An inverted light-sheet microscope for the functional investigation of signalling dynamics during development and tissue homeostasis at single cell resolution

Signalling pathways coordinate proper development and homeostasis of multicellular systems.

It has been shown in recent years that biological information can be encoded in the dynamics, i.e. the temporal change, of a signal. Encoding information in dynamics can ensure transmission of information to be specific and accurate. However, most of our knowledge comes from studies using single cells.

With new technological advancements, we are now at the stage to address the key question: What is the function of signalling dynamics in controlling cellular behaviour within three-dimensional multicellular systems?

To address this, we will use two model systems: (1) Somitogenesis has been the prime model system for the study of dynamic signal encoding at tissue level.

It describes the sequential segmentation of vertebrate embryos, which is controlled by signalling gradients and oscillations.

While the role of signalling dynamics in controlling differentiation and segmentation has been studied extensively, we do not understand how tissue growth is coordinated with segmentation. (2) Homeostasis of adult tissue is maintained by similar signalling pathways as those that govern somitogenesis.

In the small intestine some of these pathways have recently been shown to be dynamic.

We will apply a systematic approach to (A) visualize the dynamics of signalling pathways and (B) understand the function of these dynamics.

By comparing those two model systems, we will be able to derive general principles of dynamic signal encoding in multicellular systems.

I have previously established a microfluidic system which – in combination with dynamic signalling reporters – now allows us to dissect the function of signalling dynamics at tissue level.

Using entrainment of endogenous oscillators we could for instance show that the phase-relationship between two oscillating signalling pathways is critical for proper somitogenesis.

To understand how signalling dynamics impact on cellular behaviour and cell proliferation within heterogenous tissues, we now have to combine such functional studies with the investigation at single-cell resolution.

Imaging of dynamic signalling reporters within tissues and organoids has so far been limited by high phototoxicity at high spatiotemporal resolution, which prevents quantification of signalling dynamics at single-cell resolution. With this proposal I am applying for an inverted light-sheet microscope.

Using this we will be able to (A) visualize signalling dynamics within embryos and organoids at single-cell resolution and (B) study the function of these dynamics by combining light-sheet microscopy with microfluidics.

In this way, we will reach an unprecedented level of understanding of how signalling dynamics control multicellular biology.