Noise-resistant developmental reproducibility in vertebrate somite formation.

The reproducibility of embryonic development is remarkable, although molecular processes are intrinsically stochastic at the single-cell level. How the multicellular system resists the inevitable noise to acquire developmental reproducibility constitutes a fundamental question in developmental biology. Toward this end, we focused on vertebrate somitogenesis as a representative system, because somites are repeatedly reproduced within a single embryo whereas such reproducibility is lost in segmentation clock gene-deficient embryos. However, the effect of noise on developmental reproducibility has not been fully investigated, because of the technical difficulty in manipulating the noise intensity in experiments. In this study, we developed a computational model of ERK-mediated somitogenesis, in which bistable ERK activity is regulated by an FGF gradient, cell-cell communication, and the segmentation clock, subject to the intrinsic noise. The model simulation generated our previous in vivo observation that the ERK activity was distributed in a step-like gradient in the presomitic mesoderm, and its boundary was posteriorly shifted by the clock in a stepwise manner, leading to regular somite formation. Here, we showed that this somite regularity was robustly maintained against the noise. Removing the clock from the model predicted that the stepwise shift of the ERK activity occurs at irregular timing with irregular distance owing to the noise, resulting in somite size variation. This model prediction was recently confirmed by live imaging of ERK activity in zebrafish embryos. Through theoretical analysis, we presented a mechanism by which the clock reduces the inherent somite irregularity observed in clock-deficient embryos. Therefore, this study indicates a novel role of the segmentation clock in noise-resistant developmental reproducibility.

Size-reduced embryos reveal a gradient scaling-based mechanism for zebrafish somite formation.

Little is known about how the sizes of animal tissues are controlled. A prominent example is somite size, which varies widely both within an individual and across species. Despite intense study of the segmentation clock governing the timing of somite generation, how it relates to somite size is poorly understood. Here, we examine somite scaling and find that somite size at specification scales with the length of the presomitic mesoderm (PSM) despite considerable variation in PSM length across developmental stages and in surgically size-reduced embryos. Measurement of clock period, axis elongation speed and clock gene expression patterns demonstrate that existing models fail to explain scaling. We posit a 'clock and scaled gradient' model, in which somite boundaries are set by a dynamically scaling signaling gradient across the PSM. Our model not only explains existing data, but also makes a unique prediction that we confirm experimentally - the formation of periodic 'echoes' in somite size following perturbation of the size of one somite. Our findings demonstrate that gradient scaling plays a central role in both progression and size control of somitogenesis.

Time-lapse observation of stepwise regression of Erk activity in zebrafish presomitic mesoderm.

During somite segmentation, clock genes oscillate within the posterior presomitic mesoderm (PSM). The temporal information ties up with the posteriorly moving FGF gradient, leading to the formation of a presumptive somite within the PSM. We previously investigated Erk activity downstream of FGF signaling by collecting stained zebrafish embryos, and discovered that the steep gradient of Erk activity was generated in the PSM, and the Erk activity border regularly shifted in a stepwise manner. However, since these interpretations come from static analyses, we needed to firmly confirm them by applying an analysis that has higher spatiotemporal resolutions. Here we developed a live imaging system for Erk activity in zebrafish embryos, using a Förster resonance energy transfer (FRET)-based Erk biosensor. With this system, we firmly showed that Erk activity exhibits stepwise regression within the PSM. Although our static analyses could not detect the stepwise pattern of Erk activity in clock-deficient embryos, our system revealed that, in clock-deficient embryos, the stepwise regression of Erk activity occurs at an irregular timing, eventually leading to formation of irregularly-sized somites. Therefore, our system overcame the limitation of static analyses and revealed that clock-dependent spatiotemporal regulation of Erk is required for proper somitogenesis in zebrafish.