Periodic Reporting for period 2 - SignalingDynamics (Signaling dynamics in the control of cell proliferation and differentiation during development and homeostasis) Reporting period: 2021-09-01 to 2023-02-28 Summary of the context and overall objectives of the project Cells communicate with each other via signalling pathways to coordinate the development of multicellular organisms and their following maintenance. This communication ensures that every cell knows what to do when and where, whether to proliferate or to become a muscle or gut cell. Errors in this communication can lead to developmental defects or the formation of diseases such as cancer. Therefore, it is essential to understand precisely how this communication works in the healthy tissue and what goes wrong in the context of diseases. Interestingly, it has been shown that communication pathways are not static, but change in activity over time. Just like the morse code - in which information is transmitted by varying the length of sequential sounds - cells in our bodies can make use of dynamic changes in activity to transmit biological information. How this works, especially in the context of a multicellular system, is not understood. Here, we investigate how these so-called "signalling dynamics" are employed to coordinate tissue growth with the differentiation into specific cell types. We compare its role in embryonic development and in the maintenance of adult tissues. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far To study how cells communicate with each other, we use the segmenting vertebrate embryo as model system of embryonic development and the growing small intestine as model system of adult tissue maintenance. To study tissue maintenance, we make use of so-called organoids or mini-organs, culture systems that can be grown in the lab and recapitulate key aspects of the respective organ. Critical steps in this investigations are (1) the visualization of signalling activities within single cells in the context of an entire tissue and (2) the functional analysis of cell-cell communication. To address these points, we have initiated the following:1. For the visualization of this cell-cell communication we use available and have generated new signalling reporter lines that emit fluorescence when communication pathways are active. We have also generated combinations of reporters that either report on different signalling pathways and cell fate. Moreover, we have optimized real-time imaging of our model systems and detection and quantification of these fluorescent reporters. 2. To understand how cells communicate with each other and especially how cells use dynamic changes to transmit information, we have to be able to subtly modulate this communication and analyze the effect of this modulation. We have set up and optimized a microfluidic system that allows us to culture our model systems, segmenting embryo tissue and mini-organs, in small incubation chambers - termed chips - and apply pulses of pathway modulators with high precision. This allows us to subtly change the observed pathway activity and then analyze the effect on development and growth. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Here, we are setting up an experimental system that allows the precise investigation of cell-cell communication in both embryonic development and adult tissue homeostasis. Using this system, we analyze how cell differentiation and proliferation are coordinated with each other via signalling pathways to ensure the formation of an organism with the right proportions and to ensure tissue integrity in the adult organism. This comparison will allow us to draw general conclusions on how cells communicate with other and will in the future serve as basis to dissect how cell communication might be targeted for therapy of diseases such as cancer. Mini-organ of the small intestine expressing a fluorescent signalling reporter.