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.     

From dynamic expression patterns to boundary formation in the presomitic mesoderm

The segmentation of the vertebrate body is laid down during early embryogenesis. The formation of signaling gradients, the periodic expression of genes of the Notch-, Fgf- and Wnt-pathways and their interplay in the unsegmented presomitic mesoderm (PSM) precedes the rhythmic budding of nascent somites at its anterior end, which later develops into epithelialized structures, the somites. Although many in silico models describing partial aspects of somitogenesis already exist, simulations of a complete causal chain from gene expression in the growth zone via the interaction of multiple cells to segmentation are rare. Here, we present an enhanced gene regulatory network (GRN) for mice in a simulation program that models the growing PSM by many virtual cells and integrates WNT3A and FGF8 gradient formation, periodic gene expression and Delta/Notch signaling. Assuming Hes7 as core of the somitogenesis clock and LFNG as modulator, we postulate a negative feedback of HES7 on Dll1 leading to an oscillating Dll1 expression as seen in vivo. Furthermore, we are able to simulate the experimentally observed wave of activated NOTCH (NICD) as a result of the interactions in the GRN. We esteem our model as robust for a wide range of parameter values with the Hes7 mRNA and protein decays exerting a strong influence on the core oscillator. Moreover, our model predicts interference between Hes1 and HES7 oscillators when their intrinsic frequencies differ. In conclusion, we have built a comprehensive model of somitogenesis with HES7 as core oscillator that is able to reproduce many experimentally observed data in mice.     

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.     

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.     

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.     

A role for Fringe in segment morphogenesis but not segment formation in the grasshopper, Schistocerca gregaria

Studies of somitogenesis in vertebrates have identified a number of genes that are regulated by a periodic oscillator that patterns the pre-somitic mesoderm. One of these genes, hairy, is homologous to a Drosophila segmentation gene that also shows periodic spatial expression. This, and the periodic expression of a zebrafish homologue of hairy during somitogenesis, has suggested that insect segmentation and vertebrate somitogenesis may use similar molecular mechanisms and possibly share a common origin. In chicks and mice expression of the lunatic fringe gene also oscillates in the presomitic mesoderm. Fringe encodes an extracellular protein that regulates Notch signalling. This, and the finding that mutations in Notch or its ligands disrupt somite patterning, suggests that Notch signalling plays an important role in vertebrate somitogenesis. Although Notch signalling is not known to play a role in the formation of segments in Drosophila, we reasoned that it might do so in other insects such as the grasshopper, where segment boundaries form between cells, not between syncytial nuclei as they do in Drosophila. Here we report the cloning of a single fringe gene from the grasshopper Schistocerca. We show that it is not detectably expressed in the forming trunk segments of the embryo until after segment boundaries have formed. We conclude that fringe is not part of the mechanism that makes segments in Schistocerca. Thereafter it is expressed in a pattern which shows that it is a downstream target of the segmentation machinery and suggests that it may play a role in segment morphogenesis. Like its Drosophila counterpart, Schistocerca fringe is also expressed in the eye, in rings in the legs, and during oogenesis, in follicle cells. Received: 14 October 1999 / Accepted: 18 January 2000     

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.     

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.     

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.     

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.     

Interaction between X-Delta-2 and Hox genes regulates segmentation and patterning of the anteroposterior axis

In vertebrates, the paraxial mesoderm already exhibits a complex Hox gene pattern by the time that segmentation occurs and somites are formed. The anterior boundaries of the Hox genes are always maintained at the same somite number, suggesting coordination between somite formation and Hox expression. To study this interaction, we used morpholinos to knockdown either the somitogenesis gene X-Delta-2 or the complete Hox paralogous group 1 (PG1) in Xenopus laevis. When X-Delta-2 is knocked down, Hox genes from different paralogous groups are downregulated from the beginning of their expression at gastrula stages. This effect is not via the canonical Notch pathway, as it is independent of the Notch effector Su(H). We also reveal for the first time a clear role for Hox genes in somitogenesis, as loss of PG1 gene function results in the perturbation of somite formation and downregulation of the X-Delta-2 expression in the PSM. This effect on X-Delta-2 expression is also observed during neurula stages, before the somites are formed. These results show that somitogenesis and patterning of the anteroposterior axis are closely linked via a feedback loop involving Hox genes and X-Delta-2, suggesting the existence of a coordination mechanism between somite formation and anteroposterior patterning. Such a mechanism is likely to be functional during gastrulation, before the formation of the first pair of somites, as suggested by the early X-Delta-2 regulation of the Hox genes.