (muscles of the anal sphincter, pygidial cavity, pygidial ring nerve, pygidial cirri) appear at stages 1–3, and then (from stage 3) the formation of new metameres begins in front of the pygidium. Differentiation of the first newborn segment is associated with the tissue remodeling in the last old segment. Formation of the next segments resembles accelerated postlarval growth. The neural elements of the regenerative bud are developing faster than the surrounding muscles. The neurites extending from the CNS and PNS come to the surface of the wound epithelium at stage 1. Later, nerve fibers from the CNS lengthen and thicken along with the growth of the regenerative bud. Ganglion, parapodial nerves, oblique muscles and coeloms of the first segment are detected at stage 4. Longitudinal muscles regenerate in anterior to posterior progression, being constantly in contact with the corresponding fibers of the old tissues. All other muscles differentiate from blastemal cells in isolation from the old musculature of the stump. Our data promote the further using of the posterior body end regeneration in A. virens as an experimental model for resolving crucial problems of developmental biology.     

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.     

Regeneration of segment boundaries in oncopeltus: cell lineage

The segment boundary of Oncopeltus is a straight interface between the cells of two segmental compartments; it coincides with an abrupt change of pigmentation and a groove in the surface of the cuticle. When a segment boundary is burnt it is regenerated by the cells migrating into the wound. We describe the cell lineage of this process: the two masses of migrating cells first contact each other on a convoluted frontier, but this soon straightens. The new boundary forms exactly at the interface between the two cell populations, without a single cell straying across the line. Even when the cautery is asymmetric and, as a consequence, the cells from the different segments meet at an abnormal position, the segment boundary is regenerated at this interface. After extirpation or transplantation ectopic boundaries can be formed within a segment. These new boundaries also act as lineage restrictions and stop mingling of cells across them. Our results support the hypothesis that the integrity and straightness of the compartment boundary depend on cell affinities; the cells in one polyclone adhering more to each other than to the cells of the neighbouring polyclone. There may be a gradient of cell affinity in each segment, with an abrupt change at the segment boundary.     

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 ventral nerve cord signals positional information during segment formation in an annelid (Ophryotrocha puerilis,Polychaeta)

Summary Growth and regeneration of segments were recorded in the polychaeteOphryotrocha puerilis. In one experiment the ventral nerve cords (VNCs) of the animals were cut; in the other, VNCs were left intact. VNC lesion in some specimens resulted in the outgrowth of supernumerary posterior parts from the site of operation. The characteristics of outgrowth of these supernumeraries were essentially the same as in normal specimens without double tails. After removing different numbers of caudal setigers, each of the two tails of the same double-tail monster independently regenerated different segment numbers within a given time. A simple model is proposed, allowing for these results, which states that the larval body of a polychaete consists of two regions with completely different positional values (episphere — prostomium; hyposphere — pygidium). During growth, segments with intervening positional values are intercalated. The rate of segment formation is high when there is a wide gap in positional values between pygidium and adjoining budding zone and the posteriormost segment. As this gap narrows, the growth rate slows down. During caudal regeneration, first of all a new pygidium with an adjacent proliferation zone is formed and the original positional value of the posteriormost part of the body is reestablished. Segment regeneration follows the same rules as segment growth. The results presented here also demonstrate that the VNC plays an important role, not only in segment proliferation, but also in signalling positional information to the newly formed segments.     

A morphological and developmental study of Drosophila embryos ligated during nuclear multiplication

In the Drosophila embryo, determination is established at the cellular blastoderm and a mosaic type development is observed after this time. Before the blastoderm stage, however, development is not of the mosaic type, as ligation during the nuclear multiplication stage causes a change in the spatial organization of the larval pattern. An aberration in determination leads to an increase in segment size, an increase in the number of cells per segment, and a decrease in segment number. This abnormal determination of blastoderm cells has also been demonstrated experimentally by marking corresponding regions of the blastoderm in ligated (posterior fragments only) and nonligated embryos. When the blastoderms of nonligated and ligated embryos are punctured at the same site, ligated embryos produce larvae with damage in segments posterior to the segments damaged in larvae from nonligated embryos. Ultrastructurally, no abnormalities were observed in the plasma membrane at the time of ligation or later in blastoderm cells which formed in the ligation area of these embryos. Evidence from this study, as well as other sources, indicates that determination of segmentation is under maternal control.     

Segmentation in the crustacean Artemia: engrailed staining studied with an antibody raised against the Artemia protein

We have studied the process of post-embryonic segmentation in the anostracan crustacean Artemia franciscana using a specific antibody raised against the engrailed protein of this organism. Three cephalic segments are specified during embryonic development, before larval hatching, whilst trunk (thoracic) segmentation begins after the first stage free-swimming nauplius larva has emerged from the dormant cyst. Thus, cephalic and trunk segmentation seem to be at least in part independent and superimposed processes. Trunk stripes of engrailed expression are added one at a time as segments are generated from the posterior growth zone. The first detectable decision in engrailed expression is the establishment of a line of engrailed-expressing cells, interpretable as delineating the parasegmental boundary. The subsequent widening of engrailed stripes is not correlated with cell lineage events but is probably mediated by the combination of inheritance of the active state and recruitment of new cells into the engrailed-expressing stripe.     

A Stable Thoracic Hox Code and Epimorphosis Characterize Posterior Regeneration in Capitella teleta

Regeneration, the ability to replace lost tissues and body parts following traumatic injury, occurs widely throughout the animal tree of life. Regeneration occurs either by remodeling of pre-existing tissues, through addition of new cells by cell division, or a combination of both. We describe a staging system for posterior regeneration in the annelid, Capitella teleta, and use the C. teleta Hox gene code as markers of regional identity for regenerating tissue along the anterior-posterior axis. Following amputation of different posterior regions of the animal, a blastema forms and by two days, proliferating cells are detected by EdU incorporation, demonstrating that epimorphosis occurs during posterior regeneration of C. teleta. Neurites rapidly extend into the blastema, and gradually become organized into discrete nerves before new ganglia appear approximately seven days after amputation. In situ hybridization shows that seven of the ten Hox genes examined are expressed in the blastema, suggesting roles in patterning the newly forming tissue, although neither spatial nor temporal co-linearity was detected. We hypothesized that following amputation, Hox gene expression in pre-existing segments would be re-organized to scale, and the remaining fragment would express the complete suite of Hox genes. Surprisingly, most Hox genes display stable expression patterns in the ganglia of pre-existing tissue following amputation at multiple axial positions, indicating general stability of segmental identity. However, the three Hox genes, CapI-lox4, CapI-lox2 and CapI-Post2, each shift its anterior expression boundary by one segment, and each shift includes a subset of cells in the ganglia. This expression shift depends upon the axial position of the amputation. In C. teleta, thoracic segments exhibit stable positional identity with limited morphallaxis, in contrast with the extensive body remodeling that occurs during regeneration of some other annelids, planarians and acoel flatworms.     

Controversies Surrounding Segments and Parasegments in Onychophora: Insights from the Expression Patterns of Four “Segment Polarity Genes” in the Peripatopsid Euperipatoides rowelli

Arthropods typically show two types of segmentation: the embryonic parasegments and the adult segments that lie out of register with each other. Such a dual nature of body segmentation has not been described from Onychophora, one of the closest arthropod relatives. Hence, it is unclear whether onychophorans have segments, parasegments, or both, and which of these features was present in the last common ancestor of Onychophora and Arthropoda. To address this issue, we analysed the expression patterns of the “segment polarity genes” engrailed, cubitus interruptus, wingless and hedgehog in embryos of the onychophoran Euperipatoides rowelli. Our data revealed that these genes are expressed in repeated sets with a specific anterior-to-posterior order along the body in embryos of E. rowelli. In contrast to arthropods, the expression occurs after the segmental boundaries have formed. Moreover, the initial segmental furrow retains its position within the engrailed domain throughout development, whereas no new furrow is formed posterior to this domain. This suggests that no re-segmentation of the embryo occurs in E. rowelli. Irrespective of whether or not there is a morphological or genetic manifestation of parasegments in Onychophora, our data clearly show that parasegments, even if present, cannot be regarded as the initial metameric units of the onychophoran embryo, because the expression of key genes that define the parasegmental boundaries in arthropods occurs after the segmental boundaries have formed. This is in contrast to arthropods, in which parasegments rather than segments are the initial metameric units of the embryo. Our data further revealed that the expression patterns of “segment polarity genes” correspond to organogenesis rather than segment formation. This is in line with the concept of segmentation as a result of concerted evolution of individual periodic structures rather than with the interpretation of ‘segments’ as holistic units.     

Biostratinomy and functional morphology of enrollment in two Middle Devonian trilobites

Although it is common knowledge that many trilobites enrolled, behavioral and functional aspects of enrollment are not at all well understood. Taphonomic details portrayed by enrolled trilobites in the Middle Devonian Hamilton Group (New York State) indicate that enrollment was a complex and morphologically constrained behavior. The trilobites Phacops rana (Green) and Greenops boothi (Green) are frequently enrolled in Hamilton strata; biostratinomic data indicate two very different enrollment postures. Interlocking morphologies (coaptative devices) and apodeme structure and disposition indicate that these postures reflect specific behaviors which involved interaction between tergal structures, inferred musculature, and the substratum. Phacops enrolled by burrowing forward and down into the sediment; dorsal muscles, attached to prominent articulating half-rings, imbricated the thorax such that each lappet overlapped the next posterior segment and locked into a posterior pleural facet. The pygidium was brought into place as the posterior segments of the thorax were placed into vincular notches along the lateral margin of the ventral cephalon. The pygidium locked with the cephalic vincular furrow to complete ‘perfect sphaeroidal’ closure. Greenops enrolled with the cephalon in an upright position at the sediment surface; a submarginal furrow on the ventral surface of the pygidium received the anterior rim of the cephalon. Relatively narrow articulating half-rings limited pleural rotation. Segments were loosely locked into narrow facets at the anterior margin of the next posterior lappet. In spite of rudimentary lappet and half-ring structures, Greenops displays an elaborate system of thoracopygidial muscles which involved dorsoventral and longitudinal attachments along the thorax and into the pygidium. Phacops, in contrast, displays very poorly developed apodemes which occur in the thorax only. Longitudinal muscle strength was likely less important during Phacops enrollment than is evident for the Greenops enrollment procedure. Conversely, Phacops clearly relied to a great degree upon competent closure devices which are poorly developed in Greenops. Biostratinomic data reveal different enrollment behaviors which reflect the function of different enrollment-related morphologies present in each species.