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In vitro characterization of the human segmentation clock.

ABSTRACT: The segmental organization of the vertebral column is established early in embryogenesis, when pairs of somites are rhythmically produced by the presomitic mesoderm (PSM). The tempo of somite formation is controlled by a molecular oscillator known as the segmentation clock1,2. Although this oscillator has been well-characterized in model organisms1,2, whether a similar oscillator exists in humans remains unknown. Genetic analyses of patients with severe spine segmentation defects have implicated several human orthologues of cyclic genes that are associated with the mouse segmentation clock, suggesting that this oscillator might be conserved in humans3. Here we show that human PSM cells derived in vitro-as well as those of the mouse4-recapitulate the oscillations of the segmentation clock. Human PSM cells oscillate with a period two times longer than that of mouse cells (5 h versus 2.5 h), but are similarly regulated by FGF, WNT, Notch and YAP signalling5. Single-cell RNA sequencing reveals that mouse and human PSM cells in vitro follow a developmental trajectory similar to that of mouse PSM in vivo. Furthermore, we demonstrate that FGF signalling controls the phase and period of oscillations, expanding the role of this pathway beyond its classical interpretation in 'clock and wavefront' models1. Our work identifying the human segmentation clock represents an important milestone in understanding human developmental biology.

SUBMITTER: Diaz-Cuadros M

PROVIDER: S-EPMC7336868 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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In vitro characterization of the human segmentation clock.

Diaz-Cuadros Margarete M Wagner Daniel E DE Budjan Christoph C Hubaud Alexis A Tarazona Oscar A OA Donelly Sophia S Michaut Arthur A Al Tanoury Ziad Z Yoshioka-Kobayashi Kumiko K Niino Yusuke Y Kageyama Ryoichiro R Miyawaki Atsushi A Touboul Jonathan J Pourquié Olivier O

Nature 20200108 7801

The segmental organization of the vertebral column is established early in embryogenesis, when pairs of somites are rhythmically produced by the presomitic mesoderm (PSM). The tempo of somite formation is controlled by a molecular oscillator known as the segmentation clock<sup>1,2</sup>. Although this oscillator has been well-characterized in model organisms<sup>1,2</sup>, whether a similar oscillator exists in humans remains unknown. Genetic analyses of patients with severe spine segmentation d ...[more]

PMID: 31915384

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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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In vitro characterization of the human segmentation clock.

Publications

In vitro characterization of the human segmentation clock.

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