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

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 singlecell 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.

A dynamic, spatially periodic, micro-pattern of HES5 underlies neurogenesis in the mouse spinal cord

“…5b), which further reduced the parameter space of repression threshold and inter-cellular time delay that could fit the observed data (Figure 5e,f). The accepted parameter values for intercellular time delay were consistent with a Delta to HES5 transmission time of 128 min measured experimentally (Isomura et al, 2017). A Hill coefficient value larger than 2 was required for notable population dynamics to emerge (KOP>0) and only minor differences in terms of parameter selection were observed for values between 3 and 6 (Supp .…”

Light Control of the Tet Gene Expression System in Mammalian Cells

“…Previously, we adopted the PA-Gal4/UAS (upstream activation sequence) system, in which the PA transcription factor was GAVPO, to analyze the functional importance of gene expression changes of the bHLH transcription factor Ascl1 in the regulation of neural stem cells (Imayoshi et al, 2013;Kageyama, 2014a, 2014b). Because VVD, the smallest light-oxygen-voltage domain-containing protein, is used as a photocontrolled dimer-formation module and GAVPO is very small (56 kDa), it can be expressed in multiple cell types via various gene delivery methods, including lipofection and electroporation of expression plasmids, and viral vectors, such as lentivirus vectors (Imayoshi et al, 2013;Isomura et al, 2017;Wang et al, 2012). GAVPO is extremely sensitive to light, and brief pulses of weak blue light are sufficient to induce high expression levels of the downstream genes.…”

“…Image analysis and quantification Image analysis was performed using ImageJ software and custom plug-ins (Imayoshi et al, 2013;Isomura et al, 2017). The customwritten code for the ImageJ plug-ins used in this study is available upon request.…”

The clock that controls spine development modelled in a dish

“…17). This sender-receiver system can be modulated using a genetically engineered optogenetic variant of the Dll1 gene that is expressed in response to stimulation by light 18 . The authors stimulated Dll1, and compared how long it took for neighbouring cells to exhibit Hes1 upregulation in mice lacking Lunatic fringe with the time it took in controls .…”