Signalling dynamics in vertebrate segmentation.

REVIEWS Signalling dynamics in vertebrate segmentation Alexis Hubaud1,2 and Olivier Pourquié1,2

Abstract | Segmentation of the paraxial mesoderm is a major event of vertebrate development that establishes the metameric patterning of the body axis. This process involves the periodic formation of sequential units, termed somites, from the presomitic mesoderm. Somite formation relies on a molecular oscillator, the segmentation clock, which controls the rhythmic activation of several signalling pathways and leads to the oscillatory expression of a subset of genes in the presomitic mesoderm. The response to the periodic signal of the clock, leading to the establishment of the segmental pre-pattern, is gated by a system of travelling signalling gradients, often referred to as the wavefront. Recent studies have advanced our understanding of the molecular mechanisms involved in the generation of oscillations and how they interact and are coordinated to activate the segmental gene expression programme. Paraxial mesoderm The mesoderm that flanks the neural tube in the embryo and that gives rise to the skeletal muscles and axial skeleton.

Marginal zone The superficial region of the embryo that contains the precursors of the mesoderm before they ingress during gastrulation.

Primitive streak The region where cells of the epiblast ingress into the embryo to form the mesoderm during gastrulation. Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS (UMR 7104), Inserm U964, Université de Strasbourg, Illkirch, 67400, France. 2 Department of Genetics, Harvard Medical School and Department of Pathology, Brigham and Woman’s Hospital, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. Correspondence to O.P. e‑mail: pourquie@genetics. med.harvard.edu doi:10.1038/nrm3891 1

This article is dedicated to the memory of Julian Lewis (1946–2014), a pioneer of the field of quantitative biology who made major contributions to our understanding of vertebrate segmentation. Segmentation is a conserved feature that governs the organization of the adult body of many animal species. The metameric organization of the vertebrae and their associated muscles has a key role in the control of locomotion in vertebrates. For instance, altered embryonic segment numbers in different fish species lead to distinct modes of swimming that enable them to adapt to their environment 1. In vertebrate embryogenesis, the tissue that is primarily segmented is the paraxial mesoderm, which lies on each side of the neural tube (FIG. 1). The paraxial mesoderm originates from progenitors of the marginal zone in zebrafish and frogs, and from progenitors of the epiblast of the anterior primitive streak in mouse and chicken embryos2. These progenitors, which are initially located in a superficial layer of the embryo, become internalized during gastrulation and form the presomitic mesoderm (PSM). This is accompanied by the activation of genes that are specifically expressed in the posterior paraxial mesoderm and promote the PSM fate3–5. Subsequently, the primitive streak, which is equivalent to the blastopore in amphibians and fish, morphs into a mass of cells called the tail bud. The tail bud is located at the posterior tip of the embryo, which contains progenitors of the PSM that contribute to posterior tissues.

The paraxial mesoderm becomes progressively segmented into repetitive structures called somites. They form as blocks of epithelial cells in the anterior PSM enclosing a core of mesenchyme. In contrast to Drosophila melanogaster, in which all segments form simultaneously 6, the formation of vertebrate segments occurs in a sequential manner with progressive addition of new pairs of somites at the anterior end of the PSM. During its maturation, the PSM becomes progressively epithelialized, and somite generation requires the formation of a posterior boundary. Under the influence of signals from neighbouring tissues, the somite is later subdivided into the ventromedial sclerotome (which will give rise to the axial skeleton) and the dorsolateral dermomyotome (from which body skeletal muscles and dorsal dermis arise)7. The rhythm with which new segments form is usually fixed for a given species, along with the total number of segments, but these numbers can vary tremendously between species; for instance, in mice, pairs of somites bud off from the PSM every two hours8 until a total of 65 pairs are formed, whereas in zebrafish, somites form every 25 minutes9, resulting in the formation of 33 somite pairs. In humans, somites are estimated to form every 4–5 hours until a total of 38–44 pairs are formed10. The periodicity of vertebrate segmentation is thought to be imposed by a molecular oscillator called the segmentation clock, which ‘ticks’ in the posterior PSM and controls the rhythmic specification of future segments. The clock drives the rhythmic activation of several signalling pathways in the PSM, such as Notch, WNT and fibroblast

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Figure 1 | Paraxial mesoderm segmentation. The paraxial mesoderm lies on both sides of the neural tube. Periodically, epithelial blocks known as somites bud off from the anterior Nature Reviews | Molecular Cell Biology part of the presomitic mesoderm. The future segmental domains are determined more posteriorly by the coordinated activation of segmentation genes in a bilateral stripe of cells (in blue). Somitogenesis occurs concomitantly with the elongation of the axis by tissue flow and the addition of new cells at the tail-bud level.

Presomitic mesoderm (PSM). The most posterior unsegmented region of the paraxial mesoderm, where the segmentation-clock oscillations take place.

Phase of the oscillator The position of an oscillator during its cycle, similar to the position of a hand on a watch. After one period, the oscillator returns to its initial phase.

growth factor (FGF), leading to the cyclic expression of a subset of genes11,12. This pulsatile signal is converted into the periodic generation of segments in response to a system of signalling gradients, known as the wavefront, which travels posteriorly along the PSM13,14. This gradient system was proposed to control the maturation of cells and their competence to respond to the clock signal. The combined action of the clock and the wavefront results in the activation of specific segmental genes15 in a bilateral stripe of cells in the anterior PSM (FIG. 1), which define the initial segmental pre-pattern on which the morphological somite is built. The stripes subsequently refine and acquire an anterior and posterior identity in response to a complex signalling cascade16. This leads to the subdivision of the future segment into an anterior and a posterior compartment (also known as ‘rostrocaudal polarity’). This partition of the somite is essential for the formation of the future vertebrae, which form from the fusion of the posterior compartment of a given pair of somites with the anterior compartment of the consecutive pair during a process called re-segmentation.

In this Review, we address the dynamics of signalling pathways during segmentation, with a focus on how oscillations arise in the embryo and how they control its segmental patterning. We first provide an overview of the current paradigm of vertebrate segmentation, before discussing the requirements for oscillations to emerge. We then present how these oscillators are structured and control the rhythmic activation of the segmental programme. Last, we discuss the spatial control of the responses to the clock signal along the PSM.

Models of segmentation The clock-and-wavefront model17 (FIG. 2) first proposed the existence of an oscillator to explain the rhythmic formation of somites in the embryo. This theoretical model, inspired by the mathematical theory of catastrophes that was developed in the 1970s, postulates that somite forma tion results from a periodic abrupt change (a catastrophe) in cellular properties triggered by a travelling front of maturation (the wavefront). The periodicity of the catastrophe is thought to be controlled by a molecular oscillator in PSM cells. The phase of the oscillator is similar between neighbouring cells, which means that cells are locally synchronized with respect to their oscillations. Thus, when a group of cells in the right phase of the oscillator cycle is hit by the wavefront, the cells undergo an abrupt change in cellular properties, leading to somite individualization. A consequence of this model is that the size of a newly formed segment is fixed by the distance travelled by the wavefront during one period of oscillation. Although the details of the original model have not been validated, research over the past 20 years in this area has provided molecular evidence for both a molecular oscillator (the segmentation clock) and a wavefront of maturation. The segmentation clock. The clock-and-wavefront model received experimental support with the discovery of the oscillatory expression of the segmentation gene HAIRY1, which belongs to the hairy and enhancer of split (HES)/‌HES-related (HER) family of transcription factors that act mainly as Notch effectors11,18. In chicken embryos, transcription of HAIRY1 occurs in a cyclic manner in posterior PSM cells, with a period of ~90 minutes, which is identical to that of somite formation. This periodic transcription process generates waves of gene expression that travel along the PSM each time a segment forms. HAIRY1 mRNA expression first occurs in a large domain in the posterior mesoderm, then the expression domain moves anteriorly and becomes narrower, and finally the expression wave slows down and arrests in the anterior region (FIG. 2a). These findings provided evidence for a molecular oscillator known as the segmentation clock11. Subsequently, other cyclic genes were described in chicken, zebrafish, frog, snake and mouse, arguing for the conservation of the segmentation clock in the PSM of vertebrates19,20. In all of the species studied, at least one member of the HES/HER family undergoes oscillatory expression in the PSM, which suggests that this class of transcription factors is at the heart of the vertebrate clock. In mice, chicken

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Figure 2 | The clock-and-wavefront model. a | The segmentation clock. The segmentation clock comprises a set of Nature Reviews | Molecular oscillatory activities, such as gene expression and signalling pathways (orange). These periodic activities controlCell the Biology determination of new segments. The synchronization of cellular oscillators and their slowing down along the presomitic mesoderm (PSM) creates the visual impression of a travelling wave that progressively sharpens as it moves anteriorly along the PSM. However, this travelling wave is not caused by cell movements because the progression of the wave is faster than the movement of a group of cells (square). b | Gradient formation. The FGF8 gradient (and presumably the FGF4 and WNT3A gradients) is formed by an RNA decay mechanism: mRNAs are only produced in the tail bud and are then progressively degraded in the PSM. As a group of cells (square) becomes located more anteriorly in the PSM owing to posterior elongation movements, the amount of Fgf8 mRNA decreases, which creates a gradient of mRNA (in blue) that is translated into a gradient of FGF8 ligand. Over time, the group of cells experiences less FGF and WNT signalling, so that the determination front position moves posteriorly with elongation of the body axis. c | Clock topologies. Oscillations are thought to arise through delayed negative-feedback loops. Three main signalling pathways were shown to oscillate in mouse embryos: Notch, WNT and fibroblast growth factor (FGF). Binding of the WNT ligand to its receptor results in β‑catenin (β‑cat)-mediated target gene transcription. AXIN2 and Dickkopf-related protein 1 (DKK1) are negative-feedback inhibitors of this pathway and are periodically expressed in the PSM. The binding of FGF ligands to their cognate receptor results in activation of the ERK pathway. Phosphorylated ERK (pERK) activates dual specificity protein phosphatase 4 (Dusp4), Dusp6 and Sprouty 2 (Spry2), which are known negative-feedback inhibitors of the FGF pathway that are expressed rhythmically. The Notch targets lunatic fringe (Lfng) and Notch-regulated ankyrin repeat protein (Nrarp) contribute to the rhythmic production of the transcriptional effector Notch intracellular domain (NICD).The mouse oscillators interact and entrain each other. Hairy and enhancer of split 7 (HES7) is activated by the FGF–phosphorylated ERK pathway posteriorly in the PSM and by NICD more anteriorly, and represses the FGF–ERK pathway and Notch inhibitors, as well as its own expression. By contrast, in zebrafish, oscillations are mainly controlled by the availability and activities of Hes/Hes-related (Her) factors. d | Clock-and-wavefront model. Two inputs control the activation of the segmental programme: the segmentation clock (in orange) and the wavefront (in blue). The clock moves along the PSM and triggers the segmental determination of cells (pink) that are passed by the wavefront during the previous cycle. DLL1, Delta-like 1. NATURE REVIEWS | MOLECULAR CELL BIOLOGY


REVIEWS and zebrafish, the segmentation clock also comprises the rhythmic activity of the WNT, FGF and Notch pathways in the PSM12,21. The wavefront. Molecular evidence for the wavefront came from the study of posterior FGF and WNT gradients in fish, chicken and mouse embryos13,14,22,23. Several members of the FGF and WNT families, such as FGF8 and WNT3A, are distributed in a gradient along the PSM24, peaking in the posterior region. Manipulating these gradients by changing the expression levels of WNT and FGF ligands in PSM cells shifts the position of somite boundaries and thus results in the generation of somites that differ in size in a predictable fashion13,14,22,25. These findings led to a model in which the wavefront corresponds to a threshold of FGF and/or WNT signalling along the gradient. This level defines a position in the PSM at which cells become competent to respond to the segmentation clock13. In the posterior PSM, high levels of FGF and WNT signalling preclude cells from responding to the periodic clock signal. The posterior FGF gradient (and possibly also the WNT gradient) is formed through an mRNA gradient along the PSM (FIG. 2b). FGF8 and presumably FGF4 genes are transcribed only in the primitive streak or in the tail bud. After ingression into the PSM, cells stop transcribing those genes, which leads to a progressive decay of their mRNAs and the formation of the gradient 23,24,26. The signalling range of these gradients is also controlled by mechanisms such as the regulation of WNT- and FGF-receptor maturation27 and the regulation of ligand activity by secreted antagonists28. The segmental programme. On reception of the clock signal in the region immediately anterior to the determination front, genes of the mesoderm posterior (MESP) family of transcription factors become activated in a restricted domain, thus forming a bilateral stripe that marks the future segments29 (FIG. 1). MESP genes are master regulators of the segmental programme that forms the future segment boundary: inactivation of Mesp1 and Mesp2 genes in the PSM blocks segment formation15,30,31. In mice, Mesp2 (and Mesp1) is expressed as stripes in the anterior PSM; at first, the stripes are the size of one somite, but then they become restricted to the anterior half of the future somite16. This pattern creates an interface between Mesp2‑positive and Mesp2‑negative domains that defines the position of the future boundary 29. Such boundary formation relies on the action of adhesion molecules and on complex cellular rearrangements that are mainly controlled by the Eph/ephrin system32.

Determination front The level of the presomitic mesoderm where cells first acquire a defined segmental identity.

Challenging clock-based segmentation. The role of the clock in somite formation has been recently challenged33. In these experiments, the posterior primitive streak of a chicken embryo was exposed to the bone morphogenetic protein (BMP) inhibitor Noggin. Noggin promotes the paraxial mesoderm fate at the expense of the lateral plate, and Noggin exposure led to the formation of epithelial spheres that exhibit a size and rosette

organization similar to that of somites. These epithelial spheres expressed some markers that are characteristic of the anterior PSM and somites, such as the transcription factor paraxis (also known as TCF15). Furthermore, when grafted in place of a somite in a host embryo, posterior primitive streak fragments treated with Noggin gave rise to normal paraxial derivatives such as muscle and vertebrae. These structures formed almost simultaneously, and their formation did not seem to involve oscillations of the cyclic genes nor expression of Mesp2. This led the authors to suggest that the segmentation clock is dispensable for somite formation and to propose that somitogenesis is driven by cell–cell interactions33. However, these somite-like structures do not harbour all of the characteristics of somites, such as the polarized expression of anteroposterior somite markers. Epithelial rosette formation is not specific to somites: it represents a classic self-organization pattern for pseudostratified epithelial structures. Therefore, the observed epithelial structures formed in the grafts of posterior primitive streak treated with Noggin possibly reflect a self-organizing pattern of differentiating paraxial mesoderm derivatives, as is observed for neural rosettes in differentiating embryonic stem cells. Accordingly, epithelialization and segmental patterning can be experimentally separated, as shown, for instance, in paraxis-null mice, in which a segmental organization is still present despite the absence of epithelial somite formation34. Furthermore, a model based only on tissue mechanics does not explain why some mutants of the segmentation clock (for example, the zebrafish hes6‑null mutant or the mouse mutant carrying intronic Hes7 deletions) undergo changes in segment length35,36. Whether such a self-organizing process is involved in normal somito genesis in vivo remains to be established, but it is compatible with the existence of a segmentation clock. Indeed, clock-based segmentation models only aim to explain the establishment of a segmental pre-pattern of gene expression in the PSM that leads to the characteristic linear array of segments — they do not aim to explain the formation of epithelial somites, which takes place later. In the Noggin-treated grafts of primitive streak, the ectopic somites are not organized into a linear array of segments, so perhaps the segmentation clock directs the self-organizing propensity to form segments; this would be consistent with the notion that the role of the segmentation clock is to govern the metameric organization of the body axis (BOX 1).

Oscillations in the PSM In the PSM, interaction of the segmentation clock with signalling gradients enables conversion of this rhythmicity into a spatial periodicity. In the following section, we examine the mechanism generating such oscillations in the PSM. Basic elements of the oscillatory network. Negative feedback loops are a simple way to obtain cyclic expression, as shown by the oscillations of the HES/HER family. These basic helix–loop–helix (bHLH) proteins act as transcriptional repressors37,38 that can directly repress their own



REVIEWS Box 1 | Generation of periodic structures by travelling oscillators Segmented structures composed of repetitions of similar anatomical modules are widely encountered in animals or even in plants. Recent experimental evidence in vertebrates shows that segmentation relies on an oscillator travelling in space, defining successive segmental domains during each oscillation. This system is not conserved in flies, in which segmentation is established simultaneously in all segments in response to a well-characterized genetic cascade that subdivides the fly embryo into progressively smaller domains. However, a system similar to that of vertebrates — that is, a system based on a transcriptional clock driving cyclic gene expression in the terminal growth zone — has been described in the short-germ band insects, notably Tribolium142 and Periplaneta143 spp. These arthropods, as well as annelids, also undergo WNT– caudal-dependent axis elongation from a growing zone comparable with vertebrate axis formation144,145. Whether such similarities result from the inheritance of segmentation and elongation mechanisms from their common ancestor with vertebrates (Urbilateria) remains under debate146. Lateral branching of roots in plants also relies on gene oscillations and on polarized growth from a progenitor region147. Oscillations could thus be a generic signalling mechanism and have been independently selected to generate segmental patterns from elongating structures148.

expression, as shown for Her1 and Her7 in zebrafish39 and for HES1 and HES7 in mice40,41,42. Similarly, cycling of the Notch, FGF and WNT gene targets in mice is thought to be triggered by negative feedback loops that are gener ated by the periodic activation of inhibitors of these pathways12,43 (FIG. 2c). For instance, lunatic fringe (LFNG) is a Notch transcriptional target that functions as a glycosyltransferase that modifies the Notch receptor 44,45. In the PSM, LFNG contributes to periodically block the cleavage of the Notch receptor, thus generating a rhythmic production of the transcriptional effector Notch intracellular domain (NICD)46,47. Moreover, the binding of FGF ligands to their cognate receptor results in the activation of the ERK pathway. Phosphorylated ERK in turn activates the genes encoding dual specificity protein phosphatase 4 (DUSP4), DUSP6 and Sprouty 2 (SPRY2), which subsequently inhibit the FGF pathway. This mechanism can account for the reported oscillations of ERK phosphorylation in the mouse PSM48. Activation of the WNT pathway involves binding of the WNT ligand to the Frizzled–low-density lipoprotein receptor-related protein 6 (LRP6) co‑receptor complex, which results in the inhibition of β‑catenin degradation and its translocation to the nucleus, where it mediates target gene transcription. The scaffolding protein AXIN2, which is part of the β‑catenin destruction complex, and the soluble inhibitor Dickkopf-related protein 1 (DKK1) are negative feedback inhibitors that are cyclically transcribed in the PSM and could, in principle, drive rhythmic activation of WNT signalling 12,14. Oscillating levels of nuclear β‑catenin have not been detected49, which suggests that nuclear translocation of β‑catenin is not the main node of WNT signalling regulation in the PSM50. Periodic activation of WNT signalling might thus be controlled by cycling of a cofactor that remains to be identified49.

Urbilateria The common ancestor of bilaterian animals.

Time delay and clearance. Negative-feedback loops must be endowed with specific properties to generate oscillations. Mathematical simulations show that introducing a specific delay between the activation of HES/‌HER factors and their self-inhibition can lead to sustained

oscillations51,52. Time delay can arise at various levels, such as gene expression, transport or post-translational modifications of signalling molecules. Through such a delay, the activation and repression of HES/HER factors can occur sequentially, so that the system does not reach a steady state. In the PSM, it has been proposed that this delay originates from the time that is required to transcribe the HES/HER or LFNG genes, or from their splicing or their nuclear export 39. In zebrafish and mice, measuring the transcription elongation rate in the PSM suggests that the delay due to this step is not sufficient and is thus unlikely to be responsible for the generation of oscillations53,54. Consistently, increasing the length of one intron of Lfng, which is expected to increase the delay between transcription and translation, has no effect on mouse segmentation55. Other studies suggest that RNA splicing has a role in the delay between activation of the mouse Hes genes and their auto-inhibition. In mouse embryos, deletion of all Hes7 introns abolishes oscillations and leads to major defects in the segmentation of the axial skeleton56. Removing two of the three introns of this gene, and thus reducing the delay imposed by splicing, modifies the period of Hes7 oscillations by 5 minutes36. This results in an increase in the number of anterior somites and, later, to a dampening and an arrest of the oscillations. The genetic modifications of the locus required to remove the introns might affect Hes7 expression55,56, but even so, these studies support a role for the specific delay caused by splicing in the generation of oscillations. In addition, kinetic analysis of Lfng and Hes7 mRNA maturation shows that nuclear export also contributes to the transcriptional delay 54. Interestingly, the importance of splicing and nuclear export in the control of oscillations seems to vary in zebrafish, mice and chickens, which potentially provides an explanation for the interspecies difference in clock period54. Mathematical modelling predicts that sustained oscillations require rapid degradation of the cyclic mRNAs51,57 and similarly rapid clearing of mRNAs and proteins to allow alternation between pathway activation and repression. HES/HER proteins have a short half-life; for instance, in mice, the half-lives of Hes1 mRNA and HES1 protein are 24 and 22 minutes, respectively 41, and in zebrafish, the half-life of the Her7 protein was shown to be 3.5 minutes57. Such dynamics rely on the rapid degradation of proteins by the proteasome41,42. It was possible to increase the stability of HES7 by mutating Lys residues involved in ubiquitylation and proteasome-dependent degradation: this led to an increase of the protein half-life from 22 to 30 minutes, but the repressive activity remained unaffected. In mice expressing this stabilized HES7 protein, oscillations are progressively lost during somitogenesis58, consistent with the mathematical simulations of the auto-inhibitory model. Little is known about the degradation of cyclic mRNAs in the PSM. Differences in stability between mRNAs can be at least partly accounted for by their 3′ untranslated region (UTR)59–61. Recent studies have identified a role for microRNAs in cyclic mRNA degradation: miR‑125a‑5p was shown to induce the destabilization of LFNG mRNA



REVIEWS in chicken embryos62. Alleviating miR‑125a‑5p‑mediated repression of LFNG arrests the oscillations of LFNG and leads to segmentation defects. Whether this mechanism is conserved in other species is unclear. However, zebrafish and conditional mouse mutants for Dicer, an enzyme necessary for microRNA biogenesis, have no obvious defects in somitogenesis63,64. Why these requirements for cycling are fulfilled in the PSM but not in neighbouring tissues is not yet understood; for instance, Hes1 is expressed in the mouse neural tube, but not in cycles. Interestingly, the PSM-specific transcription factors mesogenin 1 (MSGN1) and T-box transcription factor 6 (TBX6) directly control the expression of some cyclic genes in mice65,66, providing a potential link between the PSM fate and the oscillatory state. Topology of the mouse segmentation clock. The segmentation clock comprises various oscillatory loops that interact with each other, including the Notch–HES (HES1, HES5 and HES7), WNT and FGF–ERK oscillators (FIG. 2c). mRNA expression of Hes1, Hes5 and Hes7 has been shown to oscillate, but only Hes7‑deletion mutants display severe skeletal defects67. In addition, the genes coding for HEY factors, which are bHLH factors that can form heterodimers with HES factors, have also been reported to show oscillating expression, but their deletion does not result in segmentation defects68. The different oscillators interact and are thought to collectively set the ‘tempo’ of somitogenesis; for example, loss of HES7 oscillation disrupts the oscillation of the Notch pathway and part of the FGF–ERK pathway 69,70. In the posterior PSM, the Notch and FGF–ERK oscillators entrain each other though the action of HES7, which connects these negative-feedback loops69: Hes7 is activated by the FGF–ERK pathway and represses the FGF–ERK inhibitors Dusp4, Spry4 and the Notch inhibitor Lfng 69,71. By contrast, WNT activity continues to cycle after loss of the Notch–HES oscillations: Hes7‑null mutants, mutants lacking the Notch ligand Delta-like 1 (Dll1) or embryos overexpressing NICD still experience oscillations of the WNT target Axin2 (REFS 14,58,70,72). Therefore, HES7 is a central player of the mouse segmentation clock, but it is unlikely to control all oscillators in the PSM. The role of the WNT oscillator has been difficult to investigate. WNT loss‑of‑function mutants generally lack PSM altogether 49,73, which limits their use for such analyses. Mutant mouse embryos in which canonical WNT signalling is constitutively activated by expressing a nondegradable form of β‑catenin in the PSM still exhibit dynamic expression of Dkk1, Hes7 or Lfng 49,73. However, pharmacological alteration of WNT signalling in mouse PSM explants seems to change the periodicity of Hes7 oscillations66,74. Whether oscillations driven by the segmentation clock are controlled by a single pacemaker circuit or by the interaction of several oscillators remains to be established43. The extensive crosstalk between signalling pathways14,69,75,76, such as the link between the Notch and FGF–ERK oscillators, supports the view that interactions between the feedback loops control the segmentation clock. Accordingly, Axin2 expression remains periodic, but its dynamic pattern is perturbed by the loss

of Hes7 (REF. 58) and by the dampening of the Notch and FGF pathways14,77. Genetic analyses could not decipher a simple epistasis relationship between the FGF, Notch and WNT pathways69,77,78. Collectively, this points towards a mutual entrainment of oscillators43 (FIG. 2c), even if the existence of an outside pacemaker that regulates the signalling circuit cannot be excluded. Thus, the genetic control of the oscillations in mice remains to be firmly established. Topology of the zebrafish segmentation clock. The core circuit of the segmentation clock differs between zebrafish and mice. In fish, only the Hes/Her factors and genes of the Notch pathway have been functionally implicated in the oscillator, although periodic expression of the Wnt target tbx16 has been reported21. It has been proposed that the Hes/Her negative-feedback loops act as a pacemaker for the segmentation clock51,79. The topology of these feedback loops in zebrafish is better characterized than in mice, with evidence of diverse combinations and activities of Hes–Her dimers53,80,81. Specifically, Her1, Hes6 and Her7 can form homodimers and hetero dimers, but only Her1–Her1 and Hes6–Her7 dimers have a strong and redundant DNA-binding affinity. These active dimers were proposed to regulate the clock period depending on their availability and stability 80. Consistent with this, loss of Her7 function is rescued by the loss of Hes6 activity, which suggests that the excess of inactive Her1–Hes6 dimers in the Her7 single mutant impedes the formation of active Her1–Her1 dimers80,81. Thus, the zebrafish oscillators were proposed to be controlled by a ‘dimer cloud’ of Hes/Her factors80 (FIG. 2c), whereas the mouse segmentation clock seems to be regulated by the coordinated activity of several oscillators. Nevertheless, it remains striking that the Her1–Hes6 double mutants, in which none of the dimers can form and therefore no oscillations of her1 or her7 are detected, are viable and retain some form of segmental organization at the level of the axial skeleton80. This phenotype could relate to a self-organizing mechanism or to the existence of another oscillator and/or pacemaker in the PSM. Other Her factors are also cyclically expressed in the PSM (that is, Her11, Her12 and Her15), but their precise role remains to be clarified81–83.

From oscillations to segments Activation of the segmental programme. The main output of the clock is the activation of genes of the MESP family of transcription factors (MESP2 and the less-studied MESP1 in mice and humans; MESO1 and MESO2 in chickens; thylacine 1 (thyl1; also known as mespaa) and thyl2 (also known as mespab) in Xenopus laevis; and mespa (also known as mespaa) and mespb (also known as mespba) in zebrafish) in a bilateral stripe of cells in the anterior PSM29. In zebrafish, chickens and mice, FGF and WNT gradients provide positional information for the spatial activation of MESP genes in response to the clock. When cells are passed by the determination front, they become competent to respond to the periodic clock signal. When the competent cells are ‘hit’ by the travelling signalling gradients that have been triggered by the clock, they



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Figure 3 | Formation of a segment. a | Signalling model. Activation of mesoderm posterior 2 (Mesp2) is central to segment determination. In mice, Mesp2 is thought to be activated by Notch signalling (through Notch intracellular domain (NICD)) and Nature Reviews ERK | Molecular Cell Biology T-box transcription factor 6 (TBX6), and repressed by fibroblast growth factor (FGF)–phosphorylated (pERK). Mesp2 is first expressed as a large stripe of one segment length, which defines the future segment boundaries. MESP2 induces the degradation of TBX6, leading to the definition of the anterior boundary of the next segment to be formed. During the next cycle, it becomes restricted to the anterior part of the future segment and suppresses Notch signalling, and thus determines the anteroposterior polarity of the segment. b | Theoretical models. In the standard model, the clock (orange) and wavefront (blue line) are independent entities that determine the segments. Only one phase of the clock (orange) triggers segment determination (pink), and the anteroposterior polarity is set subsequently; the position of the wavefront determines the position of the posterior boundary of a newly determined segment. In the two-phase model, the clock is arrested in cells that are hit by the wavefront. Both phases of the clock (dark orange and light orange stripes) determine half-segments, and thus the anteroposterior polarity is set as segments are determined. The segment boundary position is set by the clock phases, which are only ‘recorded’ by the passage of the wavefront. A third model, the phase-shift model, proposes an interaction between the clock and the wavefront, as the clock activity influences the position of the wavefront. In mice, the wavefront is proposed to involve a pERK oscillator (purple) that shifts from the Notch oscillator (orange).

respond by activating the MESP genes. This mechanism ensures the periodic and sequential activation of MESP genes in stripes that mark the future segment boundaries. In mice, Mesp2 expression requires Notch signalling, and activation of Notch correlates with the stabilization of the NICD wave in an anterior PSM stripe that defines the future segment 16,30,46,84–86. In zebrafish, Notch seems to control the expression of mespa but not mespb87,88. The role of the clock in the periodic bursts of Mesp2 expression is supported by the observation that in Hes7-mutant mice, the loss of NICD oscillations results in continuous Mesp2 expression, and thus no clear segmental boundary is formed75. In mouse embryos, the anterior limit of the Mesp2 stripe is defined by the anterior expression boundary of the gene coding for the transcription factor TBX6 (REFS 30,86) (FIG. 3a). After being induced by TBX6, MESP2 induces in turn the degradation of TBX6 protein and delimitates its next anterior boundary 30. Consequently, this feedback loop demarcates the segmental border by making the posterior boundary of MESP2 (segment N) correspond to the anterior boundary of the next MESP2 stripe (segment N + 1) (FIG. 3a).

Positioning the posterior segment boundary. Evidence suggests that the FGF–ERK gradient has a role in the formation of the posterior boundary of MESP2 (FIG. 3a). If this pathway is perturbed through the addition of ligands or chemical inhibitors, the MESP2 expression domain can shift in chicken, fish and mouse embryos13,22,75,89. In mice, the expression domains of Mesp2 and phosphorylated ERK are mutually exclusive in the anterior PSM, where they form a clear interface that marks the determination front level75. Consistent with this, loss of FGF signalling in mouse mutants leads to a posterior expansion of the Mesp2 domain23,30,75. Notch and FGF targets initially cycle in phase in the posterior PSM, but these oscillations become progressively out of phase, which results in a domain where Notch but not FGF–ERK is activated, and this enables Mesp2 activation75. In mice, the length of the PSM posterior oscillatory domain can be expanded by overriding the posterior WNT gradient through constitutive activation of WNT using non-degradable β‑catenin or ectopic expression of WNT3A in the paraxial mesoderm14,49,73.



REVIEWS

Nodal signalling A pathway downstream of the transforming growth factor-β-like growth factor Nodal that helps to control the asymmetrical development of internal organs such as the heart and liver.

In WNT gain‑of‑function mutants, the stripe of Mesp2 expression is shifted anteriorly but is still found in the domain expressing the stabilized β‑catenin, which suggests that in contrast to the FGF–ERK pathway, downregulation of WNT signalling is not required for Mesp2 activation49,73. WNT has been shown to act upstream of FGF signalling in the PSM by controlling Fgf8 expression in the tail bud14. However, the effect of WNT on the maintenance of the posterior PSM identity seems to be independent from its positive effect on FGF signalling, because compound mutants that lack FGF receptor 1 and constitutively express an activated β‑catenin construct in the PSM still experience an anterior shift of the determination front49. Thus, the WNT–β‑catenin gradient seems to be permissively required to maintain oscillations in the posterior PSM in mice, but unlike the FGF gradient, it does not seem to control the positioning of the determination front. The situation differs in zebrafish, in which quantitative analyses of Wnt and Fgf activity revealed that their main effectors (nuclear β‑catenin and phosphorylated Erk, respectively) do not form a sharp boundary posterior to the domain in which mespb is activated90. Rather, it was proposed that the targets of Wnt, such as the transcription factor Msgn1, provide positional information for mespb expression90. Nevertheless, in zebrafish, the phosphorylated Erk domain forms a sharp frontier that is located more posteriorly than the mespb stripe91. Pharmacological activation of the Fgf–Erk pathway by treatment with a Dusp6 inhibitor shifts the phosphorylated ERK domain anteriorly and affects mespb expression after the formation of three somites, leading to the formation of a smaller somite after five normal somites are produced. This experiment suggests the existence of some segmental pre-patterning that occurs in the posterior PSM before mespb activation. This notion is consistent with heat-shock experiments in amphibian and chicken embryos92,93 that revealed the existence of heatshock-sensitive segmental domains in the posterior PSM. In these experiments, the exposure of frog or chicken embryos to heat shock led to delayed segmental defects. Mapping the segmental determination of the PSM in chickens by performing microsurgical inversions of small PSM fragments also showed that the level at which PSM cells become committed to forming segments lies more posteriorly than the MESO1 stripe13. Further experiments will be required to firmly establish the existence of such a pre-pattern. Retinoic acid forms a gradient opposing the FGF and WNT gradients, and it is highly concentrated in somites and the most anterior PSM94,95. This gradient is established in response to the activity of the retinoic acid biosynthetic enzyme retinal dehydrogenase 2 (RALDH2), which is expressed in the anterior PSM and somites, and of the retinoic acid-degrading enzyme cytochrome P450 26 (CYP26), which is expressed downstream of FGF signalling in the tail bud96–98. Reducing retinoic acid levels anteriorly expands the FGF gradient and thus moves the position of the determination front anteriorly, leading to the formation of smaller somites99. Such antagonistic gradients of FGF and retinoic acid signalling

are also observed in X. laevis, but the underlying mechanism differs; in X. laevis, retinoic acid directly activates the expression of the frog MESP2 homologue, thyl1 (REFS 100,101), whereas retinoic acid is not required for Mesp2 expression in mice46,102. Whereas internal organs such as the heart or gut are clearly asymmetrical, somites exhibit a striking bilateral symmetry in amniote embryos. Retinoic acid has an important role in the control of the bilateral symmetry of oscillations and segmentation that leads to the formation of symmetrical somites25,96,97,102,103,104. In chicken, mouse or fish embryos deprived of retinoic acid, somitogenesis becomes asymmetrical between the left and right side, with somite formation being consistently delayed on one side. This lateralized effect on somitogenesis can be inverted by inverting the situs of the embryo, indicating that it is caused by the left–right machinery that controls asymmetric development of internal organs downstream of Nodal signalling105,106. Thus, in the absence of retinoic acid, the PSM responds to the asymmetrical signals involved in left–right patterning that cross the paraxial mesoderm. These observations suggest that retinoic acid buffers the action of the left–right machinery, thus helping to maintain the symmetry of somite formation. Making segments or half-segments? In clock-and-wavefront models, the clock ‘slides’ along the PSM and triggers the segmental determination of cells passed by the wavefront during the previous cycle17,107–109 (FIGS 2d,3b). Only one phase of the clock provides information for the generation of the segment (for example, when Notch is activated). This was formalized in mathematical models in which the clock controls a bi-stable transition between undetermined and determined PSM108. Such models result in the sequential definition of MESP2 stripes that predefine the future segments. The information required to determine the anteroposterior polarity of the segment has to rely on other parameters that are not necessarily dependent on the clock. A mechanistic variant of this model proposes that the wavefront is cyclic and that the progressive shift with the segmentation clock determines a full segment (the phase-shift model)75 (FIG. 3b). A second category of models, the two-phase models, considers the clock phase when oscillations are slowed and arrested at the wavefront level11,110–113. Originally, these models were based on reaction diffusion mechanisms114. Oscillations are stopped either at their peak (for example, during Notch activation) or in their trough (for example, during Notch inhibition) (FIG. 3b). In these models, both phases of the clock provide spatial information and define the anterior and posterior segmental halves at the same time as the segment itself. Differentiating these two categories requires dynamic examination of the activation of Mesp2 and the clock phase when cells are determined. In mice, Mesp2 is first expressed as a stripe of one segment size and secondarily becomes restricted to the anterior half-segment domain15, which supports the notion that segments are determined before the establishment of anteroposterior polarity. Moreover, the anteroposterior polarity of the somite can be dissociated from segment formation,



REVIEWS arguing against the second category of models. For instance, in embryos overexpressing NICD72 or in hypomorphic mutants for Mesp2 (REF. 115), segments are formed despite the absence of somite anteroposterior polarity. However, both ideas are not mutually exclusive, because the segment could first be determined by the broad stripe of Mesp2 (as in a classic clock-and-wavefront model), and then half-segments could be defined through its anterior restriction (as in a two-phase model)109,116. Accordingly, the confinement of Mesp2 to the anterior compartment seems to be first controlled by the segmentation clock itself 109; later, Mesp2 repression by RIPPLY1 and RIPPLY2 reinforces this anteroposterior polarity 117–119. MESP2 suppresses Notch signalling in the anterior half, whereas this pathway remains active in the posterior half 16,120, so the boundary of Notch signalling delimitates anterior and posterior compartments in the somite (FIG. 3a).

Spatial organization of the clock The response to the clock signal requires a tight coordination of gene expression at the cellular level in the PSM. The coordinated delivery of the clock signal to the PSM cells depends on the local synchronization of the oscillators in cells. Secondly, the location of these abrupt gene expression changes is determined by the phase profile of the oscillators — that is, they occur when the clock is in the right phase in competent cells, which is controlled by the generation and travelling of waves of clock signal.

Hypomorphic mutants Mutants showing a weaker phenotype than null mutants.

Synchronization Oscillators are synchronized when they have similar phases (that is, they are ‘in phase’). Synchronization can change the oscillation period.

Coupling strength The strength of the interaction involved in synchronizing oscillators between cells.

Travelling waves Also known as kinematic waves. Waves without matter transport or transmission of a signal.

Doppler effect The change of wavelength caused by the displacement of the source.

Synchronization of oscillators. Synchronization of the oscillations was proposed to depend on intercellular communication mediated by the Notch pathway 121. Indeed, blocking Notch signalling in zebrafish results in asynchronous and noisy oscillations that are out of phase, as shown by the ‘salt-and-pepper’ expression pattern of her genes detected by in situ hybridization in Notch pathway mutants121–123 and using live imaging of a Her1 fluorescent reporter in the PSM124. In zebrafish, oscillations of the Notch ligand Delta C have been proposed to have a role in the synchronization of oscillations among neighbouring cells51,122. This Notch-based coupling of the oscillators of individual cells was suggested to play a part in buffering the noise resulting from cell division or cell displacement 125–127. The role of Notch in the control of synchronization of oscillations has been confirmed by studies reporting a lag time corresponding to the formation of several segments between the beginning of Notch inhibition and visible segment disruption125. This time lag might represent the time taken for oscillations to fully drift out of synchrony, hence leading to segmentation defects. A role of Notch in the synchronization of oscillations seems to be conserved in mice and involves LFNG, which can repress Notch signalling in neighbouring cells by inhibiting DLL1 activity 128. Consistently, Lfngmutant mice similarly display a salt-and-pepper pattern of Hes7 expression and NICD activation128. Interestingly, the severity of segmentation defects seems to correlate with the strength of the Notch perturbation. For instance, in mice, the mutation of presenilin 1 (Psen1), which is involved in Notch cleavage, led to the formation of more anterior segments than did the double knockout of Psen1

and Psen2, which completely blocks Notch signalling 129. Moreover, overexpression of NICD in mice disrupts segmentation only after the formation of approximately 15 segments72, which suggests a progressive loss of synchrony, as described in zebrafish126. In dissociated PSM cells in culture, the expression of cyclic genes becomes disorganized, which strongly supports a role of synchronization in the generation and/‌or stabilization of oscillations130,131. As well as being involved in the generation of stable oscillations, synchronization presumably has a role in setting their period. Modelling of the segmentation clock shows that the collective period of synchronized oscillators can differ from the individual period of isolated cells113. That is, the synchronization signals sent by a neighbouring cell can influence its own clock and force it to oscillate faster or slower. Changing the coupling strength or the time delay imposed by intercellular communication can change the collective period (that is, the period of oscillations in the embryo) and thus the somitogenesis period (that is, the period of somite formation). These predictions concur with the lengthening of the somitogenesis period after partial inhibition of Notch signalling in zebrafish132. Similarly, forced synchronization through periodic pulses of Delta C expression from a heat-shock-driven promoter can entrain cellular oscillators and modify their collective period133. Maternal administration of a Notch inhibitor also alters the mouse somitogenesis period, but the direct effect of Notch inhibition on clock synchronization has not been examined134. Travelling waves along the PSM. To understand the temporal and the spatial control of segment determination, it is crucial to determine how waves of oscillator activities travel along the PSM. The travelling waves exhibit a periodicity that matches the rhythm of segmentation11. In mice and zebrafish, measuring the activity of live cyclic reporters reveals that the periodicity of oscillations in the anterior PSM exactly corresponds to the period of somite formation49,130,135: that is, a segment is formed every time a wave arrives in the anterior PSM. Surprisingly, however, recent observations in zebrafish using a fluorescent Her1–GFP reporter to image the oscillations indicate that the oscillation period is slower in the posterior PSM, where the travelling waves initiate. This striking situation can be explained by the fact that the zebrafish PSM constantly shrinks during development in a manner that modifies the actual period of waves hitting the anterior PSM, as in a Doppler effect135. The precise molecular mechanism underlying the slowing down of the travelling waves has not yet been elucidated. The oscillation period at the cellular level progressively increases towards the anterior end of the PSM: at the posterior end, the period is close to the somito genesis period, whereas at the anterior end, the oscillation becomes slower until it arrests after the determination front level11,39,75,126,136. This results in a non-linear phase gradient (or phase profile). One explanation for this slowing down of the travelling wave could lie in the control of oscillation frequencies by the levels of FGF and WNT,



REVIEWS Box 2 | Clock dynamics and metabolism A role for metabolism in the control of segmentation has recently emerged. In zebrafish, the analysis of microarray series generated from consecutive fragments of presomitic mesoderm (PSM) from the tail bud to the somite level revealed that the determinationfront level marks a major transcriptome reorganization that could also account for the changes in clock dynamics. The determination front also marks an abrupt transition at the level of cell metabolism149. Genes enriched in translation and oxidative metabolism were found to be enriched in the anterior PSM, whereas genes associated with cell cycle and DNA metabolism are preferentially enriched in the posterior PSM. Metabolic perturbations, such as hypoxia150, can affect mouse somitogenesis, particularly when applied in heterozygote mouse mutants for mutations in genes involved in the segmentation clock, such as hairy and enhancer of split 7 (Hes7) or mesoderm posterior 2 (Mesp2). In this context, hypoxia inhibits fibroblast growth factor (FGF) signalling, leading to abnormal Notch oscillation and segment formation. Such interactions between physiological stresses during pregnancy and the genetic background have been proposed to explain sporadic congenital scoliosis in humans.

which decrease as the clock slows down. In mouse mutants with constitutively active WNT signalling, multiple waves of Lfng expression that exhibit a normal phase gradient continue to travel along the expanded PSM49. A possible mechanism involves the positive regulation of HES/HER factors by the FGF pathway. In mice, HES7 is positively regulated by FGF69, and in zebrafish, the expression of Hes6, which can form heterodimers with Her7, is similarly activated by Fgf 137. Thus, the gene regulatory network could be reorganized as cells experience lower levels of FGF in the anterior PSM. This ‘rewiring’ of the signalling pathways is consistent with the regulatory differences between the anterior and posterior PSM. For instance, in mice, Hes7 is regulated by FGF–phosphorylated ERK in the posterior PSM and by the Notch pathway in the anterior PSM69. In zebrafish, the activities of Hes–Her dimers were also proposed to change between the anterior and posterior PSM80,81. Ultimately, the clock arrests after cells pass the determination front level. Below a threshold of FGF and WNT signalling, it is likely that the signalling requirements for the oscillatory state disappear, leading to the arrest of the clock. In mouse embryos, the oscillations of Notch, FGF and WNT targets display different travelling wave patterns in the PSM. For instance, Dusp4 and Axin2 exhibit a dynamic on–off expression sequence in the posterior PSM, whereas Lfng expression travels continuously anteriorly along the PSM. These different dynamics between Notch and FGF oscillators were proposed to cause a phase shift in the anterior PSM, whereby Notch and FGF oscillations are in phase in the posterior PSM but become progressively out of phase in the anterior PSM75. This results in the region anterior to the determination front becoming positive for Notch and negative for FGF, allowing Mesp2 activation (FIG. 3b). Interactions between the clock and the wavefront. According to the clock-and-wavefront model, the size of a segment is determined by the distance travelled by the wavefront during one oscillation of the clock. The clock and wavefront are often considered as independent entities, but several lines of evidence support a crosstalk between these two systems138. One of the earliest

observations in support of crosstalk was that some targets of the WNT and FGF pathways that are involved in setting the FGF–WNT posterior gradient system associated with the wavefront also oscillate12,14,139. This was integrated into the phase-shift model described above, which requires an interdependency between the clock (in this case as a periodic inductive signal) and the wavefront (in this case as a periodic competency signal)75. These interactions are also supported by experiments in zebrafish showing that the stepwise regression of the Fgf–phosphorylated Erk signalling front in the posterior PSM also depends on clock activity, as double knockdown of her1 and her7 leads to a smooth regression of the phosphorylated Erk front91. A striking illustration of this crosstalk was also recently provided using an in vitro culture system of the mouse PSM that recapitulated the oscillations and travelling waves of Lfng expression140. In this system, the posterior part of the PSM spreads in two dimensions and forms a cell monolayer that progressively becomes segmented from its periphery. The segments forming from the periphery become gradually smaller, scaling with the size of the remaining central PSM-like tissue. The authors analysed the pattern of the Lfng travelling waves and showed that the phase gradient of the Lfng waves scales with the size of the formed segments, thus predicting segment size. They propose that the spatial information required to position the segment boundary is encoded by the phase gradient of the clock, thus reducing the system to one parameter 141. This contrasts with classic models, in which the clock and wavefront are independent entities that are both required to specify positional information. However, the observations of the Doppler effect of the travelling waves in zebrafish in vivo135 argue against the scaling of the travelling waves observed in the mouse in vitro system. Whether this intriguing scaling property of the travelling waves is due to the in vitro constraints of the system or whether it reflects a difference between mice and zebrafish remains to be established.

Conclusion Vertebrate segmentation is mediated by intricate signalling processes that control the generation of oscillations. During the past decade, genetic analyses have identified the main players involved in somitogenesis, but their function has yet to be characterized in more detail, notably by using live imaging techniques and by controlled perturbations of the system. How other cellular processes affect this signalling must also be examined. Notably, a role for metabolism in the control of segmentation has recently emerged (BOX 2). Moreover, the signalling network underlying the clock has been shown to markedly vary among species21. Thus, to fully understand segmentation, we need to examine the role of each pathway and refine the concepts of ‘segmentation clock’ and ‘wavefront’. Indeed, it has become evident that there is not just one oscillator operating in the PSM, but rather distinct oscillatory loops with different dynamics and functions. Whether the pacemaker of the clock corresponds to this network of oscillators or whether these signalling networks are entrained by a still-unknown oscillator



REVIEWS (or oscillators) remains to be established. The clockand-wavefront model seemingly holds at the molecular level, as a Notch pulse and FGF–ERK signalling in mice match with the ideas of the clock and the wavefront, respectively. However, from a theoretical point of view, the cyclic nature of this wavefront and the existence of multiple interacting oscillators prompt us to revise this model and the choice of relevant parameters. The interactions and coordination of the wavefront and oscillator must be analysed through systems dynamics in order to further our understanding of segmentation. First, we must question the mechanism of oscillations: Ward, A. B. & Mehta, R. S. Axial elongation in fishes: using morphological approaches to elucidate developmental mechanisms in studying body shape. Integr. Comp. Biol. 50, 1106–1119 (2011). 2. Benazeraf, B. & Pourquie, O. Formation and segmentation of the vertebrate body axis. Annu. Rev. Cell Dev. Biol. 29, 1–26 (2013). 3. Chapman, D. L. & Papaioannou, V. E. Three neural tubes in mouse embryos with mutations in the T‑box gene Tbx6. Nature 391, 695–697 (1998). 4. Yoon, J. K., Moon, R. T. & Wold, B. The bHLH class protein pMesogenin1 can specify paraxial mesoderm phenotypes. Dev. Biol. 222, 376–391 (2000). 5. Nowotschin, S., Ferrer-Vaquer, A., Concepcion, D., Papaioannou, V. E. & Hadjantonakis, A. K. Interaction of Wnt3a, Msgn1 and Tbx6 in neural versus paraxial mesoderm lineage commitment and paraxial mesoderm differentiation in the mouse embryo. Dev. Biol. 367, 1–14 (2012). 6. Peel, A. D., Chipman, A. D. & Akam, M. Arthropod segmentation: beyond the Drosophila paradigm. Nature Rev. Genet. 6, 905–916 (2005). 7. Chal, J. & Pourquie, O. in The Skeletal System (ed. Pourquie, O.) 41–116 (Cold Spring Harbor Laboratory Press, 2009). 8. Tam, P. P. The control of somitogenesis in mouse embryos. J. Embryol. Exp. Morphol. 65 (Suppl.), 103–128 (1981). 9. Schroter, C. et al. Dynamics of zebrafish somitogenesis. Dev. Dyn. 237, 545–553 (2008). 10. Muller, F. & O’Rahilly, R. Somitic-vertebral correlation and vertebral levels in the human embryo. Am. J. Anat. 177, 3–19 (1986). 11. Palmeirim, I., Henrique, D., Ish-Horowicz, D. & Pourquié, O. Avian hairy gene expression identifies a molecular clock linked to vertebrate segmentation and somitogenesis. Cell 91, 639–648 (1997). Reports the identification of periodic expression of the cyclic gene HAIRY1 in the PSM of the chicken embryo, demonstrating the existence of an oscillator linked to vertebrate segmentation. 12. Dequeant, M. L. et al. A complex oscillating network of signaling genes underlies the mouse segmentation clock. Science 314, 1595–1598 (2006). A microarray time series spanning one oscillation of the segmentation clock is used to identify cyclic genes in the mouse at the transcriptome level. This led to the identification of a network of negative-feedback inhibitors expressed cyclically in the mouse PSM. 13. Dubrulle, J., McGrew, M. J. & Pourquie, O. FGF signaling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation. Cell 106, 219–232 (2001). Together with reference 22, reports the first identification of the molecular nature of the wavefront, showing that segment position is defined in response to a gradient of FGF signalling in the PSM. 14. Aulehla, A. et al. Wnt3a plays a major role in the segmentation clock controlling somitogenesis. Dev. Cell 4, 395–406 (2003). Identifies WNT signalling as an important element of the wavefront. 15. Saga, Y., Hata, N., Koseki, H. & Taketo, M. M. Mesp2: a novel mouse gene expressed in the presegmented mesoderm and essential for segmentation initiation. Genes Dev. 11, 1827–1839 (1997). 1.

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Acknowledgements

The authors thank members of the Pourquié laboratory and the referees for comments on the manuscript. A.H. is the recipient of a fellowship from the French Ministry of Higher Education and Research. Research in the Pourquié laboratory is funded by grants of the European Research Council (ERC), the French Muscular Dystrophy Association (AFM), the Human Frontier Science Program (HFSP) and the French National Agency for Research (ANR).

Competing interests statement

The authors declare no competing interests.


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