PhD Student The Francis Crick Institute London, England, United Kingdom
Abstract: The intestinal epithelium's rapid renewal and repetitive structure makes it an excellent model for studying stem cell homeostasis and differentiation. Intestinal stem cells (ISCs) at the crypt base renew the epithelium every 5-7 days, producing secretory and absorptive cells. While ISC regulation is well-studied, little is known about how the cell fate is established at the earliest progenitors (+4/5 cells) in the intestinal epithelium. We hypothesise that this decision is driven by a Notch-mediated gene regulatory network (GRN). By using in vivo lineage tracing, high-throughput sequencing and organoids, we investigate the regulation of this fate decision.
Lineage tracing in a mouse model allowed labelling of +4/5 cells. Followed by cell sorting and bulk RNA-seq, we compared the +4/5 cell transcriptome to ISCs and shortlisted +4/5 enriched transcriptional regulators and chromatin organisers. We identified 36 genes, which have not yet been described in ISC regulation and fate decision, alongside Mtg8/16 that have recently been discovered to be co-repressors of secretory fate. The genes were further shortlisted by investigating their expression upon Notch inhibition in organoids and mice as well as RNAscope analysis to prove expression at the +4/5 position. Three candidate genes were higher expressed after Notch inhibition in vitro and in vivo and showed +4/5 specific expression, indicating a role in the Notch-driven fate decision. Knock-out of these genes in mouse intestinal organoids showed significant changes in marker gene expression for all terminally differentiated cell types of the epithelium after Notch inhibition. To further investigate the role of Mtg8 in intestinal homeostasis, we depleted the bona fide ISC marker gene Lgr5 in Mtg8 KO mice, resulting in faster recovery when compared to wild type. At the same time Mtg8 KO mice failed to recover after in vivo Notch inhibition. These findings reveal new players in the GRN driving intestinal fate decision and strengthen the Notch-mediated role of Mtg8. However, specific molecular roles and interactions require further characterisation.
Identifying new regulators of intestinal epithelial homeostasis furthers our understanding of the complex GRN behind this process and might also unravel novel mechanisms in intestinal regeneration and disease.
