Differential cell-cycle control by oscillatory versus sustained Hes1 expression via p21.

Oscillatory Hes1 expression activates cell proliferation, while high and sustained Hes1 expression induces quiescence, but the mechanism by which Hes1 differentially controls cell proliferation depending on its expression dynamics is unclear. Here, we show that oscillatory Hes1 expression down-regulates the expression of the cyclin-dependent kinase inhibitor p21 (Cdkn1a), which delays cell-cycle progression, and thereby activates the proliferation of mouse neural stem cells (NSCs). By contrast, sustained Hes1 overexpression up-regulates p21 expression and inhibits NSC proliferation, although it initially down-regulates p21 expression. Compared with Hes1 oscillation, sustained Hes1 overexpression represses Dusp7, a phosphatase for phosphorylated Erk (p-Erk), and increases the levels of p-Erk, which can up-regulate p21 expression. These results indicate that p21 expression is directly repressed by oscillatory Hes1 expression, but indirectly up-regulated by sustained Hes1 overexpression, suggesting that depending on its expression dynamics, Hes1 differentially controls NSC proliferation via p21.

Biological Significance of the Coupling Delay in Synchronized Oscillations.

The significance of the coupling delay, which is the time required for interactions between coupled oscillators, in various oscillatory dynamics has been investigated mathematically for more than three decades, but its biological significance has been revealed only recently. In the segmentation clock, which regulates the periodic formation of somites in embryos, Hes7 expression oscillates synchronously between neighboring presomitic mesoderm (PSM) cells, and this synchronized oscillation is controlled by Notch signaling-mediated coupling between PSM cells. Recent studies have shown that inappropriate coupling delays dampen and desynchronize Hes7 oscillations, as simulated mathematically, leading to the severe fusion of somites and somite-derived tissues such as the vertebrae and ribs. These results indicate the biological significance of the coupling delay in synchronized Hes7 oscillations in the segmentation clock. The recent development of an in vitro PSM-like system will facilitate the detailed analysis of the coupling delay in synchronized oscillations.

Light Control of Gene Expression Dynamics.

The progress in live-cell imaging technologies has revealed diverse dynamic patterns of transcriptional activity in various contexts. The discovery raised a next question of whether the gene expression patterns play causative roles in triggering specific biological events or not. Here, we introduce optogenetic methods that realize optical control of gene expression dynamics in mammalian cells and would be utilized for answering the question, by referring the past, the present, and the future.

Coupling delay controls synchronized oscillation in the segmentation clock.

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 fusion3-7. 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 oscillators8. 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.

Dynamic Delta-like1 expression in presomitic mesoderm cells during somite segmentation.

During somite segmentation, the expression of clock genes such as Hes7 oscillates synchronously in the presomitic mesoderm (PSM). This synchronous oscillation slows down in the anterior PSM, leading to wave-like propagating patterns from the posterior to anterior PSM. Such dynamic expression depends on Notch signaling and is critical for somite formation. However, it remains to be determined how slowing oscillations in the anterior PSM are controlled, and whether the expression of the Notch ligand Delta-like1 (Dll1) oscillates on the surface of individual PSM cells, as postulated to be responsible for synchronous oscillation. Here, by using Dll1 fluorescent reporter mice, we performed live-imaging of Dll1 expression in PSM cells and found the oscillatory expression of Dll1 protein on the cell surface regions. Furthermore, a comparison of live-imaging of Dll1 and Hes7 oscillations revealed that the delay from Dll1 peaks to Hes7 peaks increased in the anterior PSM, suggesting that the Hes7 response to Dll1 becomes slower in the anterior PSM. These results raise the possibility that Dll1 protein oscillations on the cell surface regulate synchronous Hes7 oscillations, and that the slower response of Hes7 to Dll1 leads to slower oscillations in the anterior PSM.

Oscillations of MyoD and Hes1 proteins regulate the maintenance of activated muscle stem cells.

The balance between proliferation and differentiation of muscle stem cells is tightly controlled, ensuring the maintenance of a cellular pool needed for muscle growth and repair. We demonstrate here that the transcriptional regulator Hes1 controls the balance between proliferation and differentiation of activated muscle stem cells in both developing and regenerating muscle. We observed that Hes1 is expressed in an oscillatory manner in activated stem cells where it drives the oscillatory expression of MyoD. MyoD expression oscillates in activated muscle stem cells from postnatal and adult muscle under various conditions: when the stem cells are dispersed in culture, when they remain associated with single muscle fibers, or when they reside in muscle biopsies. Unstable MyoD oscillations and long periods of sustained MyoD expression are observed in differentiating cells. Ablation of the Hes1 oscillator in stem cells interfered with stable MyoD oscillations and led to prolonged periods of sustained MyoD expression, resulting in increased differentiation propensity. This interfered with the maintenance of activated muscle stem cells, and impaired muscle growth and repair. We conclude that oscillatory MyoD expression allows the cells to remain in an undifferentiated and proliferative state and is required for amplification of the activated stem cell pool.

Oscillatory Control of Notch Signaling in Development.

The Notch effectors Hes1 and Hes7 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner during neurogenesis and somitogenesis. These two biological events exhibit different types of oscillations: anti-/out-of-phase oscillation in neural stem cells during neurogenesis and in-phase oscillation in presomitic mesoderm (PSM) cells during somitogenesis. Accelerated or delayed Dll1 expression by shortening or elongating the size of the Dll1 gene, respectively, dampens or quenches Dll1 oscillation at intermediate levels, a phenomenon known as "amplitude/oscillation death" of coupled oscillators. Under this condition, both Hes1 oscillation in neural stem cells and Hes7 oscillation in PSM cells are also dampened. As a result, maintenance of neural stem cells is impaired, leading to microcephaly, while somite segmentation is impaired, leading to severe fusion of somites and their derivatives, such as vertebrae and ribs. Thus, the appropriate timing of Dll1 expression is critical for the oscillatory expression in Notch signaling and normal processes of neurogenesis and somitogenesis. Optogenetic analysis indicated that Dll1 oscillations transfer the oscillatory information between neighboring cells, which may induce anti-/out-of-phase and in-phase oscillations depending on the delay in signaling transmission. These oscillatory dynamics can be described in a unified manner by mathematical modeling.

Reconstitution of an Ultradian Oscillator in Mammalian Cells by a Synthetic Biology Approach.

The Notch effector gene Hes1 is an ultradian clock exhibiting cyclic gene expression in several progenitor cells, with a period of a few hours. Because of the complexity of studying Hes1 in the endogenous setting, and the difficulty of imaging these fast oscillations in vivo, the mechanism driving oscillations has never been proven. Here, we applied a "build it to understand it" synthetic biology approach to construct simplified "hybrid" versions of the Hes1 ultradian oscillator combining synthetic and natural parts. We successfully constructed a simplified synthetic version of the Hes1 promoter matching the endogenous regulation logic. By mathematical modeling and single-cell real-time imaging, we were able to demonstrate that Hes1 is indeed able to generate stable oscillations by a delayed negative feedback loop. Moreover, we proved that introns in Hes1 contribute to the transcriptional delay but may not be strictly necessary for oscillations to occur. We also developed a novel reporter of endogenous Hes1 oscillations able to amplify the bioluminescence signal 5-fold. Our results have implications also for other ultradian oscillators.

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions.

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

ES cell-derived presomitic mesoderm-like tissues for analysis of synchronized oscillations in the segmentation clock.

Somites are periodically formed by segmentation of the anterior parts of the presomitic mesoderm (PSM). In the mouse embryo, this periodicity is controlled by the segmentation clock gene Hes7, which exhibits wave-like oscillatory expression in the PSM. Despite intensive studies, the exact mechanism of such synchronous oscillatory dynamics of Hes7 expression still remains to be analyzed. Detailed analysis of the segmentation clock has been hampered because it requires the use of live embryos, and establishment of an in vitro culture system would facilitate such analyses. Here, we established a simple and efficient method to generate mouse ES cell-derived PSM-like tissues, in which Hes7 expression oscillates like traveling waves. In these tissues, Hes7 oscillation is synchronized between neighboring cells, and the posterior-anterior axis is self-organized as the central-peripheral axis. This method is applicable to chemical-library screening and will facilitate the analysis of the molecular nature of the segmentation clock.

Illuminating information transfer in signaling dynamics by optogenetics.

Cells receive diverse signaling cues from their environment that trigger cascades of biochemical reactions in a dynamic manner. Single-cell imaging technologies have revealed that not only molecular species but also dynamic patterns of signaling inputs determine the fates of signal-receiving cells; however it has been challenging to elucidate how such dynamic information is delivered and decoded in complex networks of inter-cellular and inter-molecular interactions. The recent development of optogenetic technology with photo-sensitive proteins has changed this situation; the combination of microscopy and optogenetics provides fruitful insights into the mechanism of dynamic information processing at the single-cell level. Here, we review recent efforts to visualize the flows of dynamic patterns in signaling pathways, which utilize methods integrating single-cell imaging and optogenetics.

Segmentation Genes Enter an Excited State.

The expression of segmentation clock genes is self-oscillatory due to a delayed negative-feedback mechanism. Hubaud et al. (2017), reporting in Cell, now reveal that segmentation clock genes also exhibit properties of an excitable system, with Yap signaling setting the threshold for adopting an oscillatory state and Notch signaling triggering oscillations.

Dynamics of spatiotemporal line defects and chaos control in complex excitable systems.

Spatiotemporal pattern formation governs dynamics and functions in various biological systems. In the heart, excitable waves can form complex oscillatory and chaotic patterns even at an abnormally higher frequency than normal heart beats, which increase the risk of fatal heart conditions by inhibiting normal blood circulation. Previous studies suggested that line defects (nodal lines) play a critical role in stabilizing those undesirable patterns. However, it remains unknown if the line defects are static or dynamically changing structures in heart tissue. Through in vitro experiments of heart tissue observation, we reveal the spatiotemporal dynamics of line defects in rotating spiral waves. We combined a novel signaling over-sampling technique with a multi-dimensional Fourier analysis, showing that line defects can translate, merge, collapse and form stable singularities with even and odd parity while maintaining a stable oscillation of the spiral wave in the tissue. These findings provide insights into a broad class of complex periodic systems, with particular impact to the control and understanding of heart diseases.

Optogenetic perturbation and bioluminescence imaging to analyze cell-to-cell transfer of oscillatory information.

Cells communicate with each other to coordinate their gene activities at the population level through signaling pathways. It has been shown that many gene activities are oscillatory and that the frequency and phase of oscillatory gene expression encode various types of information. However, whether or how such oscillatory information is transmitted from cell to cell remains unknown. Here, we developed an integrated approach that combines optogenetic perturbations and single-cell bioluminescence imaging to visualize and reconstitute synchronized oscillatory gene expression in signal-sending and signal-receiving processes. We found that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell level. In this way, the oscillation dynamics are transmitted through Notch signaling, thereby synchronizing the population of oscillators. Thus, this approach enabled us to control and monitor dynamic cell-to-cell transfer of oscillatory information to coordinate gene expression patterns at the population level.

Oscillatory control of Delta-like1 in cell interactions regulates dynamic gene expression and tissue morphogenesis.

Notch signaling regulates tissue morphogenesis through cell-cell interactions. The Notch effectors Hes1 and Hes7 are expressed in an oscillatory manner and regulate developmental processes such as neurogenesis and somitogenesis, respectively. Expression of the mRNA for the mouse Notch ligand Delta-like1 (Dll1) is also oscillatory. However, the dynamics of Dll1 protein expression are controversial, and their functional significance is unknown. Here, we developed a live-imaging system and found that Dll1 protein expression oscillated in neural progenitors and presomitic mesoderm cells. Notably, when Dll1 expression was accelerated or delayed by shortening or elongating the Dll1 gene, Dll1 oscillations became severely dampened or quenched at intermediate levels, as modeled mathematically. Under this condition, Hes1 and Hes7 oscillations were also dampened. In the presomitic mesoderm, steady Dll1 expression led to severe fusion of somites and their derivatives, such as vertebrae and ribs. In the developing brain, steady Dll1 expression inhibited proliferation of neural progenitors and accelerated neurogenesis, whereas optogenetic induction of Dll1 oscillation efficiently maintained neural progenitors. These results indicate that the appropriate timing of Dll1 expression is critical for the oscillatory networks and suggest the functional significance of oscillatory cell-cell interactions in tissue morphogenesis.

Deubiquitinating enzymes regulate Hes1 stability and neuronal differentiation.

Hairy and enhancer of split 1 (Hes1), a basic helix-loop-helix transcriptional repressor protein, regulates the maintenance of neural stem/progenitor cells by repressing proneural gene expression via Notch signaling. Previous studies showed that Hes1 expression oscillates in both mouse embryonic stem cells and neural stem cells, and that the oscillation contributes to their potency and differentiation fates. This oscillatory expression depends on the stability of Hes1, which is rapidly degraded by the ubiquitin/proteasome pathway. However, the detailed molecular mechanisms governing Hes1 stability remain unknown. We analyzed Hes1-interacting deubiquitinases purified from mouse embryonic stem cells using an Hes1-specific antibody, and identified the ubiquitin-specific protease 27x (Usp27x) as a new regulator of Hes1. We found that Hes1 was deubiquitinated and stabilized by Usp27x and its homologs ubiquitin-specific protease 22 (Usp22) and ubiquitin-specific protease 51 (Usp51). Knockdown of Usp22 shortened the half-life of Hes1, delayed its oscillation, and enhanced neuronal differentiation in mouse developing brain, whereas mis-expression of Usp27x reduced neuronal differentiation. These results suggest that these deubiquitinases modulate Hes1 protein dynamics by removing ubiquitin molecules, and thereby regulate neuronal differentiation of stem cells.

Ultradian oscillations and pulses: coordinating cellular responses and cell fate decisions.

Biological clocks play key roles in organismal development, homeostasis and function. In recent years, much work has focused on circadian clocks, but emerging studies have highlighted the existence of ultradian oscillators - those with a much shorter periodicity than 24 h. Accumulating evidence, together with recently developed optogenetic approaches, suggests that such ultradian oscillators play important roles during cell fate decisions, and analyzing the functional links between ultradian oscillation and cell fate determination will contribute to a deeper understanding of the design principle of developing embryos. In this Review, we discuss the mechanisms of ultradian oscillatory dynamics and introduce examples of ultradian oscillators in various biological contexts. We also discuss how optogenetic technology has been used to elucidate the biological significance of ultradian oscillations.

Oscillatory control of factors determining multipotency and fate in mouse neural progenitors.

The basic helix-loop-helix transcription factors Ascl1/Mash1, Hes1, and Olig2 regulate fate choice of neurons, astrocytes, and oligodendrocytes, respectively. These same factors are coexpressed by neural progenitor cells. Here, we found by time-lapse imaging that these factors are expressed in an oscillatory manner by mouse neural progenitor cells. In each differentiation lineage, one of the factors becomes dominant. We used optogenetics to control expression of Ascl1 and found that, although sustained Ascl1 expression promotes neuronal fate determination, oscillatory Ascl1 expression maintains proliferating neural progenitor cells. Thus, the multipotent state correlates with oscillatory expression of several fate-determination factors, whereas the differentiated state correlates with sustained expression of a single factor.

Dclk1 distinguishes between tumor and normal stem cells in the intestine.

There is great interest in tumor stem cells (TSCs) as potential therapeutic targets; however, cancer therapies targeting TSCs are limited. A drawback is that TSC markers are often shared by normal stem cells (NSCs); thus, therapies that target these markers may cause severe injury to normal tissues. To identify a potential TSC-specific marker, we focused on doublecortin-like kinase 1 (Dclk1). Dclk1 was reported as a candidate NSC marker in the gut, but recent reports have implicated it as a marker of differentiated cells (for example, Tuft cells). Using lineage-tracing experiments, we show here that Dclk1 does not mark NSCs in the intestine but instead marks TSCs that continuously produce tumor progeny in the polyps of Apc(Min/+) mice. Specific ablation of Dclk1-positive TSCs resulted in a marked regression of polyps without apparent damage to the normal intestine. Our data suggest the potential for developing a therapy for colorectal cancer based on targeting Dclk1-positive TSCs.

Oscillatory gene expression and somitogenesis.

A bilateral pair of somites forms periodically by segmentation of the anterior ends of the presomitic mesoderm (PSM). This periodic event is regulated by a biological clock called the segmentation clock, which involves cyclic gene expression. Expression of her1 and her7 in zebrafish and Hes7 in mice oscillates by negative feedback, and mathematical models have been used to generate and test hypotheses to aide elucidation of the role of negative feedback in regulating oscillatory expression. her/Hes genes induce oscillatory expression of the Notch ligand deltaC in zebrafish and the Notch modulator Lunatic fringe in mice, which lead to synchronization of oscillatory gene expression between neighboring PSM cells. In the mouse PSM, Hes7 induces coupled oscillations of Notch and Fgf signaling, while Notch and Fgf signaling cooperatively regulate Hes7 oscillation, indicating that Hes7 and Notch and Fgf signaling form the oscillator networks. Notch signaling activates, but Fgf signaling represses, expression of the master regulator for somitogenesis Mesp2, and coupled oscillations in Notch and Fgf signaling dissociate in the anterior PSM, which allows Notch signaling-induced synchronized cells to express Mesp2 after these cells are freed from Fgf signaling. These results together suggest that Notch signaling defines the prospective somite region, while Fgf signaling regulates the pace of segmentation. It is likely that these oscillator networks constitute the core of the segmentation clock, but it remains to be determined whether as yet unknown oscillators function behind the scenes.