Signalling Dynamics in Small Intestinal Homeostasis: A Crucial Role

Chapter 1 gives a general introduction about small intestinal homeostasis, signalling dynamics in systems and methods to modulate these dynamics. In Chapter 2, Weterings, van Oostrom et al., 2021[27], we highlight the shared principles between embryonic development and tissue homeostasis and propose that integrating insights from both fields will advance research in both areas. Interestingly,
homeostasis of the small intestine is maintained by the same signalling pathways that are included in somitogenesis. Notch and Wnt signalling dynamics have been shown important in cell fate decisions during somitogenesis[44]. Therefore, this raised the important question if and how signalling dynamics affect cell fate decisions during homeostasis of the small intestine. In Chapter 3, Weterings, Eto
et al., 2024[55], we demonstrate that Notch-driven signalling dynamics control cell fate decisions during small intestinal homeostasis[55]. Hes1, a Notch downstream target[56], oscillates with a period gradient from 90 minutes in the crypt base to 170 minutes near the villi. Microfluidic modulation of Hes1 frequency altered cell composition: 90- and 130-minute oscillations promoted stemness, while 170-minute oscillations did not. Additionally, 90-minute oscillations favoured Paneth cell formation, whereas 130-minute oscillations induced other secretory subtypes, revealing a key mechanism of Notch signalling dynamics in secretory cell differentiation. Chapter 4 provides a detailed protocol for the microfluidic tool we have developed. This versatile and user-friendly tool can be easily adopted and customized by researchers to explore various questions related to the role of signalling dynamics in different biological contexts. Chapter 5 highlights the crosstalk between Wnt and Notch signalling dynamics during small intestinal homeostasis on tissue wide and single cell level. As our research progressed, it became increasingly evident that accurate cell segmentation is crucial for analysing single cell signalling dynamics. In response, we developed a new mouse model, detailed in Chapter 6, featuring a plasma membrane miRFP (monomeric infra-red fluorescent protein) tag. This tag can be easily combined with other reporter lines, which make use of a blue, yellow, green or red fluorescent tag. Using a membrane reporter in the far-red channel is beneficial due to miRFP’s slow maturation. While unsuitable for tracking fast signalling dynamics, it enables high-resolution imaging with increased laser
power due to its low phototoxicity. Chapter 7 provides a comprehensive reflection on the study of signalling dynamics, including the rationale behind the chosen approaches, alternative methodologies, key findings in small intestinal homeostasis, broader implications for the field, and the overall contributions of this thesis.