The function of Notch signalling in segment formation in the crustacean Daphnia magna (Branchiopoda)

Ten years ago we showed for the first time that Notch signalling is required in segmentation in spiders, indicating the existence of similar mechanisms in arthropod and vertebrate segmentation. However, conflicting results in various arthropod groups hampered our understanding of the ancestral function of Notch in arthropod segmentation. Here we fill a crucial data gap in arthropods and analyse segmentation in a crustacean embryo. We analyse the expression of homologues of the Drosophila and vertebrate segmentation genes and show that members of the Notch signalling pathway are expressed at the same time as the pair-rule genes. Furthermore, inactivation of Notch signalling results in irregular boundaries of the odd-skipped-like expression domains and affects the formation of segments. In severe cases embryos appear unsegmented. We suggest two scenarios for the function of Notch signalling in segmentation. The first scenario agrees with a segmentation clock involving Notch signalling, while the second scenario discusses an alternative mechanism of Notch function which is integrated into a hierarchical segmentation cascade.     

Decoupling elongation and segmentation: Notch involvement in anostracan crustacean segmentation

Repeated body segments are a key feature of arthropods. The formation of body segments occurs via distinct developmental pathways within different arthropod clades. Although some species form their segments simultaneously without any accompanying measurable growth, most arthropods add segments sequentially from the posterior of the growing embryo or larva. The use of Notch signaling is increasingly emerging as a common feature of sequential segmentation throughout the Bilateria, as inferred from both the expression of proteins required for Notch signaling and the genetic or pharmacological disruption of Notch signaling. In this study, we demonstrate that blocking Notch signaling by blocking γ‐secretase activity causes a specific, repeatable effect on segmentation in two different anostracan crustaceans, Artemia franciscana and Thamnocephalus platyurus. We observe that segmentation posterior to the third or fourth trunk segment is arrested. Despite this marked effect on segment addition, other aspects of segmentation are unaffected. In the segments that develop, segment size and boundaries between segments appear normal, engrailed stripes are normal in size and alignment, and overall growth is unaffected. By demonstrating Notch involvement in crustacean segmentation, our findings expand the evidence that Notch plays a crucial role in sequential segmentation in arthropods. At the same time, our observations contribute to an emerging picture that loss‐of‐function Notch phenotypes differ significantly between arthropods suggesting variability in the role of Notch in the regulation of sequential segmentation. This variability in the function of Notch in arthropod segmentation confounds inferences of homology with vertebrates and lophotrochozoans.     

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.     

Ancestral functions of Delta/Notch signaling in the formation of body and leg segments in the cricket Gryllus bimaculatus

Delta/Notch signaling controls a wide spectrum of developmental processes, including body and leg segmentation in arthropods. The various functions of Delta/Notch signaling vary among species. For instance, in Cupiennius spiders, Delta/Notch signaling is essential for body and leg segmentation, whereas in Drosophila fruit flies it is involved in leg segmentation but not body segmentation. Therefore, to gain further insight into the functional evolution of Delta/Notch signaling in arthropod body and leg segmentation, we analyzed the function of the Delta (Gb'Delta) and Notch (Gb'Notch) genes in the hemimetabolous, intermediate-germ cricket Gryllus bimaculatus. We found that Gb'Delta and Gb'Notch were expressed in developing legs, and that RNAi silencing of Gb'Notch resulted in a marked reduction in leg length with a loss of joints. Our results suggest that the role of Notch signaling in leg segmentation is conserved in hemimetabolous insects. Furthermore, we found that Gb'Delta was expressed transiently in the posterior growth zone of the germband and in segmental stripes earlier than the appearance of wingless segmental stripes, whereas Gb'Notch was uniformly expressed in early germbands. RNAi knockdown of Gb'Delta or Gb'Notch expression resulted in malformation in body segments and a loss of posterior segments, the latter probably due to a defect in posterior growth. Therefore, in the cricket, Delta/Notch signaling might be required for proper morphogenesis of body segments and posterior elongation, but not for specification of segment boundaries.     

Delta-Notch signalling in segmentation

Modular body organization is found widely across multicellular organisms, and some of them form repetitive modular structures via the process of segmentation. It's vastly interesting to understand how these regularly repeated structures are robustly generated from the underlying noise in biomolecular interactions. Recent studies from arthropods reveal similarities in segmentation mechanisms with vertebrates, and raise the possibility that the three phylogenetic clades, annelids, arthropods and chordates, might share homology in this process from a bilaterian ancestor. Here, we discuss vertebrate segmentation with particular emphasis on the role of the Notch intercellular signalling pathway. We introduce vertebrate segmentation and Notch signalling, pointing out historical milestones, then describe existing models for the Notch pathway in the synchronization of noisy neighbouring oscillators, and a new role in the modulation of gene expression wave patterns. We ask what functions Notch signalling may have in arthropod segmentation and explore the relationship between Notch-mediated lateral inhibition and synchronization. Finally, we propose open questions and technical challenges to guide future investigations into Notch signalling in 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.     

Unravelling the evolution of neural stem cells in arthropods: Notch signalling in neural stem cell development in the crustacean Daphnia magna

The genetic regulatory networks controlling major developmental processes seem to be conserved in bilaterians regardless of an independent or a common origin of the structures. This has been explained by the employment of a genetic toolkit that was repeatedly used during bilaterian evolution to build the various forms and body plans. However, it is not clear how genetic networks were incorporated into the formation of novel structures and how homologous genes can regulate the disparate morphological processes. Here we address this question by analysing the role of Notch signalling, which is part of the bilaterian toolkit, in neural stem cell evolution in arthropods. Within arthropods neural stem cells have evolved in the last common ancestor of insects and crustaceans (Tetraconata). We analyse here for the first time the role of Notch signalling in a crustacean, the branchiopod Daphnia magna, and show that it is required in neural stem cells for regulating the time of neural precursor production and for binary cell fate decisions in the ventral neuroectoderm. The function of Notch signalling has diverged in the ventral neuroectoderm of insects and crustaceans accompanied by changes in the morphogenetic processes. In the crustacean, Notch controlled mechanisms of neuroblast regulation have evolved that are surprisingly similar to vertebrates and thus present a remarkable case of parallel evolution. These new data on a representative of crustaceans complete the arthropod data set on Notch signalling in the nervous system and allow for reconstructing how the Notch signalling pathway has been co-opted from pre-existing structures to the development of the evolving neural stem cells in the Tetraconata ancestor.     

Notch-mediated segmentation and growth control of the Drosophila leg.

The possession of segmented appendages is a defining characteristic of the arthropods. By analyzing both loss-of-function and ectopic expression experiments, we show that the Notch signaling pathway plays a fundamental role in the segmentation and growth of the Drosophila leg. Local activation of Notch is necessary and sufficient to promote the formation of joints between segments. This segmentation process requires the participation of the Notch ligands, Serrate and Delta, as well as Fringe. These three proteins are each expressed in the developing leg and antennal imaginal discs in a segmentally repeated pattern that is regulated downstream of the action of Wingless and Decapentaplegic. Our studies further show that Notch activation is both necessary and sufficient to promote leg growth. We also identify target genes regulated both positively and negatively downstream of Notch signaling that are required for normal leg development. Together, these observations outline a regulatory hierarchy for the segmentation and growth of the leg. The Notch pathway is also deployed for segmentation during vertebrate somitogenesis, which raises the possibility of a common origin for the segmentation of these distinct tissues.     

Notch-Mediated Segmentation and Growth Control of the Drosophila Leg

The possession of segmented appendages is a defining characteristic of the arthropods. By analyzing both loss-of-function and ectopic expression experiments, we show that the Notch signaling pathway plays a fundamental role in the segmentation and growth of the Drosophila leg. Local activation of Notch is necessary and sufficient to promote the formation of joints between segments. This segmentation process requires the participation of the Notch ligands, Serrate and Delta, as well as Fringe. These three proteins are each expressed in the developing leg and antennal imaginal discs in a segmentally repeated pattern that is regulated downstream of the action of Wingless and Decapentaplegic. Our studies further show that Notch activation is both necessary and sufficient to promote leg growth. We also identify target genes regulated both positively and negatively downstream of Notch signaling that are required for normal leg development. Together, these observations outline a regulatory hierarchy for the segmentation and growth of the leg. The Notch pathway is also deployed for segmentation during vertebrate somitogenesis, which raises the possibility of a common origin for the segmentation of these distinct tissues.     

The functional relationship between ectodermal and mesodermal segmentation in the crustacean, Parhyale hawaiensis

In arthropods, annelids and chordates, segmentation of the body axis encompasses both ectodermal and mesodermal derivatives. In vertebrates, trunk mesoderm segments autonomously and induces segmental arrangement of the ectoderm-derived nervous system. In contrast, in the arthropod Drosophila melanogaster, the ectoderm segments autonomously and mesoderm segmentation is at least partially dependent on the ectoderm. While segmentation has been proposed to be a feature of the common ancestor of vertebrates and arthropods, considering vertebrates and Drosophila alone, it is impossible to conclude whether the ancestral primary segmented tissue was the ectoderm or the mesoderm. Furthermore, much of Drosophila segmentation occurs before gastrulation and thus may not accurately represent the mechanisms of segmentation in all arthropods. To better understand the relationship between segmented germ layers in arthropods, we asked whether segmentation is an intrinsic property of the ectoderm and/or the mesoderm in the crustacean Parhyale hawaiensis by ablating either the ectoderm or the mesoderm and then assaying for segmentation in the remaining tissue layer. We found that the ectoderm segments autonomously. However, mesoderm segmentation requires at least a permissive signal from the ectoderm. Although mesodermal stem cells undergo normal rounds of division in the absence of ectoderm, they do not migrate properly in respect to migration direction and distance. In addition, their progeny neither divide nor express the mesoderm segmentation markers Ph-twist and Ph-Even-skipped. As segmentation is ectoderm-dependent in both Parhyale and holometabola insects, we hypothesize that segmentation is primarily a property of the ectoderm in pancrustacea.     

Composite signalling from Serrate and Delta establishes leg segments in Drosophila through Notch.

The receptor protein NOTCH and its ligands SERRATE and DELTA are involved in many developmental processes in invertebrates and vertebrates alike. Here we show that the expression of the Serrate and Delta genes patterns the segments of the leg in Drosophila by a combination of their signalling activities. Coincident stripes of Serrate and Delta expressing cells activate Enhancer of split expression in adjacent cells through Notch signalling. These cells form a patterning boundary from which a putative secondary signal leads to the development of leg joints. Elsewhere in the tarsal segments, signalling by DELTA and NOTCH is necessary for the development of non-joint parts of the leg. We propose that these two effects result from different thresholds of NOTCH activation, which are translated into different downstream gene expression effects. We propose a general mechanism for creation of boundaries by Notch signalling.     

Suppressor of Hairless and Presenilin phenotypes imply involvement of canonical Notch-signalling in segmentation of the spider Cupiennius salei

Arthropods, vertebrates, and annelids all have a segmented body. Our recent discovery of involvement of Notch-signalling in spider segmentation revived the discussion on the origin of segmented body plans and suggests the sharing of a common genetic program in a common ancestor. Here, we analysed the spider homologues of the Suppressor of Hairless and Presenilin genes, which encode components of the canonical Notch-pathway, to further explore the role of Notch-signalling in spider segmentation. RNAi silencing of two spider Suppressor of Hairless homologues and the spider Presenilin homologue causes severe segmentation phenotypes. The most prominent defect is the consistent breakdown of segmentation after the formation of three (Suppressor of Hairless) or five (Presenilin) opisthosomal segments. These phenotypes indicate that Notch-signalling during spider segmentation likely involves the canonical pathway via Presenilin and Suppressor of Hairless. Furthermore, it implies that Notch-signalling influences both the formation and patterning of the spider segments: it is required for the specification of the posterior segments and for proper specification of the segment boundaries. We argue that alternative, partly redundant, pathways might act in the formation of the anterior segments that are not active in the posterior segments. This suggests that at least some differences exist in the specification of anterior and posterior segments of the spider, a finding that may be valid for most short germ arthropods. Our data provide additional evidence for the similarities of Notch-signalling in spider segmentation and vertebrate somitogenesis and strengthen our previous notion that the formation of the segments in arthropods and vertebrates might have shared a genetic program in a common ancestor.     

The centipede Strigamia maritima: what it can tell us about the development and evolution of segmentation

One of the most fundamental features of the body plan of arthropods is its segmental design. There is considerable variation in segment number among arthropod groups (about 20-fold); yet, paradoxically, the vast majority of arthropod species have a fixed number of segments, thus providing no variation in this character for natural selection to act upon. However, the 1000-species-strong centipede order Geophilomorpha provides an exception to the general rule of intraspecific invariance in segment number. Members of this group, and especially our favourite animal Strigamia maritima, may thus help us to understand the evolution of segment number in arthropods. Evolution must act by modifying the formation of segments during embryogenesis. So, how this developmental process operates, in a variable-segment-number species, is of considerable interest. Strigamia maritima turns out to be a tractable system both at the ecological level of investigating differences in mean segment number between populations and at the molecular level of studying the expression patterns of developmental genes. Here we report the current state of play in our work on this fascinating animal, including our recent finding of a double-segment periodicity in the expression of two Strigamia segmentation genes, and its possible implications for our understanding of arthropod segmentation mechanisms in general.     

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 ancestry of segmentation

Recent studies of segmentation in the spider suggest that the ancestral vertebrate and arthropod segmentation mechanisms utilized the Notch signaling pathway and bolster the argument that segmentation is an ancestral feature of all bilaterians.     

Notch-mediated segmentation of the appendages is a molecular phylotypic trait of the arthropods

Arthropod limbs are arguably the most diverse organs in the animal kingdom. Morphological diversity of the limbs is largely based on their segmentation, because this divides the limbs into modules that can evolve separately for new morphologies and functions. Limb segmentation also distinguishes the arthropods from related phyla (e.g. onychophorans) and thus forms an important evolutionary innovation in arthropods. Understanding the genetic basis of limb segmentation in arthropods can thus shed light onto the mechanisms of macroevolution and the origin of a character (articulated limbs) that defines a new phylum (arthropods). In the fly Drosophila limb segmentation and limb growth are controlled by the Notch signaling pathway. Here we show that the Notch pathway also controls limb segmentation and growth in the spider Cupiennius salei, a representative of the most basally branching arthropod group Chelicerata, and thus this function must trace from the last common ancestor of all arthropods. The similarities of Notch and Serrate function between Drosophila and Cupiennius are extensive and also extend to target genes like odd-skipped, nubbin, AP-2 and hairy related genes. Our data confirm that the jointed appendages, which are a morphological phylotypic trait of the arthropods and the basis for naming the phylum, have a common developmental genetic basis. Notch-mediated limb segmentation is thus a molecular phylotypic trait of the arthropods.     

Parallels between the proximal–distal development of vertebrate and arthropod appendages: homology without an ancestor?

Evolutionary studies suggest that the limbs of vertebrates and the appendages of arthropods do not share a common origin. However, recent genetic studies show new similarities in their developmental programmes. These similarities might be caused by the independent recruitment of homologous genes for similar functions or by the conservation of an ancestral proximal-distal development programme. This basic programme might have arisen in an ancestral outgrowth and been independently co-opted in vertebrate and arthropod appendages. It has subsequently diverged in both phyla to fine-pattern the limb and to control phylum-specific cellular events. We suggest that although vertebrate limbs and arthropod appendages are not strictly homologous structures they retain remnants of a common ancestral developmental programme.     

Expression of Pax group III genes suggests a single-segmental periodicity for opisthosomal segment patterning in the spider Cupiennius salei

Pair-rule patterning forms a key step for segmentation in insects. The expression patterns of pair-rule gene orthologs in representatives of other arthropod groups imply that these genes were segmentation genes in the last common ancestor of the various arthropod groups, but almost nothing is known about the underlying mechanism in noninsect arthropods. Here, we cloned and analyzed members of the Pax group III genes from the spider Cupiennius salei. Pax group III genes comprise genes like the Drosophila genes paired, gooseberry, and gooseberry-neuro, as well as the vertebrate Pax 3 and Pax 7 genes. We recovered three Pax group III genes from the spider C. salei, Cs-pairberry-1, Cs-pairberry-2, and Cs-pairberry-3, and show that the combined expression of the three spider genes mimics the patterns in insects, suggesting an ancestral role for Pax group III genes in segmentation, neurogenesis, and appendage formation in arthropods. One of the genes, pairberry-3, is expressed in a segmental periodicity before overt morphological segmentation is visible, suggesting a single segmental periodicity for opisthosomal segment pattering in the spider. Comparisons among arthropods suggest that the underlying mechanisms for pair-rule gene orthologs are more diverged than the ones for the segment-polarity genes. We argue that there may be a correlation between the lower variation in patterns of segment-polarity genes and the phylotypic stage. The segment-polarity genes are required to define the segment borders of the embryo at the germ-band stage, the arthropod phylotypic stage. Pair-rule gene orthologs act more upstream and may display more variation in their action.     

Characterization of Notch-class gene expression in segmentation stem cells and segment founder cells in Helobdella robusta (Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae)

To understand the evolution of segmentation, we must compare segmentation in all three major groups of eusegmented animals: vertebrates, arthropods, and annelids. The leech Helobdella robusta is an experimentally tractable annelid representative, which makes segments in anteroposterior progression from a posterior growth zone consisting of 10 identified stem cells. In vertebrates and some arthropods, Notch signaling is required for normal segmentation and functions via regulation of hes-class genes. We have previously characterized the expression of an hes-class gene (Hro-hes) during segmentation in Helobdella, and here, we characterize the expression of an H. robusta notch homolog (Hro-notch) during this process. We find that Hro-notch is transcribed in the segmental founder cells (blast cells) and their stem-cell precursors (teloblasts), as well as in other nonsegmental tissues. The mesodermal and ectodermal lineages show clear differences in the levels of Hro-notch expression. Finally, Hro-notch is shown to be inherited by newly born segmental founder cells as well as transcribed by them before their first cell division.