Decrypting gastrointestinal development and homeostasis one cell at a time

Abstract

In this thesis, I have applied multiple single-cell techniques to advance our understanding of how tissues and organs develop and maintain homeostasis. Cells are known to have heterogeneous transcriptomes; even isogenic cells in identical culture conditions have different transcriptional profiles and exhibit distinct phenotypes. In organs, multiple cell types are exposed to diverse microenvironments, making this cellular heterogeneity even more apparent. This heterogeneity is lost when tissues are analyzed in bulk, which only yields a population average. Hence, to fully understand the complexity of an organ or tissue, single cell biology is paramount. In the first study, we developed a new method for sorting cells based on RNA abundance. By fluorescently labeling cells with dozens of probes, we are able to isolate cells by fluorescent-activated cell sorting with a resolution of 10-20 transcripts. Moreover, by preserving the quality of total RNA during probe hybridization, we are able to perform unbiased transcriptional profiling on the sorted subpopulation, e.g. by RNA-sequencing. The subsequent four studies all employ recently developed single-cell RNA sequencing to analyze single cells. By sorting and analyzing Paneth cells from the murine small intestine, we revealed the maturation trajectory of the Paneth cell from a stem cell onwards. Moreover, differential gene expression between two clusters of mature Paneth cells showed the existence of two distinct maturation states, indicated by e.g. the expression of a subset of antimicrobial defensin genes. Next, we used a similar strategy to study Lgr5+ stem cells in different gastrointestinal organs spread over three developmental time points. We isolated Lgr5+ stem cells from the stomach, small intestine and colon at E13.5, E18.5 and from adult. Our data shows large differences between stem cells in the intestine and stomach in adult, e.g. by the expression of many organ-specific differentiation markers. However, at E13.5, the transcriptional profiles of all three organs are remarkably similar, suggesting a ‘naïve’ Lgr5+ stem cell at early embryogenesis that later becomes organ specific. Preliminary data suggests a role for DNA methylation in this process. In addition to in vivo studies, we also made use of the ex vivo organoid system to study quiescence in Lgr5+ stem cells and the formation of high numbers of enteroendocrine cells. By removing EGF from the culture medium, the normally highly proliferative Lgr5+ stem cells become quiescent. Furthermore, these non-dividing stem cells demonstrate a bias towards expression of enteroendocrine markers and differentiation into the enteroendocrine lineage. Upon maturation of the cells, we found a heterogeneous population of cells, which differentially express a variety of hormones, thus mimicking the in vivo situation. In conclusion, single-cell sequencing has yielded valuable information about multiple biological processes in different organs and cell types, and with many new techniques currently being developed will continue to reveal exciting insights into cellular identity.