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The Myc transcriptional regulators are implicated in a range of cellular functions, including proliferation, cell cycle progression, metabolism and pluripotency maintenance. Here, we investigated the expression, regulation and function of Myc during mouse embryonic axis elongation and segmentation. Expression of bothcMycandMycNin the domains where neuromesodermal progenitors (NMPs) and underlying caudal pre-somitic mesoderm (cPSM) cells reside is coincident WNT and FGF signals; factors known to maintain progenitors in an undifferentiated state. Pharmacological inhibition of MYC activity, downregulates expression of WNT/FGF components. In turn, we find thatcMycexpression is WNT, FGF and NOTCH regulated, placing it centrally in the signalling circuit that operates in the tail end that both sustains progenitors and drives maturation of the PSM into somites. Interfering with MYC function in the PSM, where it displays oscillatory expression, delays the timing of segmentation clock oscillations and thus of somite formation. In summary, we identify Myc as a component that links NMP maintenance and PSM maturation during the body axis elongation stages of mouse embryogenesis.Summary StatementMYC operates in a positive feedback loop with WNT/FGF signals to maintain the progenitors which facilitate body axis elongation while its activity is crucial for timing of the segmentation clock.

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

A segmented body plan is fundamental to all vertebrate species and this bestows both rigidity and flexibility on the body. Segmentation is initiated through the process of somitogenesis. This article aims to provide a broad and balanced cross-species overview of somitogenesis and to highlight the key molecular and cellular events involved in each stage of segmentation. We highlight where our understanding of this multifaceted process relies on strong experimental evidence as well as those aspects where our understanding still relies largely on models.

The vertebrate segmentation clock is a molecular oscillator that regulates the periodicity of somite formation. Three signalling pathways have been proposed to underlie the molecular mechanism of the oscillator, namely the Notch, Wnt and Fgf pathways. Characterizing the roles and hierarchy of these three pathways in the oscillator mechanism is currently the focus of intense research. Recent publications report the first identification of a molecular mechanism involved in the regulation of the pace of this oscillator. We review these and other recent findings regarding the interaction between the three pathways in the oscillator mechanism that have significantly expanded our understanding of the segmentation clock.

Somites are formed progressively from the presomitic mesoderm (PSM) in a highly regulated process according to a strict periodicity driven by an oscillatory mechanism. The Notch and Wnt pathways are key components in the regulation of this somitic oscillator and data from Xenopus and zebrafish embryos indicate that the Notch-downstream target Nrarp participates in the regulation of both activities. We have analyzed Nrarp/nrarp-a expression in the PSM of chick, mouse and zebrafish embryos, and we show that it cycles in synchrony with other Notch regulated cyclic genes. In the mouse its transcription is both Wnt- and Notch-dependent, whereas in the chick and fish embryo it is simply Notch-dependent. Despite oscillating mRNA levels, Nrarp protein does not oscillate in the PSM. Finally, neither gain nor loss of Nrarp function interferes with the normal expression of Notch-related cyclic genes.

Notochord-derived Sonic Hedgehog (Shh) is essential for dorsoventral patterning of the overlying neural tube. Increasing concentration and duration of Shh signal induces progenitors to acquire progressively more ventral fates. We show that Notch signalling augments the response of neuroepithelial cells to Shh, leading to the induction of higher expression levels of the Shh target gene Ptch1 and subsequently induction of more ventral cell fates. Furthermore, we demonstrate that activated Notch1 leads to pronounced accumulation of Smoothened (Smo) within primary cilia and elevated levels of full-length Gli3. Finally, we show that Notch activity promotes longer primary cilia both in vitro and in vivo. Strikingly, these Notch-regulated effects are Shh independent. These data identify Notch signalling as a novel modulator of Shh signalling that acts mechanistically via regulation of ciliary localisation of key components of its transduction machinery., Highlighted article: Shh signalling controls dorso-ventral cell fate in the neural tube. Notch regulates ciliary architecture and localisation of key Shh pathway components, thus sensitising cells to Shh.

Segmentation of the vertebrate body axis is initiated early in development with the sequential formation of somites. Somitogenesis is temporally regulated by a molecular oscillator, the segmentation clock, which acts within presomitic mesoderm (PSM) cells to drive periodic expression of the cyclic genes. We have investigated the kinetics of the progression of cycling gene expression along the PSM. Here we show that c-hairy1 and c-hairy2 mRNA expression traverses the PSM in an entirely progressive manner and that both these genes and c-Lfng maintain a similar anterior limit of expression during each cycle. However, some differences are seen regarding both the onset of a new oscillation of these genes and the duration of their expression in the caudal PSM. We also investigated whether oscillating cyclic gene expression in the PSM is entirely cell autonomous. We find that while small PSM explants are still able to maintain their oscillation schedule, once they are dissociated, PSM cells are no longer able to maintain synchronous oscillations. The results imply that cell communication or a community effect is essential for the normal pattern of cyclic gene expression in these cells.

During somitogenesis an oscillatory mechanism termed the “segmentation” clock generates periodic waves of gene expression, which translate into the periodic spatial pattern manifest as somites. The dynamic expression of the clock genes shares the same periodicity as somitogenesis. Notch signaling is believed to play a role in the segmentation clock mechanism. The paper by Hirata et al.(1) identifies a biological clock in cultured cells that is dependent upon the Notch target gene Hes1, and which shows a periodicity similar to that of the segmentation clock. This finding opens the possibility that the same oscillator mechanism might also operate in other tissues or cell types. BioEssays 25:200–203, 2003. © 2003 Wiley Periodicals, Inc.

The most obvious segments of the vertebrate embryo are the trunk mesodermal somites which give rise to the segmented vertebral column and the skeletal muscles of the body. Mechanistic insights into vertebrate somitogenesis have recently been gained from observations of rhythmic expression of the avian hairy-related gene (c-hairy1) in chick presomitic mesoderm (PSM), suggesting the existence of a molecular clock linked to somite segmentation ([1]; reviewed in [2]). Here, we show that lunatic Fringe (lFng), a vertebrate homolog of the Drosophila Fringe gene, is also expressed rhythmically in PSM. The PSM expression of lFng was observed as coordinated pulses of mRNA resembling the expression of c-hairy1. We show that c-hairy1 and lFng expression in the PSM are coincident, indicating that both genes are responding to the same segmentation clock. The genes were found to differ in their regulation, however; in contrast to c-hairy1, lFng mRNA oscillations required continued protein synthesis, suggesting that lFng could be acting downstream of c-hairy1 in the clock mechanism. In Drosophila, Fringe has been shown to play a role in modulating Notch–Delta signalling [3,4], a pathway which in vertebrates has been implicated in defining somite boundaries [5–9]. These observations place the segmentation clock upstream of the Notch–Delta pathway during vertebrate somitogenesis.

Biofilm formation by the Gram-positive bacterium Bacillus subtilis is tightly controlled at the level of transcription. The biofilm contains specialized cell types that arise from controlled differentiation of the resident isogenic bacteria. DegU is a response regulator that controls several social behaviours exhibited by B. subtilis including swarming motility, biofilm formation and extracellular protease (exoprotease) production. Here, for the first time, we examine the prevalence and origin of exoprotease-producing cells within the biofilm. This was accomplished using single-cell analysis techniques including flow cytometry and fluorescence microscopy. We established that the number of exoprotease-producing cells increases as the biofilm matures. This is reflected by both an increase at the level of transcription and an increase in exoprotease activity over time. We go on to demonstrate that exoprotease-producing cells arise from more than one cell type, namely matrix-producing and non-matrix-producing cells. In toto these findings allow us to add exoprotease-producing cells to the list of specialized cell types that are derived during B. subtilis biofilm formation and furthermore the data highlight the plasticity in the origin of differentiated cells.

The most obvious manifestation of the existence of a segmented, or metameric, body plan in vertebrate embryos is seen during the formation of the somites. Somites are transient embryonic structures formed in a progressive manner from a nonsegmented mesoderm in a highly regulated process called somitogenesis. As development proceeds different compartments are formed within each somite and these progressively follow a variety of differentiation programs to form segmented organs, such as the different bones that make the axial skeleton, body skeletal muscles and part of the dermis. Transcription factors from the basic helix-loop-helix (bHLH) protein family have been described to be implicated in each of the processes involved in somite formation. bHLH proteins are a family of transcription factors characterized by the presence of a DNA binding domain and a dimerization motif that consists of a basic region adjacent to an amphipathic helix, a loop and a second amphipathic helix. In this chapter we will review a number of bHLH proteins known to play a role in somitogenesis.

Hensen's node of the chick embryo contains multipotent self-renewing progenitor cells that can contribute to either the floor plate or the notochord. Floor plate cells are a population of epithelial cells that lie at the ventral midline of the developing neural tube, whereas the notochord is a rod of axial mesoderm that lies directly beneath the floor plate. These two tissues serve as a source of a potent signalling morphogen, sonic hedgehog (Shh), which patterns the dorsoventral axis of the neural tube. We show, through both gain- and loss-of-function approaches, that Notch signalling promotes the contribution of chick axial progenitor cells to the floor plate and inhibits contribution to the notochord. Thus, we propose that Notch regulates the allocation of appropriate numbers of progenitor cells from Hensen's node of the chick embryo to the notochord and the floor plate.

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., Author Summary Vertebrate animals generate their segmented body plan during embryogenesis. These embryonic segments, or somites, form one after another from tissue at the tail end of the embryo in a highly regulated process controlled by a molecular oscillator. This oscillator drives the expression of a group of genes in this tissue and determines the periodicity of somite formation. To date the genes regulated by this molecular clock comprise components of the Notch, Wnt, and FGF pathways. Recent data in the zebrafish embryo have suggested that the only role of Notch signalling in this process is to synchronise gene oscillations between neighbouring cells and that somite formation can continue in the absence of Notch activity. However, we show that mouse embryos lacking all Notch activity do not show oscillatory activity and do not develop somites. We propose that, at least in the mouse embryo, Notch activity is absolutely essential for building a segmented body axis.

Temporal control is considered the fourth dimension in embryonic development and it sets the pace to attain the correct molecular patterning of the developing embryo. In this chapter we review one of the best-studied time dependent events in embryogenesis, which is the formation of somites. Somites are the basis of the future segmented framework of the vertebrate adult body and their reiterated appearance during the early stages of embryo development establishes the proper temporal and physical template from where other structures will develop and consequently shape the segmentation pattern of the embryo. Several models have been proposed over the last few decades to explain the mechanism(s) regulating somite periodicity, but no molecular evidence seemed to back up any of the postulated models. Remarkably, in 1997 the first evidence that the formation of the somites depended on an intrinsic molecular clock was at last provided through the description of oscillating gene expression in the tissue from which somites are generated. Since then, a huge amount of data has been and continues to be provided that is gradually revealing the ever more complex molecular mechanism underlying this segmentation clock. We are also beginning to learn about embryonic structures other than the somites which exhibit oscillations of gene expression suggesting they too are dependent upon a segmentation-like clock. This is in itself the clearest evidence that there is still a long way to go before we unveil the myriad of molecular mechanisms that lead to the time control of embryonic development.

The most obvious manifestation of the existence of a segmented, or metameric, body plan in vertebrate embryos is seen during the formation of the somites. Somites are transient embryonic structures formed in a progressive manner from a nonsegmented mesoderm in a highly regulated process called somitogenesis. As development proceeds different compartments are formed within each somite and these progressively follow a variety of differentiation programs to form segmented organs, such as the different bones that make the axial skeleton, body skeletal muscles and part of the dermis. Transcription factors from the basic helix-loop-helix (bHLH) protein family have been described to be implicated in each of the processes involved in somite formation. bHLH proteins are a family of transcription factors characterized by the presence of a DNA binding domain and a dimerization motif that consists of a basic region adjacent to an amphipathic helix, a loop and a second amphipathic helix. In this chapter we will review a number of bHLH proteins known to play a role in somitogenesis.

Somites are embryonic precursors of the ribs, vertebrae and certain dermis tissue. Somite formation is a periodic process regulated by a molecular clock which drives cyclic expression of a number of clock genes in the presomitic mesoderm. To date the mechanism regulating the period of clock gene oscillations is unknown. Here we show that chick homologues of the Wnt pathway genes that oscillate in mouse do not cycle across the chick presomitic mesoderm. Strikingly we find that modifying Wnt signalling changes the period of Notch driven oscillations in both mouse and chick but these oscillations continue. We propose that the Wnt pathway is a conserved mechanism that is involved in regulating the period of cyclic gene oscillations in the presomitic mesoderm.

Temporal control is considered the fourth dimension in embryonic development and it sets the pace to attain the correct molecular patterning of the developing embryo. In this chapter we review one of the best-studied time dependent events in embryogenesis, which is the formation ofsomites. Somites are the basis of the future segmented framework of the vertebrate adult body and their reiterated appearance during the early stages of embryo development establishes the proper temporal and physical template from where other structures will develop and consequently shape the segmentation pattern of the embryo. Several models have been proposed over the last few decades to explain the mechanism(s) regulating somite periodicity, but no molecular evidence seemed to back up any of the postulated models. Remarkably, in 1997 the first evidence that the formation of the somites depended on an intrinsic molecular clock was at last provided through the description of oscillating gene expression in the tissue from which somites are generated. Since then, a huge amount of data has been and continues to be provided that is gradually revealing the ever more complex molecular mechanism underlying this segmentation clock. We are also beginning to learn about embryonic structures other than the somites which exhibit oscillations of gene expression suggesting they too are dependent upon a segmentation-like clock. This is in itself the clearest evidence that there is still a long way to go before we unveil the myriad of molecular mechanisms that lead to the time control of embryonic development.

Ventral midline cells at different rostrocaudal levels of the central nervous system exhibit distinct properties but share the ability to pattern the dorsoventral axis of the neural tube. We show here that ventral midline cells acquire distinct identities in response to the different signaling activities of underlying mesoderm. Signals from prechordal mesoderm control the differentiation of rostral diencephalic ventral midline cells, whereas notochord induces floor plate cells caudally. Sonic hedgehog (SHH) is expressed throughout axial mesoderm and is required for the induction of both rostral diencephalic ventral midline cells and floor plate. However, prechordal mesoderm also expresses BMP7 whose function is required coordinately with SHH to induce rostral diencephalic ventral midline cells. BMP7 acts directly on neural cells, modifying their response to SHH so that they differentiate into rostral diencephalic ventral midline cells rather than floor plate cells. Our results suggest a model whereby axial mesoderm both induces the differentiation of overlying neural cells and controls the rostrocaudal character of the ventral midline of the neural tube.

In the vertebrate embryo, tissue blocks called somites are laid down in head-to-tail succession, a process known as somitogenesis. Research into somitogenesis has been both experimental and mathematical. For zebrafish, there is experimental evidence for oscillatory gene expression in cells in the presomitic mesoderm (PSM) as well as evidence that Notch signalling synchronises the oscillations in neighbouring PSM cells. A biological mechanism has previously been proposed to explain these phenomena. Here we have converted this mechanism into a mathematical model of partial differential equations in which the nuclear and cytoplasmic diffusion of protein and mRNA molecules is explicitly considered. By performing simulations, we have found ranges of values for the model parameters (such as diffusion and degradation rates) that yield oscillatory dynamics within PSM cells and that enable Notch signalling to synchronise the oscillations in two touching cells. Our model contains a Hill coefficient that measures the co-operativity between two proteins (Her1, Her7) and three genes (her1, her7, deltaC) which they inhibit. This coefficient appears to be bounded below by the requirement for oscillations in individual cells and bounded above by the requirement for synchronisation. Consistent with experimental data and a previous spatially non-explicit mathematical model, we have found that signalling can increase the average level of Her1 protein. Biological pattern formation would be impossible without a certain robustness to variety in cell shape and size; our results possess such robustness. Our spatially-explicit modelling approach, together with new imaging technologies that can measure intracellular protein diffusion rates, is likely to yield significant new insight into somitogenesis and other biological processes.

Background During vertebrate embryogenesis, somites are generated at regular intervals, the temporal and spatial periodicity of which is governed by a gradient of fibroblast growth factor (FGF) and/or Wnt signaling activity in the presomitic mesoderm (PSM) in conjunction with oscillations of gene expression of components of the Notch, Wnt and FGF signaling pathways. Principal Findings Here, we show that the expression of Sprouty4, which encodes an FGF inhibitor, oscillates in 2-h cycles in the mouse PSM in synchrony with other oscillating genes from the Notch signaling pathway, such as lunatic fringe. Sprouty4 does not oscillate in Hes7-null mutant mouse embryos, and Hes7 can inhibit FGF-induced transcriptional activity of the Sprouty4 promoter. Conclusions Thus, periodic expression of Sprouty4 is controlled by the Notch segmentation clock and may work as a mediator that links the temporal periodicity of clock gene oscillations with the spatial periodicity of boundary formation which is regulated by the gradient of FGF/Wnt activity.