Persistence, period and precision of autonomous cellular oscillators from the zebrafish segmentation clock.
ABSTRACT: In vertebrate development, the sequential and rhythmic segmentation of the body axis is regulated by a "segmentation clock". This clock is comprised of a population of coordinated oscillating cells that together produce rhythmic gene expression patterns in the embryo. Whether individual cells autonomously maintain oscillations, or whether oscillations depend on signals from neighboring cells is unknown. Using a transgenic zebrafish reporter line for the cyclic transcription factor Her1, we recorded single tailbud cells in vitro. We demonstrate that individual cells can behave as autonomous cellular oscillators. We described the observed variability in cell behavior using a theory of generic oscillators with correlated noise. Single cells have longer periods and lower precision than the tissue, highlighting the role of collective processes in the segmentation clock. Our work reveals a population of cells from the zebrafish segmentation clock that behave as self-sustained, autonomous oscillators with distinctive noisy dynamics.
Project description:Vertebrate body segmentation is controlled by the segmentation clock, a molecular oscillator involving transcriptional oscillations of cyclic genes in presomitic mesoderm cells. The rapid and highly dynamic nature of this oscillating system has proved challenging for study at the single-cell level. We achieved visualization of clock activity with a cellular level of resolution in living embryos, allowing direct comparison of oscillations in neighbor cells. We provide direct evidence that presomitic mesoderm cells oscillate asynchronously in zebrafish Notch pathway mutants. By tracking oscillations in mitotic cells, we reveal that a robust cell-autonomous, Notch-independent mechanism resumes oscillations after mitosis. Finally, we find that cells preferentially divide at a certain oscillation phase, likely reducing the noise generated by cell division in cell synchrony and suggesting an intriguing relationship between the mitotic cycle and clock oscillation.
Project description:The suprachiasmatic nucleus (SCN) is the primary circadian pacemaker in mammals. Individual SCN neurons in dispersed culture can generate independent circadian oscillations of clock gene expression and neuronal firing. However, SCN rhythmicity depends on sufficient membrane depolarization and levels of intracellular calcium and cAMP. In the intact SCN, cellular oscillations are synchronized and reinforced by rhythmic synaptic input from other cells, resulting in a reproducible topographic pattern of distinct phases and amplitudes specified by SCN circuit organization. The SCN network synchronizes its component cellular oscillators, reinforces their oscillations, responds to light input by altering their phase distribution, increases their robustness to genetic perturbations, and enhances their precision. Thus, even though individual SCN neurons can be cell-autonomous circadian oscillators, neuronal network properties are integral to normal function of the SCN.
Project description:Collective cell movement is a crucial component of embryonic development. Intercellular interactions regulate collective cell movement by allowing cells to transfer information. A key question is how collective cell movement itself influences information flow produced in tissues by intercellular interactions. Here, we study the effect of collective cell movement on the synchronization of locally coupled genetic oscillators. This study is motivated by the segmentation clock in zebrafish somitogenesis, where short-range correlated movement of cells has been observed. We describe the segmentation clock tissue by a Voronoi diagram, cell movement by the force balance of self-propelled and repulsive forces between cells, the dynamics of the direction of self-propelled motion, and the synchronization of genetic oscillators by locally coupled phase oscillators. We find that movement with a correlation length of about 2 ? 3 cell diameters is optimal for the synchronization of coupled oscillators. Quantification of cell mixing reveals that this short-range correlation of cell movement allows cells to exchange neighbors most efficiently. Moreover, short-range correlated movement strongly destabilizes nonuniform spatial phase patterns, further promoting global synchronization. Our theoretical results suggest that collective cell movement may enhance the synchronization of the segmentation clock in zebrafish somitogenesis. More generally, collective cell movement may promote information flow in tissues by enhancing cell mixing and destabilizing spurious patterns.
Project description:The mammalian circadian timing system consists of a master pacemaker in neurons of the suprachiasmatic nucleus (SCN) and clocks of a similar molecular makeup in most peripheral body cells. Peripheral oscillators are self-sustained and cell autonomous, but they have to be synchronized by the SCN to ensure phase coherence within the organism. In principle, the rhythmic expression of genes in peripheral organs could thus be driven not only by local oscillators, but also by circadian systemic signals. To discriminate between these mechanisms, we engineered a mouse strain with a conditionally active liver clock, in which REV-ERBalpha represses the transcription of the essential core clock gene Bmal1 in a doxycycline-dependent manner. We examined circadian liver gene expression genome-wide in mice in which hepatocyte oscillators were either running or arrested, and found that the rhythmic transcription of most genes depended on functional hepatocyte clocks. However, we discovered 31 genes, including the core clock gene mPer2, whose expression oscillated robustly irrespective of whether the liver clock was running or not. By contrast, in liver explants cultured in vitro, circadian cycles of mPer2::luciferase bioluminescence could only be observed when hepatocyte oscillators were operational. Hence, the circadian cycles observed in the liver of intact animals without functional hepatocyte oscillators were likely generated by systemic signals. The finding that rhythmic mPer2 expression can be driven by both systemic cues and local oscillators suggests a plausible mechanism for the phase entrainment of subsidiary clocks in peripheral organs.
Project description:During vertebrate embryonic development, the formation of axial structures is driven by a population of stem-like cells that reside in a region of the tailbud called the chordoneural hinge (CNH). We have compared the mouse CNH transcriptome with those of surrounding tissues and shown that the CNH and tailbud mesoderm are transcriptionally similar, and distinct from the presomitic mesoderm. Amongst CNH-enriched genes are several that are required for axial elongation, including Wnt3a, Cdx2, Brachyury/T and Fgf8, and androgen/oestrogen receptor nuclear signalling components such as Greb1 We show that the pattern and duration of tailbud Greb1 expression is conserved in mouse, zebrafish and chicken embryos, and that Greb1 is required for axial elongation and somitogenesis in zebrafish embryos. The axial truncation phenotype of Greb1 morphant embryos can be explained by much reduced expression of No tail (Ntl/Brachyury), which is required for axial progenitor maintenance. Posterior segmentation defects in the morphants (including misexpression of genes such as mespb, myoD and papC) appear to result, in part, from lost expression of the segmentation clock gene, her7.
Project description:Individual neurons within the suprachiasmatic nuclei (SCNs) are capable of functioning as autonomous clocks and generating circadian rhythms in the expression of genes that form the molecular clockworks. Limited information is available on how these molecular oscillations in individual clock cells are coordinated to provide for the ensemble rhythmicity that is normally observed from the entire SCN. Because calcium influx via voltage-dependent calcium channels (VDCCs) has been implicated in the regulation of gene expression and synchronization of rhythmicity across the population of SCN clock cells, we first examined the rat SCN and an immortalized line of SCN cells (SCN2.2) for expression and circadian regulation of different VDCC alpha1 subunits. The rat SCN and SCN2.2 cells exhibited mRNA expression for all major types of VDCC alpha1 subunits. Relative levels of VDCC expression in the rat SCN and SCN2.2 cells were greatest for L-type channels, moderate for P/Q- and T-type channels, and minimal for R- and N-type channels. Interestingly, both rat SCN and SCN2.2 cells showed rhythmic expression of P/Q- and T-type channels. VDCC involvement in the regulation of molecular rhythmicity in SCN2.2 cells was then examined using the nonselective antagonist, cadmium. The oscillatory patterns of rPer2 and rBmal1 expression were abolished in cadmium-treated SCN2.2 cells without affecting cellular morphology and viability. These findings raise the possibility that the circadian regulation of VDCC activity may play an important role in maintaining rhythmic clock gene expression across an ensemble of SCN oscillators.
Project description:Rhythmic and sequential subdivision of the elongating vertebrate embryonic body axis into morphological somites is controlled by an oscillating multicellular genetic network termed the segmentation clock. This clock operates in the presomitic mesoderm (PSM), generating dynamic stripe patterns of oscillatory gene-expression across the field of PSM cells. How these spatial patterns, the clock's collective period, and the underlying cellular-level interactions are related is not understood. A theory encompassing temporal and spatial domains of local and collective aspects of the system is essential to tackle these questions. Our delayed coupling theory achieves this by representing the PSM as an array of phase oscillators, combining four key elements: a frequency profile of oscillators slowing across the PSM; coupling between neighboring oscillators; delay in coupling; and a moving boundary describing embryonic axis elongation. This theory predicts that the segmentation clock's collective period depends on delayed coupling. We derive an expression for pattern wavelength across the PSM and show how this can be used to fit dynamic wildtype gene-expression patterns, revealing the quantitative values of parameters controlling spatial and temporal organization of the oscillators in the system. Our theory can be used to analyze experimental perturbations, thereby identifying roles of genes involved in segmentation.
Project description:The vertebrate segmentation clock is a gene expression oscillator controlling rhythmic segmentation of the vertebral column during embryonic development. The period of oscillations becomes longer as cells are displaced along the posterior to anterior axis, which results in traveling waves of clock gene expression sweeping in the unsegmented tissue. Although various hypotheses necessitating the inclusion of additional regulatory genes into the core clock network at different spatial locations have been proposed, the mechanism underlying traveling waves has remained elusive. Here, we combined molecular-level computational modeling and quantitative experimentation to solve this puzzle. Our model predicts the existence of an increasing gradient of gene expression time delays along the posterior to anterior direction to recapitulate spatiotemporal profiles of the traveling segmentation clock waves in different genetic backgrounds in zebrafish. We validated this prediction by measuring an increased time delay of oscillatory Her1 protein production along the unsegmented tissue. Our results refuted the need for spatial expansion of the core feedback loop to explain the occurrence of traveling waves. Spatial regulation of gene expression time delays is a novel way of creating dynamic patterns; this is the first report demonstrating such a control mechanism in any tissue and future investigations will explore the presence of analogous examples in other biological systems.
Project description:The periodic segmentation of the vertebrate body axis into somites, and later vertebrae, relies on a genetic oscillator (the segmentation clock) driving the rhythmic activity of signaling pathways in the presomitic mesoderm (PSM). To understand whether oscillations are an intrinsic property of individual cells or represent a population-level phenomenon, we established culture conditions for stable oscillations at the cellular level. This system was used to demonstrate that oscillations are a collective property of PSM cells that can be actively triggered in vitro by a dynamical quorum sensing signal involving Yap and Notch signaling. Manipulation of Yap-dependent mechanical cues is sufficient to predictably switch isolated PSM cells from a quiescent to an oscillatory state in vitro, a behavior reminiscent of excitability in other systems. Together, our work argues that the segmentation clock behaves as an excitable system, introducing a broader paradigm to study such dynamics in vertebrate morphogenesis.
Project description:The formation of reiterated somites along the vertebrate body axis is controlled by the segmentation clock, a molecular oscillator expressed within presomitic mesoderm (PSM) cells. Although PSM cells oscillate autonomously, they coordinate with neighboring cells to generate a sweeping wave of cyclic gene expression through the PSM that has a periodicity equal to that of somite formation. The velocity of each wave slows as it moves anteriorly through the PSM, although the dynamics of clock slowing have not been well characterized. Here, we investigate segmentation clock dynamics in the anterior PSM in developing zebrafish embryos using an in vivo clock reporter, her1:her1-venus. The her1:her1-venus reporter has single-cell resolution, allowing us to follow segmentation clock oscillations in individual cells in real-time. By retrospectively tracking oscillations of future somite boundary cells, we find that clock reporter signal increases in anterior PSM cells and that the periodicity of reporter oscillations slows to about ?1.5 times the periodicity in posterior PSM cells. This gradual slowing of the clock in the anterior PSM creates peaks of clock expression that are separated at a two-segment periodicity both spatially and temporally, a phenomenon we observe in single cells and in tissue-wide analyses. These results differ from previous predictions that clock oscillations stop or are stabilized in the anterior PSM. Instead, PSM cells oscillate until they incorporate into somites. Our findings suggest that the segmentation clock may signal somite formation using a phase gradient with a two-somite periodicity.