Morphogen signalling forms an activity gradient and instructs cell identities in a signalling strength-dependent manner to pattern developing tissues. However, developing tissues also undergo dynamic morphogenesis, which may produce cells with unfit morphogen signalling and consequent noisy morphogen gradients. Here we show that a cell competition-related system corrects such noisy morphogen gradients. Zebrafish imaging analyses of the Wnt/β-catenin signalling gradient, which acts as a morphogen to establish embryonic anterior-posterior patterning, identify that unfit cells with abnormal Wnt/β-catenin activity spontaneously appear and produce noise in the gradient. Communication between unfit and neighbouring fit cells via cadherin proteins stimulates apoptosis of the unfit cells by activating Smad signalling and reactive oxygen species production. This unfit cell elimination is required for proper Wnt/β-catenin gradient formation and consequent anterior-posterior patterning. Because this gradient controls patterning not only in the embryo but also in adult tissues, this system may support tissue robustness and disease prevention.

The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single-cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single-cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay.

Bioluminescence from cells is so dim that bioluminescence microscopy is performed using an ultra low-light imaging camera. Although the image sensor of such cameras has been greatly improved over time, such improvements have not been made commercially available for microscopes until now. Here, we customized the optical system of a microscope for bioluminescence imaging. As a result, bioluminescence images of cells could be captured with a conventional objective lens and colour imaging camera. As bioluminescence microscopy requires no excitation light, it lacks the photo-toxicity associated with fluorescence imaging and permits the long-term, nonlethal observation of living cells. Thus, bioluminescence microscopy would be a powerful tool in cellular biology that complements fluorescence microscopy.

Bioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin—a substrate of luciferase in promoter assays of gene expression in cultured cells—confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492–506 nm and 524–578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 μM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol‐12‐myristate‐13‐acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells. Microsc. Res. Tech. 78:715–722, 2015. © 2015 Wiley Periodicals, Inc.

Cancer cells require the regulation of organelle-specific unfolded protein responses, such as endoplasmic reticulum (ER) stress, because of their increased metabolic activity during rapid proliferation and cell growth, which are executed through the activation of diverse signaling pathways. In this study, we focused on the dynamic regulation of ER stress in accordance with cancer cellular demand, and we performed real-time monitoring of the activation of the binding immunoglobulin protein (Bip) promoter, which is one of the most responsive genes to ER stress during cancer cell growth, in two and three dimensional (2D and 3D) cell culture using bioluminescence imaging at the single-cell level. Bioluminescence images were obtained from living single cancer cells after transient transfection of the reporter gene, and we observed Bip promoter activation during cell growth. Bip promoter activation was also observed in 2D and 3D culture using stably transfected glioblastoma cancer cells with the reporter gene. The Bip promoter was activated especially in dividing cells during cell growth. We then performed real-time monitoring of Bip promoter activation by bioluminescence imaging in tissue slices obtained from U251/pBipPro-Luc tumors. Luminescence intensity was not constant and was different in individual regions of the tumor slices, and the Bip promoter was activated in several regions during monitoring in vitro. These results show that real-time monitoring by bioluminescence imaging at the single-cell level is a suitable tool for not only gene analysis of signal transduction and regulation of the dynamics of the unfolded protein response in cancer cells but also for the evaluation of the efficacy of anti-cancer agents, and could provide additional information that has been difficult to obtain using conventional assays.