Oscillatory lunatic fringe activity is crucial for segmentation of the anterior but not posterior skeleton

The Notch pathway plays multiple roles during vertebrate somitogenesis, functioning in the segmentation clock and during rostral/caudal (R/C) somite patterning. Lunatic fringe (Lfng) encodes a glycosyltransferase that modulates Notch signaling, and its expression patterns suggest roles in both of these processes. To dissect the roles played by Lfng during somitogenesis, a novel allele was established that lacks cyclic Lfng expression within the segmentation clock, but that maintains expression during R/C somite patterning (Lfng(DeltaFCE1)). In the absence of oscillatory Lfng expression, Notch activation is ubiquitous in the PSM of Lfng(DeltaFCE1) embryos. Lfng(DeltaFCE1) mice exhibit severe segmentation phenotypes in the thoracic and lumbar skeleton. However, the sacral and tail vertebrae are only minimally affected in Lfng(DeltaFCE1) mice, suggesting that oscillatory Lfng expression and cyclic Notch activation are important in the segmentation of the thoracic and lumbar axial skeleton (primary body formation), but are largely dispensable for the development of sacral and tail vertebrae (secondary body formation). Furthermore, we find that the loss of cyclic Lfng has distinct effects on the expression of other clock genes during these two stages of development. Finally, we find that Lfng(DeltaFCE1) embryos undergo relatively normal R/C somite patterning, confirming that Lfng roles in the segmentation clock are distinct from its functions in somite patterning. These results suggest that the segmentation clock may employ varied regulatory mechanisms during distinct stages of anterior/posterior axis development, and uncover previously unappreciated connections between the segmentation clock, and the processes of primary and secondary body formation.     

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.     

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.     

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.     

Hopf bifurcation in the presomitic mesoderm during the mouse segmentation

Vertebrae and ribs arise from embryonic tissues called somites. Somites arise sequentially from the unsegmented embryo tail, called presomitic mesoderm (PSM). The pace of somite formation is controlled by gene products such as hairy and enhancer of split 7 (Hes7) whose expression oscillates in the PSM. In addition to the cyclic genes, there is a gradient of fibroblast growth factor 8 (Fgf8) mRNA from posterior to anterior PSM. Recent experiments have shown that in the absence of Fgf signaling, Hes7 oscillations in the anterior and posterior PSM are lost. On the other hand, Notch mutants reduce the amplitude of posterior Hes7 oscillations and abolish anterior Hes7 oscillations. To understand these phenotypes, we delineated and simulated a logical and a delay differential equation (DDE) model with similar network topology in wild-type and mutant situations. Both models reproduced most wild-type and mutant phenotypes suggesting that the chosen topology is robust to explain these phenotypes. Numerical continuation of the model showed that even in the wild-type situation, the system changed from sustained to damped, i.e. a Hopf bifurcation occurred, when the Fgf concentration decreased in the PSM. This numerical continuation analysis further indicated that the most sensitive parameters for the oscillations are the parameters of Hes7 followed by those of Lunatic fringe (Lfng) and Notch1. In the wild-type, the damping of Hes7 oscillations was not so strong so that cells reached the new somites before they lose Hes7 oscillations. By contrast, in the fibroblast growth factor receptor 1 (Fgfr1) conditional knock-out (cKO) mutant simulation, Notch signaling was not able to maintain sustained Hes7 oscillations. Our analysis suggests that Fgf signaling makes cells enter an oscillatory state of Hes7 expression. After moving to the anterior PSM, where Fgf signaling is missing, Notch signaling compensates the damping of Hes7 oscillations in the anterior PSM.     

Fgf/MAPK signalling is a crucial positional cue in somite boundary formation.

The temporal and spatial regulation of somitogenesis requires a molecular oscillator, the segmentation clock. Through Notch signalling, the oscillation in cells is coordinated and translated into a cyclic wave of expression of hairy-related and other genes. The wave sweeps caudorostrally through the presomitic mesoderm (PSM) and finally arrests at the future segmentation point in the anterior PSM. By experimental manipulation and analyses in zebrafish somitogenesis mutants, we have found a novel component involved in this process. We report that the level of Fgf/MAPK activation (highest in the posterior PSM) serves as a positional cue within the PSM that regulates progression of the cyclic wave and thereby governs the positions of somite boundary formation.     

The vertebrate segmentation clock and its role in skeletal birth defects

The segmental structure of the vertebrate body plan is most evident in the axial skeleton. The regulated generation of somites, a process called somitogenesis, underlies the vertebrate body plan and is crucial for proper skeletal development. A genetic clock regulates this process, controlling the timing of somite development. Molecular evidence for the existence of the segmentation clock was first described in the expression of Notch signaling pathway members, several of which are expressed in a cyclic fashion in the presomitic mesoderm (PSM). The Wnt and fibroblast growth factor (FGF) pathways have also recently been linked to the segmentation clock, suggesting that a complex, interconnected network of three signaling pathways regulates the timing of somitogenesis. Mutations in genes that have been linked to the clock frequently cause abnormal segmentation in model organisms. Additionally, at least two human disorders, spondylocostal dysostosis (SCDO) and Alagille syndrome (AGS), are caused by mutations in Notch pathway genes and exhibit vertebral column defects, suggesting that mutations that disrupt segmentation clock function in humans can cause congenital skeletal defects. Thus, it is clear that the correct, cyclic function of the Notch pathway within the vertebrate segmentation clock is essential for proper somitogenesis. In the future, with a large number of additional cyclic genes recently identified, the complex interactions between the various signaling pathways making up the segmentation clock will be elucidated and refined.     

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.     

Cyclical expression of the Notch/Wnt regulator Nrarp requires modulation by Dll3 in somitogenesis

Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rhpn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function.     

The period of the somite segmentation clock is sensitive to Notch activity

The number of vertebrae is defined strictly for a given species and depends on the number of somites, which are the earliest metameric structures that form in development. Somites are formed by sequential segmentation. The periodicity of somite segmentation is orchestrated by the synchronous oscillation of gene expression in the presomitic mesoderm (PSM), termed the "somite segmentation clock," in which Notch signaling plays a crucial role. Here we show that the clock period is sensitive to Notch activity, which is fine-tuned by its feedback regulator, Notch-regulated ankyrin repeat protein (Nrarp), and that Nrarp is essential for forming the proper number and morphology of axial skeleton components. Null-mutant mice for Nrarp have fewer vertebrae and have defective morphologies. Notch activity is enhanced in the PSM of the Nrarp(-/-) embryo, where the ~2-h segmentation period is extended by 5 min, thereby forming fewer somites and their resultant vertebrae. Reduced Notch activity partially rescues the Nrarp(-/-) phenotype in the number of somites, but not in morphology. Therefore we propose that the period of the somite segmentation clock is sensitive to Notch activity and that Nrarp plays essential roles in the morphology of vertebrae and ribs.     

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.     

Molecular clocks underlying vertebrate embryo segmentation: A 10-year-old hairy-go-round

Segmentation of the vertebrate embryo body is a fundamental developmental process that occurs with strict temporal precision. Temporal control of this process is achieved through molecular segmentation clocks, evidenced by oscillations of gene expression in the unsegmented presomitic mesoderm (PSM, precursor tissue of the axial skeleton) and in the distal limb mesenchyme (limb chondrogenic precursor cells). The first segmentation clock gene, hairy1, was identified in the chick embryo PSM in 1997. Ten years later, chick hairy2 expression unveils a molecular clock operating during limb development. This review revisits vertebrate embryo segmentation with special emphasis on the current knowledge on somitogenesis and limb molecular clocks. A compilation of human congenital disorders that may arise from deregulated embryo clock mechanisms is presented here, in an attempt to reconcile different sources of information regarding vertebrate embryo development. Challenging open questions concerning the somitogenesis clock are presented and discussed, such as When?, Where?, How?, and What for? Hopefully the next decade will be equally rich in answers. Birth Defects Research (Part C) 81:65–83, 2007. © 2007 Wiley‐Liss, Inc.     

Modeling the segmentation clock as a network of coupled oscillations in the Notch, Wnt and FGF signaling pathways

The formation of somites in the course of vertebrate segmentation is governed by an oscillator known as the segmentation clock, which is characterized by a period ranging from 30 min to a few hours depending on the organism. This oscillator permits the synchronized activation of segmentation genes in successive cohorts of cells in the presomitic mesoderm in response to a periodic signal emitted by the segmentation clock, thereby defining the future segments. Recent microarray experiments [Dequeant, M.L., Glynn, E., Gaudenz, K., Wahl, M., Chen, J., Mushegian, A., Pourquie, O., 2006. A complex oscillating network of signaling genes underlies the mouse segmentation clock. Science 314, 1595-1598] indicate that the Notch, Wnt and Fibroblast Growth Factor (FGF) signaling pathways are involved in the mechanism of the segmentation clock. By means of computational modeling, we investigate the conditions in which sustained oscillations occur in these three signaling pathways. First we show that negative feedback mediated by the Lunatic Fringe protein on intracellular Notch activation can give rise to periodic behavior in the Notch pathway. We then show that negative feedback exerted by Axin2 on the degradation of β-catenin through formation of the Axin2 destruction complex can produce oscillations in the Wnt pathway. Likewise, negative feedback on FGF signaling mediated by the phosphatase product of the gene MKP3/Dusp6 can produce oscillatory gene expression in the FGF pathway. Coupling the Wnt, Notch and FGF oscillators through common intermediates can lead to synchronized oscillations in the three signaling pathways or to complex periodic behavior, depending on the relative periods of oscillations in the three pathways. The phase relationships between cycling genes in the three pathways depend on the nature of the coupling between the pathways and on their relative autonomous periods. The model provides a framework for analyzing the dynamics of the segmentation clock in terms of a network of oscillating modules involving the Wnt, Notch and FGF signaling pathways.