Chick Hairy1 protein interacts with Sap18, a component of the Sin3/HDAC transcriptional repressor complex

Background  

The vertebrate adult axial skeleton, trunk and limb skeletal muscles and dermis of the back all arise from early embryonic structures called somites. Somites are symmetrically positioned flanking the embryo axial structures (neural tube and notochord) and are periodically formed in a anterior-posterior direction from the presomitic mesoderm. The time required to form a somite pair is constant and species-specific. This extraordinary periodicity is proposed to depend on an underlying somitogenesis molecular clock, firstly evidenced by the cyclic expression of the chick hairy1 gene in the unsegmented presomitic mesoderm with a 90 min periodicity, corresponding to the time required to form a somite pair in the chick embryo. The number of hairy1 oscillations at any given moment is proposed to provide the cell with both temporal and positional information along the embryo's anterior-posterior axis. Nevertheless, how this is accomplished and what biological processes are involved is still unknown. Aiming at understanding the molecular events triggered by the somitogenesis clock Hairy1 protein, we have employed the yeast two-hybrid system to identify Hairy1 interaction partners.     

Oscillations of the snail genes in the presomitic mesoderm coordinate segmental patterning and morphogenesis in vertebrate somitogenesis

The segmented body plan of vertebrate embryos arises through segmentation of the paraxial mesoderm to form somites. The tight temporal and spatial control underlying this process of somitogenesis is regulated by the segmentation clock and the FGF signaling wavefront. Here, we report the cyclic mRNA expression of Snail 1 and Snail 2 in the mouse and chick presomitic mesoderm (PSM), respectively. Whereas Snail genes' oscillations are independent of NOTCH signaling, we show that they require WNT and FGF signaling. Overexpressing Snail 2 in the chick embryo prevents cyclic Lfng and Meso 1 expression in the PSM and disrupts somite formation. Moreover, cells mis-expressing Snail 2 fail to express Paraxis, remain mesenchymal, and are thereby inhibited from undergoing the epithelialization event that culminates in the formation of the epithelial somite. Thus, Snail genes define a class of cyclic genes that coordinate segmentation and PSM morphogenesis.     

Segmentation in vertebrates: a molecular clock linked to periodic somite formation]

In the vertebrate embryo, somites constitute the basis of the segmental body pattern. They give rise to the axial skeleton, the dermis of the back and all striated muscles of the body. In the chick embryo, a pair of somites buds off, in a highly coordinated fashion, every 90 minutes, from the cranial end of the presomitic mesoderm (PSM) while new mesenchymal cells enter the paraxial mesoderm as a consequence of gastrulation. The processes leading to the segmentation of the somite are not yet understood. We have identified and characterised c-hairy1, an avian homologue of the Drosophila segmentation gene, hairy. c-hairy1 is strongly expressed in the presomitic mesoderm where its mRNA exhibits a cyclic posterior-to-anterior wave of expression whose periodicity corresponds to the formation time of one somite (90 min). Fate mapping of the rostral half of the PSM using the quail-chick chimera technique supports a model of cryptic segmentation within the presomitic mesoderm, and indicates that c-hairy1 expression dynamics are not due to massive cell displacement. Analysis of in vitro cultures of isolated presomitic mesoderm demonstrates that rhythmic c-hairy1 mRNA production and degradation is an autonomous property of the paraxial mesoderm. Rather than resulting from the caudal-to-rostral propagation of an activating signal, it arises from pulses of c-hairy1 expression that are coordinated in time and space. Blocking protein synthesis does not alter the propagation of c-hairy1 expression, indicating that negative autoregulation of c-hairy1 expression is unlikely to control its periodic expression. Most of the segmentation models proposed for somite formation rely on the existence of an internal clock coordinating the cells to segment together to form a somite. These results provide the first molecular evidence of a developmental clock linked to segmentation and somitogenesis of the paraxial mesoderm, and support the possibility that segmentation mechanisms used by invertebrates and vertebrates have been conserved.     

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

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

Evidence for medial/lateral specification and positional information within the presomitic mesoderm.

In the vertebrate embryo, segmentation is built on repetitive structures, named somites, which are formed progressively from the most rostral part of presomitic mesoderm, every 90 minutes in the avian embryo. The discovery of the cyclic expression of several genes, occurring every 90 minutes in each presomitic cell, has shown that there is a molecular clock linked to somitogenesis. We demonstrate that a dynamic expression pattern of the cycling genes is already evident at the level of the prospective presomitic territory. The analysis of this expression pattern, correlated with a quail/chick fate-map, identifies a 'wave' of expression travelling along the future medial/lateral presomitic axis. Further analysis also reveals the existence of a medial/lateral asynchrony of expression at the level of presomitic mesoderm. This work suggests that the molecular clock is providing cellular positional information not only along the anterior/posterior but also along the medial/lateral presomitic axis. Finally, by using an in vitro culture system, we show that the information for morphological somite formation and molecular segmentation is segregated within the medial/lateral presomitic axis. Medial presomitic cells are able to form somites and express segmentation markers in the absence of lateral presomitic cells. By contrast, and surprisingly, lateral presomitic cells that are deprived of their medial counterparts are not able to organise themselves into somites and lose the expression of genes known to be important for vertebrate segmentation, such as Delta-1, Notch-1, paraxis, hairy1, hairy2 and lunatic fringe.     

Synchronized oscillation of the segmentation clock gene in vertebrate development

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

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

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

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

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

The vertebrate segmentation clock and its role in skeletal birth defects

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

FGF signaling controls somite boundary position and regulates segmentation clock control of spatiotemporal Hox gene activation

Vertebrate segmentation requires a molecular oscillator, the segmentation clock, acting in presomitic mesoderm (PSM) cells to set the pace at which segmental boundaries are laid down. However, the signals that position each boundary remain unclear. Here, we report that FGF8 which is expressed in the posterior PSM, generates a moving wavefront at which level both segment boundary position and axial identity become determined. Furthermore, by manipulating boundary position in the chick embryo, we show that Hox gene expression is maintained in the appropriately numbered somite rather than at an absolute axial position. These results implicate FGF8 in ensuring tight coordination of the segmentation process and spatiotemporal Hox gene activation.     

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

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

A proposed mechanism for the interaction of the segmentation clock and the determination front in somitogenesis

Background

Recent discoveries in the field of somitogenesis have confirmed, for the most part, the feasibility of the clock and wavefront model. There are good candidates for both the clock (various genes expressed cyclically in the tail bud of vertebrate embryos have been discovered) and the wavefront (there are at least three different substances, whose expression levels vary along the presomitic mesoderm [PSM], that have important effects on the formation of somites). Nevertheless, the mechanisms through which the wavefront interacts with the clock to arrest the oscillations and induce somite formation have not yet been fully elucidated.

Principal Findings

In this work, we propose a gene regulatory network which is consistent with all known experimental facts in embryonic mice, and whose dynamic behaviour provides a potential explanation for the periodic aggregation of PSM cells into blocks: the first step leading to the formation of somites.

Significance

To our knowledge, this is the first proposed mechanism that fully explains how a block of PSM cells can stop oscillating simultaneously, and how this process is repeated periodically, via the interaction of the segmentation clock and the determination front.     

Cyclic expression of esr9 gene in Xenopus presomitic mesoderm

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

The segmentation clock in mice: interaction between the Wnt and Notch signalling pathways

In the last few years, the efforts to elucidate the mechanisms underlying the segmentation clock in various vertebrate species have multiplied. Early evidence suggested that oscillations are caused by one of the genes under the Notch signalling pathway (like those of the her or Hes families). Recently, Aulehla et al. [Wnt3a plays a major role in the segmentation clock controlling somitogenesis. Dev. Cell 4, 395-406] discovered that Axin2 (a gene under the Wnt3a signalling pathway) also oscillates in the presomitic mesoderm (PSM) of mice embryos and proposed some mechanisms through which the Notch and Wnt3a pathways may interact. They further suggested that a decreasing concentration of Wnt3a along the PSM may be the gradient the segmentation clock interacts with to form somites. These results were reviewed by Rida et al. [A notch feeling of somite segmentation and beyond. Dev. Biol. 265, 2-22], who introduced a complex clockwork comprising genes Hes1, Lfng (under the Notch pathway), and Axin2, as well as their multiple interactions. In the present work we develop a mathematical model based on the Rida et al. review and use it to tackle some of the questions raided by the Aulehla et al. paper: can the Axin2 feedback loop constitute a clock? Could a decreasing Wnt3a signaling constitute the wavefront, where phase is recorded and the spatial pattern laid down? What is the master oscillator?     

The clock and wavefront model revisited

The currently accepted interpretation of the clock and wavefront model of somitogenesis is that a posteriorly moving molecular gradient sequentially slows the rate of clock oscillations, resulting in a spatial readout of temporal oscillations. However, while molecular components of the clocks and wavefronts have now been identified in the pre-somitic mesoderm (PSM), there is not yet conclusive evidence demonstrating that the observed molecular wavefronts act to slow clock oscillations. Here we present an alternative formulation of the clock and wavefront model in which oscillator coupling, already known to play a key role in oscillator synchronisation, plays a fundamentally important role in the slowing of oscillations along the anterior-posterior (AP) axis. Our model has three parameters which can be determined, in any given species, by the measurement of three quantities: the clock period in the posterior PSM, somite length and the length of the PSM. A travelling wavefront, which slows oscillations along the AP axis, is an emergent feature of the model. Using the model we predict: (a) the distance between moving stripes of gene expression; (b) the number of moving stripes of gene expression and (c) the oscillator period profile along the AP axis. Predictions regarding the stripe data are verified using existing zebrafish data. We simulate a range of experimental perturbations and demonstrate how the model can be used to unambiguously define a reference frame along the AP axis. Comparing data from zebrafish, chick, mouse and snake, we demonstrate that: (a) variation in patterning profiles is accounted for by a single nondimensional parameter; the ratio of coupling strengths; and (b) the period profile along the AP axis is conserved across species. Thus the model is consistent with the idea that, although the genes involved in pattern propagation in the PSM vary, there is a conserved patterning mechanism across species.