In this study, we document the origin of M cells, a cell type contained within the follicle-associated epithelium of Peyer's patches. The FAE is a continuation of the epithelium that forms the luminal lining of the small intestine and the colon. Outside the FAE, this lining consists of repeating crypt-villus units. At the bottoms of crypts, Lgr5+
stem cells are intermingled with Paneth cells. These cycling Lgr5 stem cells give rise to all epithelial cell types of the small intestine: enterocytes and much smaller numbers of accessory goblet, enteroendocrine, tuft, and Paneth cells (2
). The quiescent +4 stem cell population, not studied here, is also capable of producing all cell types of the epithelium and appears to be directly connected to the Lgr5 stem cell population (26
The FAE clearly differs in composition from the epithelial lining that is organized into crypts and villi. Differentiation in the FAE is limited to two morphologically and functionally distinguishable cell types: FAE-specific enterocytes and so-called M cells. The M cells specialize in monitoring the content of the intestinal lumen for immunogenic substances and deliver antigens at their basal side to the immune cells of the follicles. We asked whether the crypt-based Lgr5 stem cells are able to generate the typical FAE cell types. Lineage-tracing experiments (3
), based on Lgr5-driven activation of the Rosa26-LacZ locus, demonstrate that Lgr5 stem cells residing in crypts adjacent to PP generate the FAE cells. A subset of the epithelial cells within the LacZ+
ribbons overlying PPs, moreover, are decorated with the M cell-specific substrate for the lectin UAE-1. This clearly shows that M cells constitute the sixth cell type derived from Lgr5 stem cells.
From our studies on SpiB null mice, it appears that this Ets family transcription factor is essential for development of M cells in a cell-autonomous fashion. SpiB, like the closely related PU.1 gene product, was known to induce effector functions in mesodermally derived lymphoid and myeloid cells (7
). In situ
hybridizations on small intestinal sections showed expression of SpiB in M cells. The assumed role of SpiB in generating these immune-regulating endodermal cells is consistent with microarray data from FACS-sorted M cells (27
). A complete absence of M cells, based on extensive EM analysis and staining for the lectin UEA-1, annexin V, and GP2, in SpiB mutant mice demonstrated the absolute requirement of this transcription factor in M cell development in adult animals. Wild-type bone marrow transplantations into SpiB-null mice did not rescue the M cell defect, showing that it is specifically due to the lack of SpiB expression in the epithelial layer.
The notion that M cells arise from Lgr5 stem cells and that the transcriptional regulator SpiB turns on M cell-specific genes could be confirmed in our recently developed 3-dimensional intestinal organoid culture system. We exploited the previously reported dependence of M cell development on the cytokine RankL (14
). First, we found that the mere addition of this cytokine to the standard three-growth-factor (Rspondin1, epithelial growth factor, and Noggin) cocktail resulted in organoids in which the majority of the differentiated cells expressed M cell markers. Of note, M cells never appear in standard minigut cultures. Increase in the levels of mRNA for SpiB and Rank occurred within 24 h under these conditions. A concomitant increase of mRNA for the M cell marker annexin V was detected. GP2 messengers were first detected at day 3. When SpiB mutant organoids were exposed to RankL, M cell marker expression did not occur. The in vitro
-induced M cells appeared to be functionally competent, as measured in a bead uptake experiment. In the absence of SpiB, no RankL-induced bead uptake was detected. Microarray analysis of RankL-activated organoids convincingly confirmed the upregulation of SpiB and the known M cell markers GP2 and annexin V. Moreover, it generated a list of 161 other genes whose expression was previously reported to be specifically enhanced in PP-derived M cells (27
). Thus, SpiB is necessary for M cell differentiation. Unlike RankL, it appears not to be sufficient for M cell differentiation, as forced retroviral expression of SpiB in organoids did not induce GP2 expression (data not shown).
Taken together, these observations sketch the following scenario. Lgr5 stem cells at crypt bottoms generate naïve progenitors. When leaving the crypts, these progenitors adopt one of six differentiated cell fates. Only when progenitors move into FAE rather than a villus flank will these cells sense RankL secreted by underlying reticular cells. Exposure to this cytokine suffices for these cells to adopt the M cell fate. An early downstream regulator in this process is the SpiB transcription factor. If its expression cannot be induced, no M cells are formed. We predict that RankL will induce, parallel to SpiB, one or more regulator genes.
M cells have been difficult to study, based on their exceedingly low numbers within the intestinal epithelium and their presence in architecturally complex structures, i.e., the PPs. In this study, we describe a simple method to produce large numbers of M cells in vitro and in isolation of all nonepithelial elements of PPs. This technology will allow detailed functional studies on the mechanism of antigen recognition, processing, and transport by M cells.