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.     

Notch Is a Critical Component of the Mouse Somitogenesis Oscillator and Is Essential for the Formation of the Somites

Segmentation of the vertebrate body axis is initiated through somitogenesis, whereby epithelial somites bud off in pairs periodically from the rostral end of the unsegmented presomitic mesoderm (PSM). The periodicity of somitogenesis is governed by a molecular oscillator that drives periodic waves of clock gene expression caudo-rostrally through the PSM with a periodicity that matches somite formation. To date the clock genes comprise components of the Notch, Wnt, and FGF pathways. The literature contains controversial reports as to the absolute role(s) of Notch signalling during the process of somite formation. Recent data in the zebrafish have suggested that the only role of Notch signalling is to synchronise clock gene oscillations across the PSM and that somite formation can continue in the absence of Notch activity. However, it is not clear in the mouse if an FGF/Wnt-based oscillator is sufficient to generate segmented structures, such as the somites, in the absence of all Notch activity. We have investigated the requirement for Notch signalling in the mouse somitogenesis clock by analysing embryos carrying a mutation in different components of the Notch pathway, such as Lunatic fringe (Lfng), Hes7, Rbpj, and presenilin1/presenilin2 (Psen1/Psen2), and by pharmacological blocking of the Notch pathway. In contrast to the fish studies, we show that mouse embryos lacking all Notch activity do not show oscillatory activity, as evidenced by the absence of waves of clock gene expression across the PSM, and they do not develop somites. We propose that, at least in the mouse embryo, Notch activity is absolutely essential for the formation of a segmented body axis.     

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.     

Zebrafish segmentation and pair-rule patterning

Segmentation in the vertebrate embryo is evident within the paraxial mesoderm in the form of somites, which are repeated structures that give rise to the vertebrae and muscle of the trunk and tail. In the zebrafish, our genetic screen identified two groups of mutants that affect somite formation and pattern. Mutations of one class, the fss-type mutants, disrupt the formation of the anterior-posterior somite boundaries during somitogenesis. However, segmentation within the paraxial mesoderm is not completely eliminated in these mutants. Irregular somite boundaries form later during embryogenesis and, strikingly, the vertebrae are not fused. Here, we show that formation of the irregular somite boundaries in these mutants is dependent upon the activity of a second group of genes, the you-type genes, which include sonic you, the zebrafish homologue of the Drosophila segment polarity gene, sonic hedgehog. Further to characterize the defects caused by the fss-type mutations, we examined their effects on the expression of her1, a zebrafish homologue of the Drosophila pair-rule gene hairy. In wild-type embryos, her1 is expressed in a dynamic, repeating pattern, remarkably similar to that of its Drosophila and Tribolium counterparts, suggesting that a pair-rule mechanism also functions in the segmentation of the vertebrate paraxial mesoderm. We have found that the fss-type mutants have abnormal pair-rule patterning. Although a her1 mutant could not be identified, analysis of a double mutant that abolishes most her1 expression suggests that a her1 mutant may not display a pair-rule phenotype analogous to the hairy phenotype observed in Drosophila. Cumulatively, our data indicate that zebrafish homologues of both the Drosophila segment polarity genes and pair-rule genes are involved in segmenting the paraxial mesoderm. However, both the relationship between these two groups of genes within the genetic heirarchy governing segmentation and the precise roles that they play during segmentation likely differ significantly between the two organisms. Dev. Genet. 23:65–76, 1998. © 1998 Wiley-Liss, Inc.     

Onset of the segmentation clock in the chick embryo: evidence for oscillations in the somite precursors in the primitive streak

Vertebrate somitogenesis is associated with a molecular oscillator, the segmentation clock, which is defined by the periodic expression of genes related to the Notch pathway such as hairy1 and hairy2 or lunatic fringe (referred to as the cyclic genes) in the presomitic mesoderm (PSM). Whereas earlier studies describing the periodic expression of these genes have essentially focussed on later stages of somitogenesis, we have analysed the onset of the dynamic expression of these genes during chick gastrulation until formation of the first somite. We observed that the onset of the dynamic expression of the cyclic genes in chick correlated with ingression of the paraxial mesoderm territory from the epiblast into the primitive streak. Production of the paraxial mesoderm from the primitive streak is a continuous process starting with head mesoderm formation, while the streak is still extending rostrally, followed by somitic mesoderm production when the streak begins its regression. We show that head mesoderm formation is associated with only two pulses of cyclic gene expression. Because such pulses are associated with segment production at the body level, it suggests the existence of, at most, two segments in the head mesoderm. This is in marked contrast to classical models of head segmentation that propose the existence of more than five segments. Furthermore, oscillations of the cyclic genes are seen in the rostral primitive streak, which contains stem cells from which the entire paraxial mesoderm originates. This indicates that the number of oscillations experienced by somitic cells is correlated with their position along the AP axis.     

Differential Axial Requirements for Lunatic Fringe and Hes7 Transcription during Mouse Somitogenesis

Vertebrate segmentation is regulated by the “segmentation clock”, which drives cyclic expression of several genes in the caudal presomitic mesoderm (PSM). One such gene is Lunatic fringe (Lfng), which encodes a modifier of Notch signalling, and which is also expressed in a stripe at the cranial end of the PSM, adjacent to the newly forming somite border. We have investigated the functional requirements for these modes of Lfng expression during somitogenesis by generating mice in which Lfng is expressed in the cranial stripe but strongly reduced in the caudal PSM, and find that requirements for Lfng activity alter during axial growth. Formation of cervical, thoracic and lumbar somites/vertebrae, but not sacral and adjacent tail somites/vertebrae, depends on caudal, cyclic Lfng expression. Indeed, the sacral region segments normally in the complete absence of Lfng and shows a reduced requirement for another oscillating gene, Hes7, indicating that the architecture of the clock alters as segmentation progresses. We present evidence that Lfng controls dorsal-ventral axis specification in the tail, and also suggest that Lfng controls the expression or activity of a long-range signal that regulates axial extension.     

Homeotic gene expression in the locust Schistocerca: An antibody that detects conserved epitopes in ultrabithorax and abdominal-A proteins

To investigate what role homeotic genes may play in morphological evolution, we are comparing homeotic gene expression in two very different insects, Drosophila (Diptera) and Schistocerca (Orthoptera). In this paper we describe a monoclonal antibody, FP6.87, that recognizes the products of both the Ultrabithorax (Ubx) and abdominal-A (abd-A) genes in Drosophila, via an epitope common to the carboxy terminal region of these two proteins. This antibody recognizes nuclear antigens present in the posterior thorax and abdomen of Schistocerca. We infer that it recognizes the Schistocerca homolog of UBX protein, and probably also of ABD-A. As the distribution of Schistocerca ABD-A protein is already known, we can use this reagent to map the expression of Schistocerca UBX in the thorax and anterior abdomen, where ABD-A is not expressed. Both the general domain, and many of the details, of UBX exp ression are remarkably conserved compared with Drosophila. Thus UBX expression extends back from T2 in the ectoderm (including the CNS), but only from A1 in the mesoderm. As noted for other bithorax complex genes in Schistocerca, expression begins in the abdomen, at or shortly before the time of segmentation. It only later spreads anteriorly to the thorax. For much of embryogene-sis, the expression of UBX in the thoracic epidermis is largely restricted to the T3 limb. Inthis limb, UBX is strikingly regulated, in a complex pattern that reflects limb segmentation. Reviewing these and earlier observations, we conclude that evolutionary changes affect both the precise regulation of homeotic genes within segments, and probably also the spectrum of downstream genes that respond to homeotic gene expression in a given tissue. Overall domains of homeotic gene expression appear to be well conserved between different insect groups, though a change in the extent and timing of homeotic gene expression may underlie the modification of the posterior abdomen in different insect groups. © 1994 Wiley-Liss, Inc.     

Cyclic expression of esr9 gene in Xenopus presomitic mesoderm

During somitogenesis, the cycling expression of members of the Notch signalling cascade is involved in a segmentation clock that regulates the periodic budding of somites in chicken, mouse, and zebrafish. In frog, genes with cycling expression in the presomitic mesoderm have not been reported. Here, we describe the expression of Xenopus esr9 and esr10, two new members of the Hairy/Enhancer of split related family of bHLH proteins. We show that they are expressed in a highly dynamic fashion, with their mRNA levels oscillating periodically in the presomitic mesoderm during somitogenesis. This dynamic expression is independent of de novo protein synthesis. Thus, expression of esr9 and esr10 is an indicator of the segmentation clock in the amphibian embryo. This confirms the evolutionary conservation of a molecular pathway involved in vertebrate segmentation clock.     

Notch signalling synchronizes the zebrafish segmentation clock but is not needed to create somite boundaries

Somite segmentation depends on a gene expression oscillator or clock in the posterior presomitic mesoderm (PSM) and on read-out machinery in the anterior PSM to convert the pattern of clock phases into a somite pattern. Notch pathway mutations disrupt somitogenesis, and previous studies have suggested that Notch signalling is required both for the oscillations and for the read-out mechanism. By blocking or overactivating the Notch pathway abruptly at different times, we show that Notch signalling has no essential function in the anterior PSM and is required only in the posterior PSM, where it keeps the oscillations of neighbouring cells synchronized. Using a GFP reporter for the oscillator gene her1, we measure the influence of Notch signalling on her1 expression and show by mathematical modelling that this is sufficient for synchronization. Our model, in which intracellular oscillations are generated by delayed autoinhibition of her1 and her7 and synchronized by Notch signalling, explains the observations fully, showing that there are no grounds to invoke any additional role for the Notch pathway in the patterning of somite boundaries in zebrafish.     

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.     

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.     

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.     

Synchronized oscillation of the segmentation clock gene in vertebrate development

In vertebrate somitogenesis, “segmentation clock” genes (her in zebrafish, hes in mouse, and hairy in chick) show oscillation, synchronized over nearby cells through intercellular interaction. In zebrafish, neighboring cells interact by Delta-Notch signaling to realize synchronization. Under Delta-Notch, however, a cell with a high expression of the segmentation clock gene tends to suppress its expression in adjacent cells, which might produce spatial heterogeneity instead of synchronized oscillation. Here we studied the conditions under which pre-somitic mesoderm cells show synchronized oscillation of gene expression mathematically. We adopted a model that explicitly considers the kinetics of the mRNA and proteins of the segmentation clock gene and cell–cell interaction via Delta-Notch signaling. From statistical study of a model with randomly generated parameters, we revealed how the likelihood that the system generates stable synchronized oscillation depends on the rate of each reaction in the gene–protein kinetics.     

The role of presenilin 1 during somite segmentation

The Notch signalling pathway plays essential roles during the specification of the rostral and caudal somite halves and subsequent segmentation of the paraxial mesoderm. We have re-investigated the role of presenilin 1 (Ps1; encoded by Psen1) during segmentation using newly generated alleles of the Psen1 mutation. In Psen1-deficient mice, proteolytic activation of Notch1 was significantly affected and the expression of several genes involved in the Notch signalling pathway was altered, including Delta-like3, Hes5, lunatic fringe (Lfng) and Mesp2. Thus, Ps1-dependent activation of the Notch pathway is essential for caudal half somite development. We observed defects in Notch signalling in both the caudal and rostral region of the presomitic mesoderm. In the caudal presomitic mesoderm, Ps1 was involved in maintaining the amplitude of cyclic activation of the Notch pathway, as represented by significant reduction of Lfng expression in Psen1-deficient mice. In the rostral presomitic mesoderm, rapid downregulation of the Mesp2 expression in the presumptive caudal half somite depends on Ps1 and is a prerequisite for caudal somite half specification. Chimaera analysis between Psen1-deficient and wild-type cells revealed that condensation of the wild-type cells in the caudal half somite was concordant with the formation of segment boundaries, while mutant and wild-type cells intermingled in the presomitic mesoderm. This implies that periodic activation of the Notch pathway in the presomitic mesoderm is still latent to segregate the presumptive rostral and caudal somite. A transient episode of Mesp2 expression might be needed for Notch activation by Ps1 to confer rostral or caudal properties. In summary, we propose that Ps1 is involved in the functional manifestation of the segmentation clock in the presomitic mesoderm.     

Three novel Notch genes in zebrafish: implications for vertebrate Notch gene evolution and function

 Notch genes encode transmembrane receptors that interact with numerous signal transduction pathways and are essential for animal development. To facilitate analysis of vertebrate Notch gene function, we isolated cDNA fragments of three novel Notch genes from zebrafish (Danio rerio), Notch1b, Notch5 and Notch6. Notch1b is a second zebrafish Notch1 gene. From analysis of the Notch1b sequence we argue that the various vertebrate Notch gene subfamilies encode receptors with different signalling specificities. Notch5 and Notch6 represent novel vertebrate Notch gene subfamilies. Remarkably, Notch1b lacks expression in presomitic mesoderm, Notch5 is expressed in a metameric pattern within the presomitic mesoderm whilst Notch6 expression is excluded from the nervous system. The expression patterns of these genes suggest important roles in gastrulation, somitogenesis, tail bud extension, myogenesis, heart development and neurogenesis. We discuss the implications of our observations for Notch gene evolution and function. Received: 20 January 1997 / Accepted: 12 February 1997     

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.     

Vertebrate segmentation: is cycling the rule?

Vertebrate segmentation initiates with the subdivision of the paraxial mesoderm into a regular array of somites. Recent evidence suggests that the segmentation clock - a biochemical oscillator acting in the unsegmented paraxial mesoderm cells in most vertebrates - controls cyclic Notch signalling, resulting in periodic formation of somite boundaries.     

Notch around the clock.

The establishment of a segmental pattern within the vertebrate body plan is achieved during embryogenesis by the somitogenesis process. Two molecular systems have been implicated in this phenomenon: a molecular clock linked to vertebrate segmentation and the Notch signalling pathway. Rhythmic expression of the Lunatic Fringe gene in the presomitic mesoderm has now provided a link between these two systems.     

Balancing segmentation and laterality during vertebrate development

Somites are the mesodermal segments of vertebrate embryos that become the vertebral column, skeletal muscle and dermis. Somites arise within the paraxial mesoderm by the periodic, bilaterally symmetric process of somitogenesis. However, specification of left–right asymmetry occurs in close spatial and temporal proximity to somitogenesis and involves some of the same cell signaling pathways that govern segmentation. Here, we review recent evidence that identifies cross-talk between these processes and that demonstrates a role for retinoic acid in maintaining symmetrical somitogenesis by preventing impingement of left–right patterning signals upon the paraxial mesoderm.