In vitro characterization of the human segmentation clock

Margarete Diaz-Cuadros, Daniel E Wagner, Christoph Budjan, Alexis Hubaud, Oscar Tarazona, Sophia Donelly, Arthur Michaut, Ziad Al Tanoury, Kumiko Yoshioka-Kobayashi, Yusuke Niino, Ryoichiro Kageyama, Atsushi Miyawaki, Jonathan Touboul, and Olivier Pourquié

Abstract

The vertebral column is characterized by the periodic arrangement of vertebrae along the anterior-posterior (AP) axis. This segmental organization 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. While this oscillator has been well characterized in model organisms, whether a similar oscillator exists in humans remains unknown. We have previously showed that both mouse embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) differentiate in vitro into PSM upon activation of the Wnt signaling pathway combined with BMP inhibition3. Using transcriptional reporters for the cyclic gene Hes7, here we show that in vitro-derived mouse and human PSM cells recapitulate oscillations of the segmentation clock. We further found that human PSM cells oscillate twice slower than mouse cells (5-hour vs. 2.5 hour period), but similarly depend on the Fgf, Wnt, Notch and YAP4 signaling pathways for the maintenance of oscillatory behavior. Single cell RNA-sequencing comparison of differentiating mouse ESCs and human iPSCs with in vivo mouse PSM reveals that cells differentiating in vitro follow a similar path and exhibit a remarkably coordinated trajectory. Furthermore, we demonstrate that FGF signaling controls the phase and period of the oscillator. This contrasts with classical segmentation models such as the “Clock and Wavefront”, where FGF merely implements a signaling threshold specifying where oscillations stop. Overall, our work identifying the human segmentation clock represents an important breakthrough for human developmental biology.


Get the data

The full dataset is deposited in NCBI GEO under accession number GSE114186.
Counts tables generated by inDrops.py can be downloaded here.
ScanPy versions of the processed datasets can be accessed here:   MouseE9.5   MouseES   HumanIPS
Jupyter notebooks for analyzing the data in ScanPy can be found on Github.


Explore the data using SPRING

SPRING is a tool for interactive exploration of single-cell data. We provide four SPRING-based interfaces:

Mouse E9.5

Mouse E9.5 Neural/PSM


Mouse ESC

Human iPSC


Bugs, questions, or comments? Please send an email to wagnerde@gmail.com.