Curated Optogenetic Publication Database

Abstract: Tight coordination of cell-cell signaling in space and time is vital for self-organization in tissue patterning. During vertebrate development, the segmentation clock drives oscillatory gene expression in the presomitic mesoderm (PSM), leading to the periodic formation of somites. Oscillatory gene expression is synchronized at the cell population level; inhibition of Delta-Notch signaling results in the loss of synchrony and the fusion of somites. However, it remains unclear how cell-cell signaling couples oscillatory gene expression and controls synchronization. Here, we report the reconstitution of synchronized oscillation in PSM organoids by synthetic cell-cell signaling with designed ligand-receptor pairs. Optogenetic assays uncovered that the intracellular domains of synthetic ligands play key roles in dynamic cell-cell communication. Oscillatory coupling using synthetic cell-cell signaling recovered the synchronized oscillation in PSM cells deficient for Delta-Notch signaling; non-oscillatory coupling did not induce recovery. This study reveals the mechanism by which ligand-receptor molecules coordinate the synchronization of the segmentation clock, and provides direct evidence of oscillatory cell-cell communication in the segmentation clock.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.