an-optogenetic-method-to-control-analyze-gene-expression-patterns

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 ...

Kyoto University

PRESTO

The unpinning of spiral waves by the application of high-frequency wave trains was studied in cultured cardiac myocytes. Successful unpinning was observed when the frequency of the paced waves exceeded a critical level. The unpinning process was analyzed by a numerical simulation with a model of cardiac tissue. The mechanism of unpinning by high-frequency stimuli is discussed in terms of local entrainment failure, through a reduction of the two-dimensional spatial characteristics into one dimension.

Eliminating spiral waves pinned to an anatomical obstacle in cardiac myocytes by high-frequency stimuli.

The unpinning of a spiral wave from an anatomic obstacle by the application of a single stimulus near the core of the rotating wave was studied experimentally in a cell culture of cardiomyocyte monolayers as well as by computer simulations. It is shown that, with suitable positioning and timing, a single stimulus is sufficient for the successful unpinning of a pinned spiral wave. Successful unpinning is achieved when two conditions are fulfilled: (1) The stimulus is delivered in the vulnerable window of the rotating wave, and (2) the stimulus is delivered in a spatial zone in proximity to the obstacle, where the shape of the zone is defined by the phase of the anchored spiral wave. Two different scenarios for successful unpinning are discussed, which are distinguished by the distance to the stimuli applied to the obstacle.

Liberation of a pinned spiral wave by a single stimulus in excitable media.

It is well known that spiral waves are often stabilized by anchoring to a local heterogeneity ("pinning") and that such pinned waves are rather difficult to eliminate. In the present report, we show that pinned spiral waves can be eliminated through collision with a wave train arriving from the outer region, as confirmed in experiments on the Belousov-Zhabotinsky (BZ) reaction as well as in cardiomyocyte tissue culture. A numerical simulation using the Oregonator, a mathematical model for the BZ reaction, provides the parameter area for successful unpinning. The scenario of unpinning is discussed in terms of the dispersion relation of the wave train by taking into account the curvature effect of the excitation wave.

Unpinning of a spiral wave anchored around a circular obstacle by an external wave train: common aspects of a chemical reaction and cardiomyocyte tissue.

Obstacle-anchored vortices can be terminated by the application of high-frequency wave trains in excitable media. We theoretically derived the dependency between the obstacle radius and the maximum unpinning period through reinterpretation of the well-known eikonal equation. Our theoretical result was confirmed by experiments with cardiomyocyte monolayers. This result may be useful for improving the stimulation protocol of implantable cardiac pacemakers.

Utilizing the eikonal relationship in strategies for reentrant wave termination in excitable media.

Somitogenesis is controlled by cyclic genes such as Notch effectors and by the wave front established by morphogens such as Fgf8, but the precise mechanism of how these factors are coordinated remains to be determined. Here, we show that effectors of Notch and Fgf pathways oscillate in different dynamics and that oscillations in Notch signaling generate alternating phase shift, thereby periodically segregating a group of synchronized cells, whereas oscillations in Fgf signaling released these synchronized cells for somitogenesis at the same time. These results suggest that Notch oscillators define the prospective somite region, while Fgf oscillators regulate the pace of segmentation.

Different types of oscillations in Notch and Fgf signaling regulate the spatiotemporal periodicity of somitogenesis.

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.

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

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.

Oscillatory gene expression and somitogenesis.

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.

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

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.

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

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.

Deubiquitinating enzymes regulate Hes1 stability and neuronal differentiation.

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.

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

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.

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

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.

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

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.

Segmentation Genes Enter an Excited State.

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.

Illuminating information transfer in signaling dynamics by optogenetics.

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, which exhibits wave-like oscillatory expression in the PSM. Despite intensive studies, the exact mechanism of such synchronous oscillatory dynamics ofexpression 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 anculture system would facilitate such analyses. Here, we established a simple and efficient method to generate mouse ES cell-derived PSM-like tissues, in whichexpression oscillates like traveling waves. In these tissues,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.

Article	Year
Eliminating spiral waves pinned to an anatomical obstacle in cardiac myocytes by high-frequency stimuli. Physical review. E, Statistical, nonlinear, and soft matter physics\| PubMed ID: 19256934	2008
Liberation of a pinned spiral wave by a single stimulus in excitable media. Physical review. E, Statistical, nonlinear, and soft matter physics\| PubMed ID: 19391831	2009
Unpinning of a spiral wave anchored around a circular obstacle by an external wave train: common aspects of a chemical reaction and cardiomyocyte tissue. Chaos (Woodbury, N.Y.)\| PubMed ID: 20059210	2009
Utilizing the eikonal relationship in strategies for reentrant wave termination in excitable media. Physical review. E, Statistical, nonlinear, and soft matter physics\| PubMed ID: 20866302	2010
Different types of oscillations in Notch and Fgf signaling regulate the spatiotemporal periodicity of somitogenesis. Genes & development\| PubMed ID: 21632822	2011
Dclk1 distinguishes between tumor and normal stem cells in the intestine. Nature genetics\| PubMed ID: 23202126	2013
Oscillatory gene expression and somitogenesis. Wiley interdisciplinary reviews. Developmental biology\| PubMed ID: 23799565	2012
Oscillatory control of factors determining multipotency and fate in mouse neural progenitors. Science (New York, N.Y.)\| PubMed ID: 24179156	2013
Ultradian oscillations and pulses: coordinating cellular responses and cell fate decisions. Development (Cambridge, England)\| PubMed ID: 25249457	2014
Deubiquitinating enzymes regulate Hes1 stability and neuronal differentiation. The FEBS journal\| PubMed ID: 25846153	2015
Oscillatory control of Delta-like1 in cell interactions regulates dynamic gene expression and tissue morphogenesis. Genes & development\| PubMed ID: 26728556	2016
Optogenetic perturbation and bioluminescence imaging to analyze cell-to-cell transfer of oscillatory information. Genes & development\| PubMed ID: 28373207	2017
Dynamics of spatiotemporal line defects and chaos control in complex excitable systems. Scientific reports\| PubMed ID: 28798384	2017
Segmentation Genes Enter an Excited State. Developmental cell\| PubMed ID: 29065304	2017
Illuminating information transfer in signaling dynamics by optogenetics. Current opinion in cell biology\| PubMed ID: 29175322	2017
ES cell-derived presomitic mesoderm-like tissues for analysis of synchronized oscillations in the segmentation clock. Development (Cambridge, England)\| PubMed ID: 29437832	2018

Akihiro Isomura

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