Self-organisation of intestinal organoids is driven by a YAP1-dependent regeneration process

Serra, Denise. Self-organisation of intestinal organoids is driven by a YAP1-dependent regeneration process. 2021, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: https://edoc.unibas.ch/84931/

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Self-organisation is a phenomenon in which ordered structures arise from local
interactions of disordered elements. The collective behaviour of the elements confers properties to the system that are more than the sum of the properties of the elements. Indeed, the spontaneous formation of ordered structures can be explained by the dynamic and non-linear interactions of the distinct elements.
In multicellular organisms, cells constantly adjust their state according to the signals received by the neighbouring cells and the environment, leading to events of self-organisation that are observed e.g. during embryogenesis, tissue development and regeneration.
Cellular diversification, or symmetry breaking, appears within uniform starting condition through stochastic fluctuations and cell interactions that are sustained and amplified in feedback loops. Symmetry breaking brings the system from a homogeneous and disordered state to a state that is stable and well-defined. Exactly how the cells make distinct choices and influence the collective behaviour is still an open question. During my PhD I have investigated this pivotal point, fundamentally defining higher organisms, and thereby contributed to the understanding of how multicellular asymmetric structures arise from the self-organising behaviour of single stem cells.
Cellular self-organisation can be modelled in vitro through organoid culture. In the main study presented in this thesis “Self-organization and symmetry breaking in intestinal organoid development” (Serra et al., 2019) we characterize the formation of intestinal organoids from single cells. Intestinal organoids resemble the structure and function of the intestinal epithelium by containing all the tissue characteristic cell types which are similarly spatially distributed in crypts and villi. Intestinal organoids develop from a single proliferating cell that gives rise to an initially round and symmetric sphere; the sphere then break the symmetry by the differentiation of a first Paneth cell, one of the cell types of the intestinal epithelium. This event allows the succeeding formation of a stem cell niche and specification of crypt and villus domains.
The ability of a single cell to spatially and temporally organise into a functional organoid under homogeneous culture conditions was poorly elucidated, as were the driving transcriptional programs and the mechanisms leading to symmetry breaking. The absence of studies to describe this initial phase of organoid development has in part been due to the lack of technologies with adequate spatial and temporal resolution. In this manuscript, we progress and integrate several technologies for the use in organoid cultures, ranging from automated high-content imaging, multiplexed imaging and light sheet microscopy. These methods are combined with mRNA sequencing in order to allow us to characterize morphologically and molecularly the steps of organoid formation. Previous studies have revealed that several cell types of the intestinal epithelium dedifferentiate in vivo following injury. In this pluripotent state they are able to restore tissue homeostasis and these cells are also able to form into intestinal organoids. In our work we show that the different populations of stem cells and nonstem cells share a stereotypic pattern of organoid growth. The process of organoid formation is driven by the transient activation of the mechanosensor and transcriptional regulator YAP1 in single cells. YAP1 initiates a regenerative response by reprogramming cells with diverse levels of differentiation into fast proliferating cells, and exploiting the plasticity of the intestinal epithelium. These cells, cultured in homogeneous growth-promoting conditions, divide and generate a round cyst that lacks expression of known marker genes of intestinal cell types. The emergence of intercellular variable activity of YAP1 in the cyst, probably due to local changes in cell crowding, enables the establishment of Notch-Delta lateral inhibition, which is responsible for cell fate specification in the intestinal epithelium. Cells with YAP1 activity express the Notch ligand DLL1 and differentiate into Paneth cells. This is the event that denote the break of the symmetry. Paneth cells release Wnt molecules that sustain cell proliferation and self-maintenance
of the organoids. The formation of a Wnt gradient around Paneth cells allows the diversification of two functional regions in the organoid: 1) the highly proliferative crypt — containing transient amplifying cells and stem cells intermingled with Paneth cells. 2) the villus — populated by the differentiated enterocytes, Goblet and enteroendocrine cells. Failure of symmetry breaking prevents the formation of the stem cell niche and causes the differentiation of the cyst into a short-living enterocyst composed uniquely of enterocytes.
In conclusion, we describe the molecular mechanisms underlying the capacity of single cells to generate multicellular ordered and functional systems, such as the intestinal organoid. These findings can help shedding light into the principles governing formation, tissue organisation, as well as regeneration of multicellular organisms. Our findings reveal that the process of organoid formation recapitulates the regeneration of the intestinal epithelium YAP1-mediated following injury. This new knowledge will influence the future applications of intestinal organoids in research, and the interpretation of the experiments done with this model system.
Advisors:Liberali, Prisca and Grapin-Botton, Anne
Faculties and Departments:09 Associated Institutions > Friedrich Miescher Institut FMI > Quantitative Biology > Cellular heterogeneity during collective cell behavior (Liberali)
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:14540
Thesis status:Complete
Number of Pages:72
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss145401
edoc DOI:
Last Modified:10 Dec 2021 11:05
Deposited On:10 Dec 2021 11:05

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