A Microfluidics Approach for the Functional Investigation of Signaling Oscillations Governing Somitogenesis

Abstract

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and gradients are thought to control the timing and spacing of segment formation, respectively. While the involved signaling pathways have been studied extensively over the last decades, direct evidence for the function of signaling oscillations in controlling somitogenesis has been lacking. To enable the functional investigation of signaling dynamics, microfluidics is a previously established tool for the subtle modulation of these dynamics. With this microfluidics-based entrainment approach endogenous signaling oscillations are synchronized by pulses of pathway modulators. This enables modulation of, for instance, the oscillation period or the phase-relationship between two oscillating pathways. Furthermore, spatial gradients of pathway modulators can be established along the tissue to study how specific changes in the signaling gradients affect somitogenesis.The present protocol is meant to help establish microfluidic approaches for the first-time users of microfluidics. The basic principles and equipment needed to set up a microfluidic system are described, and a chip design is provided, with which a mold for chip generation can conveniently be prepared using a 3D printer. Finally, how to culture primary mouse tissue on a microfluidic chip and how to entrain signaling oscillations to external pulses of pathway modulators are discussed.This microfluidic system can also be adapted to harbor other in vivo and in vitro model systems such as gastruloids and organoids for functional investigation of signaling dynamics and morphogen gradients in other contexts.

Live imaging of adult zebrafish cardiomyocyte proliferation ex vivo

“…Microfluidic experiments were performed as described previously ( van Oostrom et al, 2021 ) with minor adaptions. Briefly, microfluidic chips were generated by casting PDMS (polydimethylsiloxane) in a mold.…”

An automated do-it-yourself system for dynamic stem cell and organoid culture in standard multi-well plates

“…The setup presented here includes PDMS microfluidic modules assembled by multi-layer soft lithography and cast from molds patterned with SU-8 and AZ photoresist. While the generation of molds using photolithography techniques requires access to clean-room facilities, which are available at most major research universities, custom molds can be obtained commercially ( https://www.flowjem.com , https://www.su8masters.com ) or can be 3D printed as an alternative to photolithography ( de Almeida Monteiro Melo Ferraz et al., 2020 ; van Oostrom et al., 2021 ). PDMS device fabrication can be performed in a non-specialized laboratory setting, requiring minimal equipment such as a spin-coater, stereomicroscope, and oven.…”

“…However, the required know-how, expertise, and design complexity of state-of-the-art microfluidic cell-culturing devices limit adaptation and modification by non-expert laboratories. Lately, a simpler-to-establish microfluidic system was presented for the culture and pulsed stimulation of primary mouse tissue in ex vivo cultures ( Sanchez et al., 2021 ; Sonnen et al., 2018 ; van Oostrom et al., 2021 ). Neither culture system, however, enables the exploration of how complex stimulation profiles and signaling dynamics, such as step-function concentration changes or oscillatory pulses, impact cellular systems, while necessitating culturing of cellular aggregates in polydimethylsiloxane (PDMS) chambers.…”

Arnold tongue entrainment reveals dynamical principles of the embryonic segmentation clock

“…We used in this study a microfluidics-based experimental entrainment platform with a general protocol elaborated in a recent publication (75). The PDMS microfluidics device was made using standard soft lithography (76) and as previously described (1).…”