Extended Data Fig. 7: Identification of rare cell types in the mini-guts.

a, Dot plot highlighting genes relevant to the identification of the small cluster designated as microfold-like (M-like) cells that share similarities with M cells residing in follicle associated epithelia (FAE) in vivo. M-like cells express the canonical immature M-cell markers Anxa5 and Marcksl1⁴⁵, involved in gram-negative bacteria binding/endocytic transport^45,46 and regulation of cytoskeleton/adhesion, respectively⁴⁷. Other genes related to bacterial sampling are also expressed, including Prnp⁴⁶, Cd14⁴⁸ and Aif1l⁴⁹. M-like cells also selectively express additional phagocytosis-related markers such as Myadm and Cyba. Notably, transcripts marking mucus secretion (sum of Muc1, Muc2, Muc3, Muc3a, Muc4 and Muc13, here referred to as ‘Mucins’) and IgA transcytosis (Pigr) are missing, which is another trait of FAE⁴⁷. Several other genes related to cytoskeleton and adhesion are also strongly upregulated in this population, including the FAE/M-cell markers Actn1^12,50 and Itgb1⁴⁹. Additional similarities to transcripts marking M-cells include the tight junction marker Cldn4 (Claudin 4) involved in antigen sampling/endocytosis^45,51, the caveolae marker Cav1⁵² and the cytokine Cxcl16⁵³ that mediates lympho-epithelial interaction in gut associated lymphoid tissue⁵⁴, as well as several upregulated NFκB target genes⁵¹. Several other known FAE and M-cell markers are missing in these M-like cells, including Spib, the master controller of M-cell differentiation acting downstream of RANKL signalling⁵⁵. This suggested that M-like cells in mini-gut tubes are only partially analogous to M-cells. We noted that our M-like cell population also shared many transcriptional similarities with two recently described, rare cell populations in the intestine, namely ‘revival stem cells’ (RSCs)^14,56 and regenerative fetal-like stem cell^15,56. In particular, M-like cells in mini-guts were found to selective express the RSC markers Clu and Msln^14,56, previously reported as FAE/M-cell markers^9,46,53, and Ly6a (Sca1), that also defines regenerative fetal-like epithelial cells^15,56. A characteristic feature of both RSCs and fetal-like stem cells is the activation of the YAP pathway, mediated by focal adhesions, inflammation or prostaglandin E2. Both YAP target genes and prostaglandin-related genes were found to be strongly and selectively expressed in our M-like cell population as well. b, Fluorescence confocal images of representative 15 days-old mini-gut tube, showing an entire tissue (left column) and a higher magnification view (right column) containing GP2⁺ (red) M-like cells. Data are representative of two replicates. Scale bar, 100 μm. c, Expression of the key enteroendocrine genes in the mini-guts tubes. Neurog3, a marker of immature enteroendocrine cells, forms a gradient towards Chga, Chgb and Neurod1, marking mature enteroendocrine cells. Furthermore, a subpopulation of the enterochromaffin cells defined by hormone substance P (Tac1) and Tph1, encoding the rate-limiting enzyme in serotonin synthesis, can be detected. A subpopulation of cholecystokinin producing I-cells (Cck) was found, co-expressing proglucagon products (Gcg), and varying levels of peptide YY (Pyy), ghrelin (Ghrl) and gastrin (Gast). Enteroendocrine cells were also found to highly express Wnt3, which may partially contribute to the observed higher number of stem cells in mini-guts.