Cinquin Lab

Understanding the somitogenesis clock: what’s missing? Full Text

Olivier Cinquin
Howard Hughes Medical Institute and Department of Biochemistry, University of Wisconsin-Madison
433 Babcock Drive, Madison, WI 53706, USA

Mechanisms of Development 124(7-8), pp501-517 (2007)


Abstract

The segmentation of vertebrate embryos depends on a complex genetic network that generates highly dynamic gene expression. Many of the elements of the network have been identified, but their interaction and their influence on segmentation remain poorly understood. A few mathematical models have been proposed to explain the dynamics of subsets of the network, but the mechanistic bases remain controversial. This review focuses on outstanding problems with the generation of somitogenesis clock oscillations, and the ways they could regulate segmentation. Proposals that oscillations are generated by a negative feedback loop formed by Lunatic fringe and Notch signaling are weighed against a model based on positive feedback, and the experimental basis for models of simple negative feedback involving Her/Hes genes or Wnt targets is evaluated. Differences are then made explicit between the many `clock and wavefront’ model variants that have been proposed to explain how the clock regulates segmentation. An understanding of the somitogenesis clock will require addressing experimentally the many questions that arise from the study of simple models.

Keywords: somitogenesis clock; mathematical models; notch; lunatic fringe; her1; her7; her13.2; hes1; hes7; wnt; fgf; retinoic acid; clock and wavefront

1. Introduction

Somitogenesis is the process by which vertebrate and cephalochordate embryos acquire a segmented structure (Saga and Takeda, 2001). Somites form by budding off from the anterior end of the presomitic mesoderm (PSM), at regular intervals of about 2 hours in mouse, 90 minutes in chick, and 30 minutes in zebrafish. Although it loses cells anteriorly to somite formation, the PSM is replenished both by cell proliferation and ingression and grows posteriorly. The PSM is thus a dynamic structure that can for many purposes be considered to be at a steady state (even if its length and the period of somitogenesis vary with age, Packard, 1976, Tam, 1981). There is limited cell migration within the PSM (Kulesa, 2002, Stern, 1988), but cells do undergo a relative movement within the PSM, as PSM anterior and posterior borders move posteriorly.

The mechanism regulating the timing of new somite segmentation and somite size has been the object of much attention. A somitogenesis clock has been identified in chick, mouse, zebrafish, and frog, which consists of oscillatory gene expression in the PSM. For many of these clock genes, waves of expression originate at the posterior end and spread anteriorly (see Figure 1). The period of oscillatory expression is the same as the period of somitogenesis, and a new somite is formed when a wave of expression reaches the anterior end of the PSM.

* Figure 1:Schematic representation of the PSM, and of clock and wavefront models based on boundary specification (A) and on whole somite specification (B). Manipulation of the wavefront by grafted beads coated with FGF8 disrupts somite size regulation for both models (C). In the original clock and wavefront model (Cooke and Zeeman, 1976), wavefront progression was controlled by the clock (D). Color coding represents cell maturity (with posterior, blue cells being the least mature), and cross-hatching expression of a clock gene such as c-hairy1 (expression sweeps through the whole PSM during one clock cycle, three snapshots of which are shown; see section 3.2 for expression of other PSM genes). In (A) and (B), a new prospective somite or prospective boundary are defined periodically by the represented clock gene interacting with a `determination front’. Actual somite segmentation occurs a few cycles after specification (the four specified but yet-unsegmented somites depicted here correspond to the situation in chick). Note that the prospective boundaries do not define compartments, as cells can cross from one to the other (Kulesa, 2002, Stern, 1988).

Two major questions are how somitogenesis clock oscillations are generated, and how they regulate segmentation. Oscillating genes have mainly been found to belong to the Notch and Wnt signaling pathways, and more recently also the FGF pathway (Dequéant, 2006). The Notch pathway has been proposed to generate oscillations by forming a negative feedback loop with its modulator Lunatic fringe, but a positive feedback loop is another possibility (section 2.1). Her/Hes genes, which are downstream targets of Notch signaling, have also been proposed to generate oscillations by forming a simple negative feedback loop (section 2.2). The Wnt pathway, also potentially generating the oscillations (section 2.3), offers numerous possibilities for crosstalk between the signaling pathways active in the PSM (section 2.4). A question closely related to the generation of oscillations is how oscillations are coordinated between cells (section 2.5).

According to the `clock and wavefront’ model, first proposed by Cooke and Zeeman (1976), segmentation is regulated by the interaction of a clock and of a wavefront of cell maturity, with the clock determining the time of segmentation and the wavefront determining the spatial extent of the somite. Molecular identities of suitable clock and wavefront became known only after the model was formulated: the clock is that described above, and a possible wavefront depends on a gradient of FGF8 signaling within the PSM (Sawada, 2001, Dubrulle, 2001). There are many variants of the clock and wavefront model that it is important to distinguish (section 3). The wavefront corresponds to a boundary between anterior and posterior PSM, which can be defined in terms of the time segmentation is determined (section 3.1) and in terms of gene expression (section 3.2); the speed of wavefront progression is thought to determine somite size (section 3.3). Finally, some very intriguing phenomena remain unexplained (section 3.4).

2. Mechanism of the clock

Three main mechanisms have been proposed for the generation of somitogenesis clock oscillations: modulation of Notch signaling by Lunatic fringe, Her/Hes auto-repression, and negative feedback of Axin2 on the Wnt pathway. The proposed wiring of the zebrafish, chick and mouse clocks is summarized in Figure 21, and the mechanisms are reviewed in detail below.

*

Figure 2: Simplified networks that have been proposed to drive the oscillation in zebrafish (A), chick (B), and mouse (C) clocks. Zebrafish her1 and her7 genes are summarized as her; see Cinquin (2007) for a more detailed diagram. Chick and mouse hairy, hey, and hes genes are summarized as hes. NICD: Notch Intra-Cellular Domain. See main text for the detail of the mechanisms.

The three main proposed `modules’ are theoretically capable of driving oscillations on their own. They can be studied independently by making use of the relevant mutants or morpholino knockdowns, and they have been mainly considered independently. They have however been shown to interact in the mouse clock, and understanding this interaction will require more than knowledge of the mechanism of the individual parts.

It is not known, in any species, which set of genes `drives’ the oscillations, i.e. which set of genes must have oscillatory expression (in a wild-type background or in a mutant background in which expression of a set of other clock genes is forced to be constant) for other oscillatory genes to be expressed normally. Expression of some non-oscillatory genes is required for clock oscillations; these genes have a role that could be described as `permissive’ for the oscillations. It is all but impossible with present experimental techniques to determine which oscillating genes are driving the oscillations rather than permitting them. This is because it is relatively easy to knock out a gene or misexpress it, but not to enforce expression at any arbitrary level chosen by the experimenter; it is therefore impossible, for an oscillatory gene whose knocking out disrupts the clock, to test whether there is a level of constant expression of that gene that would allow the rest of the clock to continue oscillating normally.

2.1 Notch – Lunatic fringe: positive or negative feedback?

The Notch receptor is activated when its extracellular domain binds one of the Notch ligands. In the canonical pathway, proteolysis then releases the intracellular domain from the membrane, activating downstream signaling. Direct visualization of the cleaved receptor reveals that Notch signaling occurs cyclically in the PSM (Huppert, 2005, Morimoto, 2005). Lunatic fringe is a gene that is cyclically expressed in chick and mouse PSM (McGrew, 1998, Forsberg, 1998, Aulehla, 1999)2, and whose promoter is directly upregulated by Notch signaling (Morales et al., 2002, Cole et al., 2002)3. Lunatic fringe modifies the Notch receptor post-translationally, making it more sensitive to activation by its ligand Delta (Panin, 1997), and can also act on the Notch ligands (Panin, 2002). Disruption of Lunatic fringe expression disrupts that of other clock components (some stop oscillating, Dale, 2003, Serth, 2003, while Axin2 oscillates with an abnormal pattern, Aulehla, 2003).

Both negative and positive feedback loops can drive oscillations (see for example Goldbeter, 2002, for a review). Both types of feedback loops have been proposed to explain how the interaction between Notch and Lunatic fringe could drive the somitogenesis clock. In the negative feedback model, Lunatic fringe modifies Notch to make it less sensitive to activation by Delta. Oscillations would be generated by alternation between activation of Lunatic fringe expression by Notch signaling, and repression of Notch signaling by Lunatic fringe (Dale, 2003). Oscillations could also arise from a positive feedback loop between Notch signaling and Lunatic fringe, with Notch signaling driving Lunatic fringe transcription, and Lunatic fringe in turn modifying Notch to enhance its signaling activity (Cinquin, 2003)4. These latter oscillations are based on `substrate depletion’ (Tyson, 2003). Lunatic fringe and modified Notch build up until they reach a point where the oscillator `fires’ and most of the Notch receptor has been modified by Lunatic fringe. The reaction then dies off because of the lack of unmodified Notch for Lunatic fringe to act on, and rapid decay of the modified receptor, which in turn causes Lunatic fringe transcription to shut off, and the oscillator to return to its original state.

Manipulation of Lunatic fringe expression has been used to investigate regulatory interactions. Misexpression of Lunatic fringe results in downregulation of endogenous Lunatic fringe expression, with loss of oscillations or oscillations of reduced amplitude (Dale, 2003, Serth, 2003). It has more recently been observed that Notch signaling becomes constantly active in Lunatic fringe mutants (Morimoto, 2005). The negative feedback model can account for these data: misexpression of Lunatic fringe would reduce Notch signalling and thus transcription of endogenous Lunatic fringe. Loss of Lunatic fringe would lead to the loss of cyclic repression of Notch signaling. However, the positive feedback model can also explain the experimental phenotypes. Indeed, depending on the level of Lunatic fringe misexpression, the model predicts either damped oscillations of endogenous Lunatic fringe expression or oscillation arrest and expression of the endogenous gene at a low baseline. This is exactly what is observed experimentally, with oscillation arrest in chick (with a misexpression system that is geared toward high levels of misexpression, Dale, 2003), and oscillation damping in mouse (with a misexpression system that is likely providing weaker misexpression, Serth, 2003). In the positive feedback model, this results from Lunatic fringe expression depleting the pool of unsensitized Notch by converting it to the sensitized form, leading to an overall depletion of Notch levels because the sensitized form is assumed to have a shorter half-life than the unsensitized one (because it is more readily used up for signaling). The level of Notch signaling is reduced to intermediary levels between climaxes and troughs of its normal oscillatory activity, leading to reduced Lunatic fringe expression. Conversely, knocking out Lunatic fringe in the model positive feedback oscillator leads to constant Notch signaling, at a level intermediary between troughs and peaks of normal signaling (O. Cinquin, unpublished); this seems consistent with the observations made by Morimoto (2005).

The positive feedback model also accounts for data that do not fit in the negative feedback model. Knocking in a lacZ reporter into the Lunatic fringe locus leads to a smear of expression in the homozygote, at levels which seem to be lower than peak expression levels in heterozygotes carrying one wild-type copy of Lunatic fringe and one lacZ knockin copy (Chen, 2005).

It has been proposed that interlocked positive and negative feedback loops could contribute to the robustness of circadian oscillations (Cheng, 2001, Ueda, 2001, Hong, 2007). It would therefore not be surprising if there were positive feedback circuits in the somitogenesis clock. The circuitry of the circadian clock has proven to be complex, but most interestingly the first in vitro reproduction of circadian oscillations has suggested a possible positive feedback mechanism (Nakajima, 2005, Mehra, 2006), rather than negative transcriptional feedback as previously thought (Ishiura, 1998). Also, although negative transcriptional feedback has long been considered the basis for circadian rhythmicity, much evidence is accumulating that questions this dogma (reviewed by Lakin-Thomas, 2006). It is important to distinguish between positive and negative feedback in the somitogenesis clock, because the two mechanisms are expected to impart very different properties to the oscillators (for example in terms of responses to perturbations in biochemical rates and delays).

There are many elements in the somitogenesis clock, and it is possible that Lunatic fringe does not mediate its effects on the Notch receptor (or its ligands) directly. It is also a possibility that Lunatic fringe really does inhibit Notch activation by Delta, contrary to what has been observed more directly in other experimental systems. However, a model with a positive feedback loop between Notch and Lunatic fringe seems to be the most parsimonious explanation of current experimental data.

Finally, transcription of the Notch ligand Dll1 has recently been observed to be oscillatory in mouse PSM (Maruhashi, 2005), similar to oscillatory transcription of the Notch ligand DeltaC in zebrafish PSM. This might not provide a mechanism for autonomous oscillations of the Notch pathway, as it is not straightforward for a simple positive feedback loop to drive oscillations without substrate depletion, even when delays are taken into account (Smolen, 1999, Smith, 1987). However, since Dll1 transcription is under control of Wnt signaling (Hofmann, 2004, Galceran, 2004), Dll1 could provide a molecular link between the Wnt and Notch pathways. It is thus possible that oscillatory activation of the mouse Notch pathway is downstream of Wnt pathway oscillations, as proposed by Aulehla (2003), but the disruption of Notch oscillations by mutations of members of the Notch pathway suggests that Notch pathway oscillations are not just a passive response of periodic stimulation by Wnt signaling.

2.2 Her/Hes

A number of Her/Hes genes, which encode transcriptional repressors of the basic helix-loop-helix (bHLH) family often downstream of Notch signaling (Iso, 2003), are expressed dynamically in the PSM (Holley, 2000, Oates, 2002, Bessho et al., 2001b, Jouve, 2000, Leimeister, 2000, Palmeirim et al., 1997). The expression of one of these genes, mouse Hes1, oscillates in cultured cells in response to serum shock (Masamizu, 2006, Hirata, 2002; see also William, 2007, for human cells), and it has been proposed that delayed Hes1 autorepression could be the basis for this oscillation (Lewis, 2003, Jensen, 2003, Barrio, 2006, Monk, 2003). Since additionally the period of Hes1 oscillations is the same in cultured cells and in mouse PSM, this raises the possibility that Hes1 self-repression is also a mechanism for driving the somitogenesis clock. However, Hes1 mouse knockouts have no somitogenesis phenotype (Ishibashi, 1995, Ohtsuka, 1999), showing that Hes1 autorepression, if it does occur, is not necessary; Hes1 could be redundant with other factors of the clock.

Hes7 is a related protein that is essential for mouse somitogenesis (Hirata, 2004, Bessho et al., 2001b), and it has been proposed that delayed self-repression of Hes7 is responsible for its oscillatory expression (Hirata, 2004; see also Bessho, 2003). The difference in the phenotypes of Hes1 and Hes7 knockouts could be due to differences in dimers they form in vivo. Hes7 has been shown to be unable by itself to significantly repress transcription from its own promoter, in a model system involving cultured cells (Chen, 2005, Bessho et al., 2001a; it is however a possibility that Hes7 can repress its own expression in combination with other factors, present in the PSM but not in cultured cells, or that Hes7 is part of a negative feedback loop involving other factors). Interestingly however, Hes7 can block Notch-mediated activation of its promoter (Chen, 2005, Bessho, 2003). Because Notch activation is cyclic in the PSM and depends on other factors such as Lunatic fringe, potential self-repression of Hes7 cannot be considered independently of the rest of the clock. Stabilization of Hes7 protein (engineered by a point mutation) leads to a disruption of oscillations, which can be explained within the framework of a simple self-repression model (Hirata, 2004), provided that strict requirements are met on the precise mechanism of Hes7 self-repression (Zeiser, 2006). Another possibility is that the average level of Hes7 repression must be within a certain range to allow functioning of the clock.

Two zebrafish her genes, her1 and her7, show oscillatory expression in the PSM and are essential for clock oscillations and normal segmentation (Oates, 2002, Henry, 2002, Holley, 2002). Her1 represses expression of its promoter in a cell-culture assay (Kawamura, 2005), and self-repression of her genes has been proposed as a basis for the oscillations of the zebrafish clock (Lewis, 2003, Oates, 2002, Holley, 2002). A problem with that model is that morpholino blocking of her1 or her7 mRNA translation can lead to downregulation of their transcription (Gajewski, 2003), while a simple negative feedback loop would predict upregulation of transcription if the repressor protein cannot be translated. Her1 and Her7 are bHLH proteins, a family whose members normally function as dimers (Davis, 2001), and reproduction of knockdown phenotypes can be achieved by taking into account the formation of Her dimers that repress transcription with different potencies (Cinquin, 2007).

In summary, it is possible that Hes autorepression is part of the `core’ mechanism of the mouse somitogenesis clock. Hes7 autorepression has however only been shown to occur in the presence of Notch signaling, which is itself cyclic, and it is therefore not possible at present to assume that Hes7 self-repression is by itself the core mechanism of the somitogenesis clock. Her autorepression in zebrafish does not carry that complication, at least in the case of Her1, but it has not been established whether Notch signaling is necessary for the existence of the oscillations or for intercellular synchronization (Holley, 2000, Jiang, 2000, Holley, 2002). In vivo imaging of oscillations in Delta mutants (Jiang, 2000) could resolve that latter question.

2.3 Wnt signaling

Wnt/ -catenin signaling is cyclic in mouse PSM (Aulehla, 2003), and induces cyclic transcription of the Wnt signaling inhibitors Axin2 (Aulehla, 2003) and Nkd1 (Ishikawa, 2004); the Dact1 (Suriben, 2006), and Dkk1 (Dequéant, 2006) inhibitors also show oscillatory expression. Axin2, Dact1, and Dkk1 phases of oscillation are closely related, and distinct from the phases of Notch-related genes and Nkd1. Wnt3a mRNA is expressed in the mouse tailbud and it has been proposed that Wnt3a protein forms a posterior-anterior gradient in the PSM, because Axin2 is downstream of Wnt signaling and oscillates with a higher amplitude in posterior PSM than in anterior PSM (Aulehla, 2003).

Axin2 is related to Axin, a critical component of the Wnt signaling pathway that acts as a scaffold for the -catenin destruction complex (Logan, 2004). Axin and Axin2 are functionally equivalent (Chia, 2005) but their expression is differentially regulated: Axin is ubiquitous while Axin2 expression is tissue-specific and downstream of Wnt signaling (Leung, 2002, Jho, 2002). Based on the persistence of some Axin2 oscillation in the Notch pathway mutant Dll1, and on the disruption of Notch pathway oscillations in the Wnt3a hypomorphic mutant vestigial tail, Aulehla (2003) proposed that a Wnt signaling – Axin2 negative feedback loop is responsible for driving the mouse somitogenesis clock. Wnt signaling would drive transcription of Axin2 mRNA, and Axin2 protein would inhibit Wnt signaling. However, Axin2 mutants have no somitogenesis phenotype (Yu, 2005), which suggests that either Axin2 does not play a significant role in the generation of the oscillations, or that it functions in the clock redundantly with other genes.

Nkd1 inhibits Wnt signaling by binding to the mediator Dishevelled (Wharton, 2001, Rousset, 2001), and stands out among oscillatory Wnt signaling inhibitors in that it oscillates in phase with Notch-related genes (Ishikawa, 2004). Its expression is dependent on Wnt signaling (as assayed with the vestigial tail hypomorph), but oscillatory expression is disrupted in the Notch pathway mutant Hes7, suggesting that Wnt oscillations might not be independent of the Notch pathway. Mouse Nkd1 knockouts have no reported somitogenesis phenotype (Li, 2005), which is not due to redundancy with the similar gene Nkd2 (Zhang, 2007).

Dact1, also known as Dpr1/Frd1, also interacts with Dishevelled (Cheyette, 2002) and can promote its degradation (Zhang, 2006), antagonizing Wnt signaling. In different contexts Dact genes can however promote Wnt signaling (Waxman, 2004, Gloy, 2002), and it has been proposed that members of the Dact family could regulate expression of Wnt pathway genes in parallel to the canonical pathway (Hikasa, 2004).

Finally, Dkk1 is a secreted inhibitor of Wnt signaling (Glinka, 1998; reviewed by Niehrs, 2006), whose expression is induced by Wnt signaling (Niida, 2004, Chamorro, 2005). Oscillation of Dkk1 mRNA could only be detected by the use of an intronic probe (Dequéant, 2006), suggesting that even though transcription is rhythmic, stability of the mRNA might prevent it from undergoing high-amplitude concentration oscillations. Mouse Dkk1 knockouts have segmentation defects whose causes have not yet been investigated (MacDonald, 2004).

Wnt signaling thus offers a number of simple negative feedback loops, any of which could theoretically be able to single-handedly drive the somitogenesis clock (similar negative feedback loops exist for the Notch and FGF pathways, with oscillation of the inhibitors Nrarp and Sprouty2, Dequéant, 2006). However, even though Wnt signaling is critical for normal functioning of the clock, only one of the Wnt inhibitors tested so far (Dkk1) has proven necessary by itself. Negative feedback is a ubiquitous feature of signaling pathways (Freeman, 2000), and it would probably be undesirable for all feedback inhibitors to cause their signaling pathway to enter an oscillatory state. It is possible that a combination of Wnt inhibitors acts redundantly to cause Wnt signaling to be oscillatory in the mouse PSM, that some members of the pathway have not been identified, that the mouse Wnt pathway is in fact unable to generate oscillations in isolation, or that it is able to generate oscillations in isolation by an unknown mechanism that could be much more sophisticated than a single negative feedback loop. The latter possibility is intriguing given the great complexity of Wnt signaling. A biochemical study of Wnt signaling has for example shown that paradoxical effects can occur, with high concentrations of Axin stabilizing -catenin rather than promoting its destruction (Lee, 2003). Also, oscillations in the PSM have been almost exclusively studied at the mRNA level, but Wnt signaling components such as Axin are heavily regulated post-translationally (Yamamoto, 1999, Willert, 1999). Inhibition of protein synthesis blocks the chick somitogenesis clock after at most one round of oscillation (depending on the gene readout; McGrew, 1998, Palmeirim et al., 1997), but this does not exclude a role for post-translational modifications having a role in the generation or shaping of the oscillations. This is because a block in protein synthesis could arrest the clock by leading to a depletion of short-lived proteins, rather than by breaking an essential transcription – translation feedback loop.

2.4 Crosstalk

Crosstalk between the pathways involved in the somitogenesis clock has been extensively documented in different contexts. ERK — downstream of the FGF pathway in chick, zebrafish, and frog PSM — can phosphorylate Groucho, a cofactor in the repression of Notch and Wnt targets in the absence of signaling, and thereby attenuate its activity (Hasson, 2005; reviewed by Hasson, 2006 and Sundaram, 2005). ERK also regulates cleavage of the Notch receptor (Kim, 2006). Within the context of the zebrafish clock, FGF drives transcription of her13.2, which in turn influences her autorepression (Kawamura, 2005). The kinase Akt, downstream of FGF signaling in mouse (Dubrulle, 2004), can influence Wnt signaling in a variety of ways (Gherzi, 2006, Fang, 2007, Fukumoto, 2001). Thus, FGF signaling can influence Notch and Wnt signaling.

Notch signaling can regulate -catenin activity, through a non-canonical pathway in conjunction with Axin (Hayward, 2006,2005), or through induction of Hes1 (Deregowski, 2006). Notch signaling can induce expression of MAPK phosphatases (Lee, 2006, Kondoh, 2007, Berset, 2001), potentially antagonizing FGF signaling. Within the context of the zebrafish clock, the Notch pathway has been implicated in the maintenance of Cyp26 expression (Echeverri, 2007), providing a potential interaction with the FGF pathway (see section 3.2).

Wnt signaling can impinge on activation of MAPK (Park, 2006, Yun, 2005, Jeon, 2007). Dishevelled can also inhibit Notch signaling by physical interaction with Notch (Axelrod, 1996, reviewed by Blair, 1996, and Ramain, 2001). Thus, Wnt signaling can influence FGF and Notch signaling. Within the context of the mouse clock, Wnt signaling has been proposed to control expression of FGF8 (Aulehla, 2003), and Wnt pathway manipulation alters Notch pathway activation (Aulehla, 2003), although the molecular mechanism is not clear. A possible molecular mechanism involves Snail genes, which are transcriptional repressors5 involved in epithelial to mesenchymal transitions (Barrallo-Gimeno, 2005). Snail1 (mouse) and Snail2 (chick) expression is oscillatory, and depends on Wnt and FGF signaling (Dale, 2006), consistent with dependence of the Snail1 promoter on ERK and PI3 signaling (Peinado, 2003, Barberà, 2004). Snail2 overexpression represses Lunatic fringe cell-autonomously (and leads to loss of PSM identity). Interestingly, Snail1 represses its own expression, adding to the number of negative feedback loops that have been proposed to drive the somitogenesis clock (Peiró, 2006).

Finally, the Ras-binding protein Canoe, also known as AF6 or Afadin, is expressed in PSM (Ikeda, 1999) and modulates the Notch, Wnt, and Ras pathways in Drosophila (Carmena, 2006). There are thus numerous ways in which the three signaling pathways known to be active in the PSM could be linked. It will be essential to determine exhaustively which molecular interactions actually occur in the PSM, and how they contribute to the generation or shaping of the clock oscillations.

2.5 Intercellular clock synchronization

Early experiments on the clock showed that oscillatory expression could go on after sectioning of chick and mouse PSM and separate incubation (Forsberg, 1998, McGrew, 1998, Palmeirim et al., 1997; this has been confirmed for many genes of the clock, and more recently investigated in detail by cutting chick PSM into more than two pieces, Maroto, 2005). This was first interpreted as suggesting that the clock was cell-autonomous; there are however a number of reasons to expect some form of coupling (Cinquin, 2003, Jiang, 2000).

It has recently been shown that dissociation of mouse PSM cells does not disrupt individual oscillations, but leads to a loss of synchrony between cells (Masamizu, 2006). This strongly suggests that local coupling keeps cells oscillating in synchrony, which has been shown directly in zebrafish by the synchronization of PSM grafts (Horikawa, 2006).

The potential mechanism for intercellular synchronization of the zebrafish clock seems reasonably straightforward, as oscillations of the Notch ligand Delta could bring neighboring cells into synchrony (as shown in silico by Lewis, 2003 and Cinquin, 2007). This is also a possibility in the mouse clock, as transcription of the Notch ligand Dll1 is oscillatory (Maruhashi, 2005). In zebrafish, it has been proposed that DeltaC, whose expression is oscillatory, acts to synchronize adjacent cells while DeltaD, whose expression is not oscillatory, would prime expression of clock genes in progenitors (Mara, 2007).

Another potential mechanism for the mouse and chick clocks is provided by Lunatic fringe, which potentiates Notch activation by Delta, has oscillatory expression, and has been observed to be secreted in some contexts (Wu, 1996, Panin, 1997). It has been suggested that Lunatic fringe only functions intracellularly, based on genetic analysis of the Drosophila wing disc (Irvine, 1994, Kim, 1995), on the fact that Lunatic fringe localizes predominantly to the Golgi (Hicks, 2000), as does Drosophila Fringe (Munro, 2000), and on the facts that forcibly-secreted Fringe is less active than wild-type Fringe (Munro, 2000) and secreted Fringe does not have detectable activity when applied to cells cultured in vitro (Brückner, 2000). However, predominant localization of Lunatic fringe to the Golgi does not preclude activity at other sites (perhaps with lower activity), and extracellular activity could be context-dependent, especially since the activity of Fringe proteins requires UDP-N-acetylglucosamine to transfer to acceptor proteins (Moloney, 2000). Medium conditioned by cultured cells secreting Fringe protein has inductive activities on Xenopus embryos (Wu, 1996), and misexpression in the Drosophila wing disc of Golgi-tethered Fringe (which is not detectably secreted in cultured cells) and wild-type Fringe does not have the same effect (compare Figures 2f and 2g of Brückner, 2000).

Secretion and extracellular activity of Lunatic fringe in chick and mouse PSM thus seems a possibility, which can lead to local cell synchronization in simulations of the somitogenesis clock based on the interaction of Lunatic fringe and the Notch receptor (Cinquin, 2003). It is however also possible that synchronization is mediated by some other factor, or combination of factors (perhaps involving Wnt signaling in mouse).

The problem of synchronization is not straightforward to address experimentally. It seems that at present there is no method to visualize individual cellular oscillations in vivo, which would make it possible to study the correlation between the dynamics of expression of neighboring cells. Visualization of individual, dissociated cells is possible, but the loss of cell-cell contact probably leads to the loss of intercellular communication, and PSM dissociation probably also alters FGF8 signaling (see for example Kuroda, 2005), and therefore possibly also the clock (see below). PSM grafts, as performed by Horikawa (2006), could be coupled to real-time in vivo imaging to provide detailed kinetic data that could be used to test mathematical models.

Spatiotemporal pattern of the clock

The relative timing of cellular oscillators in the PSM is such that, for many of the oscillatory genes, waves of expression propagate from the posterior end to the anterior end, shrinking in the process. As noted above, the propagation of these waves is not detectably affected, at least for a few cycles, by sectioning of the PSM. It is likely that oscillations are shaped by positional information in the PSM, the FGF8 gradient being an obvious candidate.

Modulation of the strength of intercellular coupling, by an unspecified molecular mechanism, can lead to in silico reproduction of the spatiotemporal pattern of the clock (Cinquin, 2003). More interestingly, a molecular link has been uncovered in zebrafish between the Notch and FGF signaling pathways: Her1 autorepression is enhanced by heterodimerization with Her13.2, whose transcription is FGF-responsive (Kawamura, 2005). In a molecular model of the zebrafish clock, this link is sufficient to establish the clock spatiotemporal pattern (Cinquin, 2007). Testing of that model will require further experimental data on dimerization of Her proteins.

3. Clock & wavefront

The clock and wavefront model was proposed by Cooke and Zeeman (1976), based on experimental data from Xenopus. One of the major goals of the model was to explain the powerful regulation of somite number: that number varies very little with changes in embryo size, whether natural or experimentally-induced (Cooke, 1975); somite cell number, rather than somite count or cell size, changes in response to alterations in total body size. In the model, the progression of a wavefront of cell maturity was supposed to be alternatively inhibited and accelerated by an oscillator, so that cells destined to segment together would be reached by the wavefront, and undergo a change in adhesive properties, within a short time-frame. Regulation of somite size (and therefore somite number) was proposed to depend on an early positional information gradient, formed by the diffusion from a source and the destruction by a sink of a morphogen molecule. The positional information gradient would set up the original pattern of cell maturity, and ensure that the PSM contains the same number of prospective somites, irrespective of its size.

The regulative abilities of PSM have been argued to be much weaker in species other than Xenopus (Primmett et al., 1988), and the question of size regulation has not been addressed after the molecular identities of somitogenesis regulators were established. The clock and wavefront model has however become very popular after the discovery of candidates for the clock (Palmeirim et al., 1997) and the wavefront (Sawada, 2001, Dubrulle, 2001). It is crucial to note that what is referred to as the `clock and wavefront’ model in the recent literature is conceptually very different from the original model described by Cooke and Zeeman (1976). Two differences are that

  • the original clock and wavefront model supposes that segmentation happens at the level of the whole somite, by a change in locomotory and/or adhesive properties of all cells (Figure 1A), while for other models (such as that of Dubrulle, 2001) it is the somite boundaries, rather than the somites themselves, that are specified: cells are induced to become boundary cells if they are reached by the wavefront at a specific phase of their oscillation (Figure 1B). This latter model, in which a border is formed prior to somite compaction, would be compatible with data from zebrafish mutants that show border specification without somite compaction (Henry et al., 2000). Experiments in chick suggest however that somite border formation is induced by the juxtaposition of Lunatic fringe-expressing and non-expressing cells (Sato, 2002), and would therefore not be directly specified by the clock and wavefront.
  • the original clock and wavefront model, contrary to a common assumption, supposes that the wavefront of maturity is pushed posteriorly by each clock pulse (Figure 1D); this is somewhat similar to the progressive activation of Hox genes by clock pulses proposed by Zákány (2001). In more recent incarnations of the model (for example that of Dubrulle, 2001) wavefront and oscillator do not influence each other, and interact only to regulate segmentation: the wavefront is supposed to progress independently of the clock, at constant speed.

Local manipulation of FGF8 signaling does affect the spatiotemporal pattern of clock gene expression (Sawada, 2001, Dubrulle, 2001), which shows that clock and wavefront are not independent. Retinoic acid signalling, thought to oppose FGF8 signaling, has been shown to have a role in the setup or maintenance of oscillation synchrony between the left and right sides of the PSM (Kawakami, 2005, Vermot et al., 2005, Vermot, 2005). Even stronger evidence for FGF signaling acting on the clock is the direct molecular link identified in zebrafish (Kawamura, 2005).

Importantly, shaping of clock gene expression by FGF8 signaling does not mean global inhibition of FGF signaling should result in short-term disruption of clock oscillations. This is because if changes in levels of FGF8 signaling result in phase-shifting of the clock (as observed in silico by Cinquin, 2007), uniform inhibition of FGF8 signaling in the whole PSM could merely block phase-shifting or cause global phase shifting, resulting in a spatiotemporal pattern that is grossly similar, at least for a few cycles, to a wild-type pattern.

A complication about the wavefront is that transcription of elements of the FGF8 signaling pathway has been reported to oscillate in the posterior PSM in phase with that of Notch targets, blurring the distinction between clock and wavefront (Dequéant, 2006; oscillations of ERK phosphorylation have however not been reported by Sawada, 2001, or Delfini, 2005). This does not seem to preclude the FGF8 pathway from playing its role in the `clock and wavefront’ models, if for example its activity needs to drop over a period of time greater than the oscillation period for PSM cells to switch from posterior to anterior identities.

3.1 Determination front

It was first observed in Xenopus and Rana that heat shocks disrupt somitogenesis, but only after a few normal somites have formed (Elsdale, 1976, Pearson, 1979). In Rana, around 3 to 4 normal somites are formed before the onset of the perturbations (the number is 5 in Xenopus); importantly, that number varies little with the somitogenesis stage at which the heat shock is applied. This led the authors to propose the existence of a `prior wave’ of somite determination, similar to the wavefront of somite formation but located more caudally. Cells reached by that wave would be distributed into somites, but that distribution would only become visible at the actual time of segmentation (Figure 1A). Heat shocks were proposed to affect somite delimitation by the `prior wave’. Disruption of somitogenesis by heat shocks is a phenomenon shared (with some differences) across species; the first defects occur after 6 to 7 somites in chick (Stern, 1988), 4 to 5 somites in zebrafish (Roy, 1999), 4 somites in rats (Cuff, 1993), but less than 2 somites in mouse (Li, 1999).

The existence of a chick `determination front’ has been addressed more directly by Dubrulle (2001), who inverted the anteroposterior axis of one-somite long PSM regions. Such inversions disrupt segmentation only when performed anterior to somite -IV (see Figure 1A for prospective somite numbering after Pourquié, 2001). Consistent with this, grafting of FGF8 coated beads can affect segmentation only of cells located posterior to somite -IV (Figure 1C), even though anterior cells do respond to FGF8 beads, at least in terms of ERK phosphorylation (as shown in Figure 1 of Delfini, 2005). A similar zebrafish determination front has been proposed by Sawada (2001), who observed normal segmentation of 4 to 5 somites in zebrafish after soaking embryos in an FGF8 inhibitor.

It follows from the data in the previous paragraphs that if an FGF8 wavefront is to specify chick somite boundaries in conjunction with the clock, that specification must occur around somite -IV (in chick), and that is where the wavefront relevant to segmentation models must be. This is consistent with the concentration of FGF8 (at least at the mRNA level) varying quickly in that region, compared to other places in the PSM (Figure 1E-H of Dubrulle, 2001): for specification to occur precisely and to lead to reproducible somite lengths, the FGF8 concentration should show a sufficiently steep gradient. Downregulation of FGF and Wnt receptors has been found to be necessary in frog for correct positioning of the wavefront (Nagano, 2006), which is interesting given that receptors can interfere with morphogen gradient formation (Kerszberg, 1998; see Lander, 2007, for a review).

3.2 Spatially-regulated expression in the PSM

Anterior and posterior PSM can be distinguished by differential gene expression. Posterior PSM expresses pMesogenin1 (mouse, with an anterior border around somite -II; Yoon, 2000), also known as Mespo (frog, with an anterior border around somite -III, Joseph, 1999; zebrafish, Yoo, 2003) and cMespo (chick, with an anterior border around somite -III, Buchberger, 2000). The domain of pMesogenin1 expression abuts that of Paraxis (Yoon, 2000), a bHLH gene required for somite epithelialization (Burgess, 1996) and expressed in anterior PSM (Burgess, 1995).

Anterior PSM expresses pairs of homologous bHLH genes whose expression patterns closely overlap: Mesp1 and Mesp2 (1 stripe in mouse, Takahashi, 2007, Saga, 1997), also known as Thylacine1 and Thylacine2 (2 stripes in frog, Sparrow, 1998), c-meso1 and c-meso2 (1 to 2 stripes in chick, Buchberger, 1998), mesp-a and mesp-b (1 to 3 stripes in zebrafish, Sawada, 2000).

The border between anterior and posterior PSM, as defined by the genetic expression just described, apparently correlates with the determination front mentioned above: the segmentation of anterior cells seems to be determined, and unaffected by heat shocks. In zebrafish, the T-box transcription factor fss/tbx24 has a role in mediating the transition between posterior and anterior PSM (Holley, 2000, Nikaido, 2002, Holley, 2002).

Control of the wavefront

Reduced FGF8 signaling is thought to be the crucial cue causing the transition from posterior identity to anterior identity. FGF8 mRNA is expressed in a gradient in the PSM, with strong posterior expression (Sawada, 2001, Dubrulle, 2001). Grafting of FGF8 beads, or misexpressing FGF8 throughout the PSM, enforces a posterior identity (Dubrulle, 2001). Dubrulle (2004) showed that FGF8 mRNA is transcribed in PSM progenitors but not in the PSM itself, and proposed that decay of the mRNA underlies the gradient: because there is a loose correlation between the anteroposterior position of a cell within the PSM and the time it has been in the PSM, a longer time has elapsed since the stop of FGF8 mRNA transcription in anterior cells than in posterior cells, and anterior cells therefore have a smaller remaining pool of FGF8.

Interestingly however, neither a zebrafish FGF8 mutant nor a mouse conditional knockout that loses FGF8 expression in the PSM show a somitogenesis phenotype (Perantoni, 2005, Reifers, 1998). Potential explanations for the lack of a phenotype include ligand redundancy, diffusion of FGF8 from other sources in the embryo, or perhaps redundancy with Wnt signaling. Wnt signaling has been proposed to control the FGF8 gradient in mouse (Aulehla, 2003), but experiments in frog suggest that Wnt signaling might be able to act in parallel to FGF to position the wavefront (Nagano, 2006).

Antagonism between FGF8 and retinoic acid

FGF8 signaling has been proposed to act in opposition with retinoic acid signaling in the limb bud (Mercader, 2000) and in PSM (Diez del Corral, 2003). Chick PSM contains high levels of retinoic acid during early somitogenesis; retinoic acid is subsequently downregulated in posterior PSM (Maden, 1998). The situation in mouse is comparable, with retinoic acid signaling activity first present in anterior PSM, but retracting further anteriorly after the first 10 somites are formed (Sirbu, 2006). Retinoic acid can be synthesized from retinaldehyde (itself derived from vitamin A) by any of a handful of enzymes (Chambers, 2007, Duester, 2003, Lin, 2003). Retinoic acid in the PSM is thought to be produced by RALDH2, which is expressed in PSM and somites and follows a similar anterior retraction during early somitogenesis as observed for retinoic acid signaling activity (Chen, 2001, Grandel, 2002, Xavier-Neto, 2000, Blentic, 2003, Swindell, 1999, Sirbu, 2006). Cyp26 degrades retinoic acid and is expressed in posterior PSM in chick during early somitogenesis (Blentic, 2003, Swindell, 1999), in mouse (Fujii, 1997), in zebrafish (Dobbs-McAuliffe, 2004), and in frog (de Roos, 1999). Expression of the retinoic acid synthesizing and degrading enzymes in distinct domains — source and sink — has been proposed as a mechanism to establish a morphogen gradient, in a different context (Swindell, 1999, Maden, 1999).

Treatment of chick PSM explants with retinoic acid downregulates FGF8 (Diez del Corral, 2003); in frog, treatment with retinoic acid actually induces FGF8, but also induces MKP3 — an inhibitor of FGF signaling — and does expand PSM of anterior character at the expense of posterior PSM (Moreno, 2004). Ectopic retinoic acid signaling in the posterior PSM of mouse Cyp26 knockouts downregulates the posterior markers FGF8 and Wnt3a (Sakai, 2001, Abu-Abed, 2003). Conversely, disruption of retinoic acid synthesis by targeting of Raldh2 expression, by incubation with chemical inhibitors, or by vitamin A deprivation, results in upregulation of FGF8 in mouse (Vermot et al., 2005, Molotkova, 2005), chick (Vermot, 2005, Diez del Corral, 2003) and zebrafish (Kawakami, 2005). Retinoic acid signaling is thus likely to repress FGF8 signaling.

Ectopic FGF8 signaling, or mimicking constitutively-active FGF signaling, prevents expression of Raldh2 (Delfini, 2005, Diez del Corral, 2003). FGF8 signaling is thus likely to repress retinoic acid signaling.

Sirbu (2006) have recently brought crucial precisions to the model of antagonism between FGF8 and retinoic acid. Firstly, retinoic acid signaling is only necessary for normal somitogenesis during early stages; thus, even though experimental manipulation of the retinoic acid pathway affects FGF8 expression, such regulation seems to happen in vivo only at a specific stage. This is compatible with normal segmentation of posterior PSM that has been physically separated from anterior PSM and should therefore not have any anterior source of retinoic acid (Packard, 1978). Secondly, endogenous retinoic acid acts to limit FGF8 expression in the ectoderm that overlies the PSM, rather than in the PSM itself (the PSM was proposed to be less sensitive to retinoic acid because of the expression of COUP-TFII, which antagonizes the action of retinoic acid receptors). This suggests that diffusion of FGF8 from the ectoderm might impact the PSM. Finally, it is noteworthy that mouse reporters suggest that retinoic signaling is not graded along the PSM; rather, there is a sharp boundary of signaling in anterior PSM (Vermot et al., 2005). This sharp boundary could be due to the ability of retinoic acid receptors to provide sharp thresholds in their transcriptional readout of retinoic acid concentrations (Kerszberg, 1996).

3.3 Control of somite size

According to the clock and wavefront model, somite size along the anteroposterior axis is determined by the distance traveled by the wavefront during one period of oscillation of the somitogenesis clock (whether it is the whole somite or its boundaries that are specified). Supposing that the PSM is at steady state, the distance traveled by the wavefront is the same as the distance the PSM grows posteriorly in the same interval. According to a model of cell-autonomous decay of FGF8 mRNA defining the wavefront (see above), for a given rate of decay of FGF8 mRNA and a given starting pool when cells join the PSM, the speed of posterior progression of PSM sets the size of somites. Thus a way to test that model (at a stage where retinoic acid does not counteract FGF signaling) would be to alter the speed of posterior progression of the PSM, without affecting the rate of the clock; unfortunately there does not seem to be a known way of doing that at present.

Manipulation of the wavefront has been achieved by disruption of retinoic acid synthesis (Maden, 2000, Vermot et al., 2005, Niederreither, 1999, Sirbu, 2006, Vermot, 2005, Diez del Corral, 2003), incubation with exogenous retinoic acid (Moreno, 2004), disruption of FGF signaling with a chemical inhibitor or grafting of FGF8-coated beads (Delfini, 2005, Sawada, 2001, Dubrulle, 2001), disruption of Wnt signaling (Nagano, 2006), or grafting of Wnt-secreting cells (Aulehla, 2003). Results consistently support the idea that increased (respectively decreased) FGF8 or Wnt signaling pushes the wavefront anteriorly, resulting in smaller (respectively larger) somites, and that the opposite is true for retinoic acid. The shift of sites of retinoic acid signaling during early somitogenesis offers a complication of the clock and wavefront model that has not yet been investigated in detail. It could be related to the difference in the segmentation mechanism of early somites that makes them more resilient to clock perturbations (Jülich, 2005), and to the segregation of PSM progenitors depending on the time at which they contribute to the PSM (Szeto, 2006; reviewed by Holley, 2006).

3.4 Unexplained phenomena

A particularly intriguing feature of PSM cell response to FGF8 signaling is that cells located anterior to a grafted FGF8 coated bead respond much more strongly than cells situated caudal to the bead, at the same distance (Delfini, 2005). This complicates the interpretation of bead grafting experiments, and could be related to the asymmetrical effect on segmentation of FGF8 beads (Dubrulle, 2001), with smaller somites anterior to the bead, and a bigger one posterior to the bead.

Another intriguing phenomena is the occurrence of spatially periodic perturbations of somitogenesis after heat shocks in chick (Primmett et al., 1988) and zebrafish (Roy, 1999). These perturbations have been shown to be linked to cell cycling in chick PSM (Primmett et al., 1989). Based on local cell cycle synchrony in the PSM, the authors proposed that segmentation is controlled by the cell cycle, with a permissive phase of the cell cycle, during which cells are competent to segment, and a later instructive phase of the cycle, which induces cells to segment and to produce a diffusible molecule that induces competent cells to also segment. This model is unique in accounting for the periodicity of segmentation defects after heat shocks (cells were proposed to be sensitive to heat shocks only when at a particular phase of their cycle, which should occur at regular spatial intervals in the PSM). The model proposed by Primmett et al. (1989) does not call for an oscillatory gene expression pattern like that of the Notch pathway, but it is also not incompatible with it.

In summary, data seems to be lacking at present for the formulation of a detailed model of clock and wavefront interaction to control segmentation. Major unknowns are the detail and kinetics of response of the FGF8 signaling pathway to its ligand (and the reason for the asymmetric response to FGF8 beads), the distribution of FGF8 protein in the PSM, the interaction between FGF and Wnt signaling, the detail of clock-gene response to changes in FGF8 signaling, molecular detail of joint control by the clock and wavefront of targets that specify somites or somite boundaries at the determination front, and fine-grained mapping of gene expression patterns in the PSM to somite positions (live imaging of reporter expression, as performed by Masamizu, 2006, is a big step in that direction).

4. Conclusion

A major hurdle in the investigation of segmentation mechanisms is that it is technically very challenging to achieve precise spatiotemporal control of expression of the relevant genes, rather than just knocking down a gene or overexpressing it at a constant, poorly-controlled level. Dynamic perturbations of the clock and wavefront would go a long way to investigate the wiring of the network, which remains poorly characterized; the problem of whether Lunatic fringe potentiates or inhibits Notch signaling shows that it might not be sufficient to examine steady-state gene expression of a disrupted clock. Controlled perturbations would also make it possible to ask both how the clock oscillates, and how it interacts with the wavefront to regulate segmentation.

Finally, it is interesting to note that although disruption of the clock correlates with disruption of segmentation, and somite segmentation occurs in synchrony with the anterior arrival of waves of clock gene expression, it has not formally been shown that the arrival of the waves causes segmentation. It is possible that the role of the clock is to set up somite anteroposterior polarity (see Kerszberg, 2000, for a possible mechanism); the polarity of somites is an essential aspect of their function (Keynes and Stern, 1988) and has been proposed to underlie segmentation (Meinhardt, 1982). Finally, it is also not known whether the spatiotemporal pattern of gene expression, with waves sweeping from posterior to anterior, is of functional significance.

Note added in proof

It has recently been shown that oscillatory expression of Cryptochrome proteins is not necessary for oscillation of the circadian clock (Fan et al, 2007), contrary to what was previously thought. This provides a striking illustration of the difficulty of distinguishing between oscillating genes whose expression does not actually need to be oscillatory to permit functioning of a clock (although a requirement for cyclic post-translational modifications is possible), from genes whose expression must be oscillatory and is therefore driving the oscillations.

Acknowledgements

I am grateful to Amanda Albazerchi and Judith Kimble for discussions, help with producing figures, and support, and to an anonymous referee for helpful comments.

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Footnotes

11
A proposed frog clock is not shown because, even though genes with dynamic expression have been identified (Jen, 1997, Li, 2003, Jen, 1999), less seems to be known about the generation of the oscillations.
22
Although a zebrafish homologue is also expressed in PSM, it has a very different pattern suggesting that it does not have a role in the generation of the clock oscillations (Prince, 2001).
33
The Notch cofactor CBF-1 can repress Notch targets in the absence of Notch signaling (reviewed by Bray, 2001); repression of Lunatic fringe in the absence of Notch signaling or activation in the presence of signaling are likely to be similar in the way they could generate oscillations.
44
Note that this does not contradict the requirement of a negative feedback cycle of length greater than 1 for the existence of oscillations in a system without delays (Snoussi, 1998), as the `biological’ positive feedback described above in fact leads to negative feedback circuits, as defined mathematically by the Jacobian matrix of the set of ordinary differential equations describing the dynamics of the system.
55
Note however that Snail2 has been shown to activate its own promoter (Sakai, 2006).