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Real-time imaging of the somite segmentation clock: revelation of unstable oscillators in the individual presomitic mesoderm cells.

ABSTRACT: Notch signaling components such as the basic helix-loop-helix gene Hes1 are cyclically expressed by negative feedback in the presomitic mesoderm (PSM) and constitute the somite segmentation clock. Because Hes1 oscillation occurs in many cell types, this clock may regulate the timing in many biological systems. Although the Hes1 oscillator is stable in the PSM, it damps rapidly in other cells, suggesting that the oscillators in the former and the latter could be intrinsically different. Here, we have established the real-time bioluminescence imaging system of Hes1 expression and found that, although Hes1 oscillation is robust and stable in the PSM, it is unstable in the individual dissociated PSM cells, as in fibroblasts. Thus, the Hes1 oscillators in the individual PSM cells and fibroblasts are intrinsically similar, and these results, together with mathematical simulation, suggest that cell-cell communication is essential not only for synchronization but also for stabilization of cellular oscillators.

SUBMITTER: Masamizu Y

PROVIDER: S-EPMC1345707 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Pluripotent stem cells (PSCs) have increasingly been used to model different aspects of embryogenesis and organ formation. Despite recent advances in the in vitro induction of major mesodermal lineages and mesoderm-derived cell types experimental model systems that can recapitulate more complex biological features of human mesoderm development and patterning are largely missing. Here, we utilized induced pluripotent stem cells (iPSCs) for the stepwise in vitro induction of presomitic mesoderm (PSM) and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modeling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We succeeded to observe oscillatory expression of core segmentation clock genes, including HES7 and DKK1, determined the period of the human segmentation clock to be around five hours and showed the presence of dynamic traveling wave-like gene expression within in vitro induced human PSM. We furthermore identified and compared oscillatory genes in human and murine PSC-derived PSM, which revealed species-specific as well as common molecular components and novel pathways associated with the mouse and human segmentation clocks. Utilizing CRISPR/Cas9-based genome editing technology, we then targeted genes, for which mutations in patients with segmentation defects of vertebrae (SDV) such as spondylocostal dysostosis (SCD) have been reported (e.g. HES7, LFNG, DLL3 and MESP2 DLL3). Subsequent analysis of patient-like knock-out and point-mutation lines as well as patient-derived iPSCs together with their genetically corrected isogenic controls revealed gene-specific alterations in oscillation, synchronization or differentiation properties, validating the overall utility of our model system, to provide novel insights into the human segmentation clock as well as diseases associated with the formation of the human axial skeleton.

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Real-time imaging of the somite segmentation clock: revelation of unstable oscillators in the individual presomitic mesoderm cells.

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