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Individual cellular activities fluctuate but are constantly coordinated at the population level via cell–cell coupling. A notable example is the somite segmentation clock, in which the expression of clock genes (such as Hes7 ) oscillates in synchrony between the cells that comprise the presomitic mesoderm (PSM) 1 , 2 . This synchronization depends on the Notch signalling pathway; inhibiting this pathway desynchronizes oscillations, leading to somite fusion 3 – 7 . However, how Notch signalling regulates the synchronicity of HES7 oscillations is unknown. Here we establish a live-imaging system using a new fluorescent reporter (Achilles), which we fuse with HES7 to monitor synchronous oscillations in HES7 expression in the mouse PSM at a single-cell resolution. Wild-type cells can rapidly correct for phase fluctuations in HES7 oscillations, whereas the absence of the Notch modulator gene lunatic fringe ( Lfng ) leads to a loss of synchrony between PSM cells. Furthermore, HES7 oscillations are severely dampened in individual cells of Lfng -null PSM. However, when Lfng -null PSM cells were completely dissociated, the amplitude and periodicity of HES7 oscillations were almost normal, which suggests that LFNG is involved mostly in cell–cell coupling. Mixed cultures of control and Lfng -null PSM cells, and an optogenetic Notch signalling reporter assay, revealed that LFNG delays the signal-sending process of intercellular Notch signalling transmission. These results—together with mathematical modelling—raised the possibility that Lfng -null PSM cells shorten the coupling delay, thereby approaching a condition known as the oscillation or amplitude death of coupled oscillators 8 . Indeed, a small compound that lengthens the coupling delay partially rescues the amplitude and synchrony of HES7 oscillations in Lfng -null PSM cells. Our study reveals a delay control mechanism of the oscillatory networks involved in somite segmentation, and indicates that intercellular coupling with the correct delay is essential for synchronized oscillation. Monitoring cells of the mouse presomitic mesoderm using the Achilles reporter fused to HES7 sheds light on the mechanisms that underpin synchronous oscillations in the expression of clock genes between neighbouring cells.

During embryogenesis, the processes that control how cells differentiate and interact to form particular tissues and organs with precise timing and shape are of fundamental importance. One prominent example of such processes is vertebrate somitogenesis, which is governed by a molecular oscillator called the segmentation clock. The segmentation clock system is initiated in the presomitic mesoderm in which a set of genes and signaling pathways exhibit coordinated spatiotemporal dynamics to establish regularly spaced boundaries along the body axis; these boundaries provide a blueprint for the development of segment-like structures such as spines and skeletal muscles. The highly complex and dynamic nature of this in vivo event and the design principles and their regulation in both normal and abnormal embryogenesis are not fully understood. Recently, live-imaging has been used to quantitatively analyze the dynamics of selected components of the circuit, particularly in combination with well-designed experiments to perturb the system. Here, we review recent progress from studies using live imaging and manipulation, including attempts to recapitulate the segmentation clock in vitro. In combination with mathematical modeling, these techniques have become essential for disclosing novel aspects of the clock.

The methyl cycle is a universal metabolic pathway providing methyl groups for the methylation of nuclei acids and proteins, regulating all aspects of cellular physiology. We have previously shown that methyl cycle inhibition in mammals strongly affects circadian rhythms. Since the methyl cycle and circadian clocks have evolved early during evolution and operate in organisms across the tree of life, we sought to determine whether the link between the two is also conserved. Here, we show that methyl cycle inhibition affects biological rhythms in species ranging from unicellular algae to humans, separated by more than 1 billion years of evolution. In contrast, the cyanobacterial clock is resistant to methyl cycle inhibition, although we demonstrate that methylations themselves regulate circadian rhythms in this organism. Mammalian cells with a rewired bacteria-like methyl cycle are protected, like cyanobacteria, from methyl cycle inhibition, providing interesting new possibilities for the treatment of methylation deficiencies. Fustin et al. reveal the evolutionarily conserved link between methyl metabolism and biological clocks. This study suggests the possibility of translating fundamental understanding of methylation deficiencies to clinical applications.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The segmented body plan of vertebrates is established during somitogenesis, a well-studied process in model organisms; however, the details of this process in humans remain largely unknown owing to ethical and technical limitations. Despite recent advances with pluripotent stem cell-based approaches 1 – 5 , models that robustly recapitulate human somitogenesis in both space and time remain scarce. Here we introduce a pluripotent stem cell-derived mesoderm-based 3D model of human segmentation and somitogenesis—which we termed ‘axioloid’—that captures accurately the oscillatory dynamics of the segmentation clock and the morphological and molecular characteristics of sequential somite formation in vitro. Axioloids show proper rostrocaudal patterning of forming segments and robust anterior–posterior FGF–WNT signalling gradients and retinoic acid signalling components. We identify an unexpected critical role of retinoic acid signalling in the stabilization of forming segments, indicating distinct, but also synergistic effects of retinoic acid and extracellular matrix on the formation and epithelialization of somites. Comparative analysis demonstrates marked similarities of axioloids to the human embryo, further validated by the presence of a Hox code in axioloids. Finally, we demonstrate the utility of axioloids for studying the pathogenesis of human congenital spine diseases using induced pluripotent stem cells with mutations in HES7 and MESP2 . Our results indicate that axioloids represent a promising platform for the study of axial development and disease in humans. A 3D model of human segmentation and somitogenesis derived from induced pluripotent stem cells captures the oscillatory dynamics of the segmentation clock as well as morphological and molecular features of the developing embryonic axis and tail.

The segmental organization of the vertebral column is established early in embryogenesis, when pairs of somites are rhythmically produced by the presomitic mesoderm (PSM). The tempo of somite formation is controlled by a molecular oscillator known as the segmentation clock 1 , 2 . Although this oscillator has been well-characterized in model organisms 1 , 2 , whether a similar oscillator exists in humans remains unknown. Genetic analyses of patients with severe spine segmentation defects have implicated several human orthologues of cyclic genes that are associated with the mouse segmentation clock, suggesting that this oscillator might be conserved in humans 3 . Here we show that human PSM cells derived in vitro—as well as those of the mouse 4 —recapitulate the oscillations of the segmentation clock. Human PSM cells oscillate with a period two times longer than that of mouse cells (5 h versus 2.5 h), but are similarly regulated by FGF, WNT, Notch and YAP signalling 5 . Single-cell RNA sequencing reveals that mouse and human PSM cells in vitro follow a developmental trajectory similar to that of mouse PSM in vivo. Furthermore, we demonstrate that FGF signalling controls the phase and period of oscillations, expanding the role of this pathway beyond its classical interpretation in ‘clock and wavefront’ models 1 . Our work identifying the human segmentation clock represents an important milestone in understanding human developmental biology. Human presomitic mesoderm cells derived in vitro demonstrate oscillations of the segmentation clock, thus providing a window into an otherwise inaccessible stage of human development.

The segmented body plan of vertebrates is established during somitogenesis, a well-studied process in model organisms, but remains largely elusive in humans due to ethical and technical limitations. Despite recent advances with pluripotent stem cell (PSC)-based approaches1–5, a system that robustly recapitulates human somitogenesis in both space and time remains missing. Here, we introduce a PSC-derived mesoderm-based 3D model of human segmentation and somitogenesis, which we termed Axioloids, that captures accurately the oscillatory dynamics of the segmentation clock as well as the morphological and molecular characteristics of segmentation and sequential somite formation in vitro. Axioloids show proper rostrocaudal patterning of forming segments and robust anterior-posterior FGF/WNT signaling gradients and Retinoic Acid (RA) signaling components. We identify an unexpected critical role of RA signaling in the stabilization of forming segments, indicating distinct, but also synergistic effects of RA and extracellular matrix (ECM) on the formation and epithelialization of somites. Importantly, comparative analysis demonstrates striking similarities of Axioloids to the human embryo, further validated by the presence of the HOX code in Axioloids. Lastly, we demonstrate the utility of our Axioloid system to study the pathogenesis of human congenital spine diseases, by using patient-like iPSC cells with mutations in HES7 and MESP2, which revealed disease-associated phenotypes including loss of epithelial somite formation and abnormal rostrocaudal patterning. These results suggest that Axioloids represent a promising novel platform to study axial development and disease in humans.

While the mechanisms of embryonic development are similar between mouse and human, the tempo is in general slower in human. The cause of interspecies differences in developmental time remains a mystery partly due to lack of an appropriate model system. Since murine and human embryos differ in their sizes, geometries, and nutrients, we use in vitro differentiation of pluripotent stem cells (PSCs) to compare the same type of cells between the species in similar culture conditions. As an example of well-defined developmental time, we focus on the segmentation clock, oscillatory gene expression that regulates the timing of sequential formation of body segments. In this way we recapitulate the murine and human segmentation clocks in vitro, showing that the species-specific oscillation periods are derived from cell autonomous differences in the speeds of biochemical reactions. Presomitic mesoderm (PSM)-like cells induced from murine and human PSCs displayed the oscillatory expression of HES7, the core gene of the segmentation clock, with oscillation periods of 2-3 hours (mouse PSM) and 5-6 hours (human PSM). Swapping HES7 loci between murine and human genomes did not change the oscillation periods dramatically, denying the possibility that interspecies differences in the sequences of HES7 loci might be the cause of the observed period difference. Instead, we found that the biochemical reactions that determine the oscillation period, such as the degradation of HES7 and delays in its expression, are slower in human PSM compared with those in mouse PSM. With the measured biochemical parameters, our mathematical model successfully accounted for the 2-3-fold period difference between mouse and human. We further demonstrate that the concept of slower biochemical reactions in human cells is generalizable to several other genes, as well as to another cell type. These results collectively indicate that differences in the speeds of biochemical reactions between murine and human cells give rise to the interspecies period difference of the segmentation clock and may contribute to other interspecies differences in developmental time.

The methyl cycle is a universally conserved metabolic pathway operating in prokaryotes and eukaryotes. In this pathway, the amino acid methionine is used to synthesize S-adenosylmethionine, the methyl donor co-substrate in the methylation of nucleic acids, histone and non-histone proteins and many other molecules within the cell. The methylation of nucleic acids and proteins is the foundation of epigenetic and epitranscriptomic regulations of gene expression, but whether the methyl cycle centrally regulates gene expression and function by controlling the availability of methyl moieties is poorly understood. From cyanobacteria to humans, a circadian clock that involves an exquisitely regulated transcription-translation-feedback loop driving oscillations in gene expression and orchestrating physiology and behavior has been described. We reported previously that inhibition of the methyl cycle in mammalian cells caused the lengthening of the period of these oscillations, suggesting the methyl cycle may indeed act as a central regulator of gene expression, at least in mammals. Here, we investigated whether the methyl cycle, given its universal presence among living beings, regulates the circadian clock in species across the phylogenetic tree of life. We reveal a remarkable evolutionary conservation of the link between the methyl cycle and the circadian clock. Moreover, we show that the methyl cycle also regulates the somite segmentation clock, another transcription-translation negative feedback loop-based timing mechanism that orchestrate embryonic development in vertebrates, highlighting the methyl cycle as a master regulator of biological clocks. Here we reveal that the methyl cycle, a universal metabolic pathway leading to the synthesis of S-adenosylmethionine, the methyl donor co-substrate in virtually all transmethylation reactions within the cell, is a conserved regulator of biological clocks. These discoveries highlight the methyl cycle as a metabolic hub that regulates gene expression via the availability of methyl moieties for the methylation of nucleic acids, proteins and many other molecules with the cell.