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Oscillatory signaling dynamics – a quantitative approach to reveal their origin and function in development

Periodic Reporting for period 4 - Oscillations (Oscillatory signaling dynamics – a quantitative approach to reveal their origin and function in development)

Reporting period: 2020-03-01 to 2020-08-31

Summary: This project aims to reveal the origin and functions of spatiotemporal signalling oscillations in the context of embryonic development. Vertebrate embryo segmentation offers a particularly suitable context to study an assembly of ultradian, genetic oscillators, which in addition, exhibit striking synchronization that generates periodic, wave-like patterns.
Understanding signalling dynamics, such as oscillations, is of fundamental importance. It is increasingly appreciated that the encoding of information at the level signalling dynamics is a general principle found across biology. The overall objective of this project is hence to elucidate the working principles of oscillation dynamics first in one specific context, segmentation, during vertebrate embryo development.

The questions we are addressing are:
• How are genetic oscillators synchronized within a tissue?
• How do spatiotemporal wave patterns originate and what function do they play during embryonic segmentation?
• What is the role of relative timing (i.e. phase-shift) between different oscillatory pathways?
• How doe genetic oscillators self-organize?
The ERC Grant has enabled several major discoveries:

• What is the role of relative timing (i.e. phase-shift) between different oscillatory pathways?
(Sonnen et al., 2018, Cell 172(5)
We have established a novel approach based on synchronization theory and implemented a microfluidics based entrainment strategy that allows, for the first time, direct experimental control of the rhythm of segmentation clock oscillations. This approach, which exploits the principles of entrainment, opens the possibility to specifically enquire the role of dynamics and rhythm, without affecting overall signalling levels. We are very actively engaged in testing the consequences of altered timing between the relative timing of Notch- and Wnt-signalling oscillations on the segmentation process.

A major milestone has been achieved in this activity, published in 2018 in Cell (Sonnen et al., 2018, Cell 172(5)). In this work, we were able to use synchronization Theory/entrainment to control the relative rhythm between Notch and Wnt signalling pathways in the presomitic mesoderm (PSM). Importantly, this approach reveals, for the first time, a functional role of the relative signaling timing for proper PSM patterning.
• How are genetic oscillators synchronized within a tissue? (Tsiairis and Aulehla, Cell 2016)
The findings regarding the self-organization of genetic oscillators have been published in 2016 in the CELL (Tsiairis and Aulehla, Cell 2016). The main findings are that a randomized ensemble of oscillating cells (oscillations are seen at the level of Notch-signalling pathway activity) will re-synchronize de novo, showing a collective, emerging frequency that corresponds to the average frequency of the input. In addition, even spatio-temporal order and wave patterns re-emerge de novo. This setup exhibiting self-organization and collective synchronization serves as new experimental model to further reveal the underlying principles.

• How do periodic wave patterns originate, either during self-organization or during embryonic development?
We have established a novel experimental setup that enables, for the first time, to visualize the onset of oscillatory wave patterns during early gastrulation, i.e. during the time period when mesoderm cells are first generated. To this end, we have implemented light-sheet microscopy methods that we currently use to visualize and quantify signalling dynamics in early mouse embryo development.
A manuscript summarizing the key results will appear in 2021. The key novel finding relates to the onset of signaling dynamics and oscillations. With the new imaging setup that we developed, we are now able to quantify signalling dynamics also during the early stages of mouse embryo gastrulation. As we have increased the sample throughput (see patent No. EP 17205938.8 ), the analysis of genetic mutants is now feasible and ongoing. The goal is to identify the underlying mechanisms leading to the emergence of coherent spatiotemporal wave patterns during development, directly in gastrulating mouse embyos.

• We have established Medaka as a novel experimental fish model to study segmentation clock dynamics by generating knock-in reporter fish lines.
We have now successfully generated the first segmentation clock reporter medaka fish line. The first results are very encouraging, since signalling dynamics and oscillations can be quantified with very high quality within the physiological in vivo context. Clearly, this avenue is very suitable to transfer the insight gained in simplified in vitro systems to ask the relevance during embryonic in vivo development.
We have established a microfluidics-based entrainment approach that enables to control the rhythm of segmentation clock oscillations. This is an important step forward for the field, as we can now specifically address the role of timing of Notch-, Wnt- and Fgf-signalling pathway oscillations, including the role of their relative timing within cells in the PSM. In addition, we have established a novel system that enables to monitor signalling oscillations during early mouse gastrulation, using light-sheet microscopy, with high spatial and temporal resolution. This is another major achievement that now enables to address the origin of synchronized oscillatory signalling behaviour in the multicellular developmental context.
Signaling Oscillations in Embryonic Patterning