Here, we used a meta-analysis approach to identify a co-expressed transcriptional signature that distinguished the FAE from a large collection of mouse primary cells, tissues and cell lines. A co-expressed gene cluster (cluster 65) was identified which was highly expressed only in the FAE and by M cells and was enriched with known M-cell- (Gp2
) or FAE-specific (Anxa10
genes. Such clusters of co-expressed genes provide an opportunity to identify novel lineage markers and regulators of cell function, as well as functions for uncharacterized genes. For example, recent studies show that the broad complex, tramtrack, bric-a-brac, and zinc finger transcription factor Zbt46 is specifically expressed by all classical DC lineages.27,28
Retrospective analysis of data from our previous comparison of the transcriptomes of different mouse lymphocyte and leukocyte populations shows that Zbtb46
was co-expressed in a cluster of 12 genes whose expression was restricted to classical DC (cluster 79 in Mabbott et al
). By applying the principle of ‘guilt-by-association’, this suggested that the other genes co-expressed within cluster 65 in the current study were also FAE-related. These novel FAE-related genes could be separated into two groups: those expressed highly within the FAE and by M cells (Calcb
) and those expressed in the FAE, but not by M cells (Ascl2
). These genes could also be further subdivided depending on their requirement for RANKL-stimulation for their expression. The potential functions of the FAE-related genes identified in this study are summarized in Fig. and examples discussed below.
Figure 8. Cartoon illustrating the putative functions of the FAE- and M-cell-related genes identified in this study. Genes were classified into groupings of related cellular function based on published data from literature searches. Red font, RANKL-dependent genes; (more ...)
Cells in the FAE express high levels of the glycosylphosphatidylinisotol (GPI)-anchored proteins GP2 and cellular prion protein (PrPC
, encoded by Prnp
GPI inositol deacylation by post-GPI attachment to proteins 1 (PGAP1, encoded by Pgap1
) is important for the efficient transport of GPI-anchored proteins from the endoplasmic reticulum to the Golgi.29
was co-expressed with Gp2
within cluster 65 implying an important role in the expression of GPI-anchored proteins in the FAE. GP2 and PrPC
may each act as a transcytotic receptors for mucosal antigens on M cells.15,30
did not cluster with other genes across this data set, most likely reflecting its ubiquitous cellular expression.
Potassium channels control the apical recycling of potassium ions. Our analysis of the novel genes within cluster 65 revealed that Kcnj15
was specific to the FAE, but was not induced in the villous epithelium after RANKL stimulation. The ion channels Cftr
were also co-expressed within cluster 65, and others (Clca4
) were significantly induced by RANKL stimulation. The role of KCNJ15 and the other ion channels in the FAE is uncertain. Enterocytes within the FAE, unlike those in the villous epithelium, contain large LAMP1+
These genes may help maintain ion concentrations in the large endosomes of FAE enterocytes.
Although RANKL stimulation is important for the maturation and maintenance of M cells, Tnfrsf11a
(which encodes RANK) did not cluster with any other gene across this data set. This is most likely due to the additional roles for RANK–RANKL signalling in the function of osteoclasts31
and lymphoid tissue-inducer cells32
that were included in the analysis. Since RANKL may also promote the survival of BM-derived DC and stimulate their expression of pro-inflammatory cytokines,33
our subtraction analysis identified only those RANKL-induced genes which were highly expressed in the FAE and also by Peyer's patch M cells when compared with the villous epithelium. This approach identified 48 genes, many of which were known to be expressed in the FAE and by M cells including Anxa5
or were also identified in the current study in cluster 65 (Clca2
This implies that the remaining 32 genes represent novel RANKL-dependent FAE-related genes.
The genes induced by RANKL stimulation encode proteins with a range of FAE- and M-cell-related activities and are summarized in Fig. . Many were associated with cytoskeletal regulation (Epb4.4l2
) and may play a role in the adaptation of the cell towards one specialized for transcytosis.4,21,36–39
The expression of Anxa5
may encode proteins that bind to and regulate the host's response to antigen and pathogens in the intestinal lumen.2,15,25,40,41
Similarly, the expression of Ctsh
suggests a role in antigen processing or pathogen degradation. Chemokines and chemokine receptors mediate the attraction of lymphocytes and leukocytes to lymphoid tissues and control their positioning within them. The chemokine genes Ccl9
were up-regulated by RANKL stimulation indicating a role in the attraction of specific cell populations towards the FAE. For example, in Peyer's patches, the CCL20-mediated recruitment of a specific B cell subset towards the FAE is important for maintaining M differentiation.22,42
Consistent with this role, Ccl20
was co-expressed with the other FAE-related genes in cluster 65.
Expression of MFG-E8 by the FAE had not previously been reported. The expression of MFG-E8 was significantly up-regulated by RANKL treatment and was preferentially co-localized to the large LAMP1+
endosomes of FAE enterocytes. In the B cell follicles, MFG-E8 produced by stromal follicular DCs aids the phagocytosis of apoptotic cells by acting as a bridging molecule between phosphatidylserine residues on apoptotic cells and integrins on mononuclear phagocytes.12,25
However, the engulfment of apoptotic cells by mononuclear phagocytes within the SED beneath the FAE was not impaired in MFG-E8-deficient mice implying an alternative role for FAE-derived MFG-E8. Data show that MFG-E8 can also act as an anti-inflammatory modulator by inhibiting the LPS-mediated release of pro-inflammatory cytokines.43
The phosphatidylserine-binding protein ANXA5 is also expressed highly in the FAE by M cells.20
In addition to its role in regulating the phagocytosis of apoptotic cells, ANXA5 can bind to Gram-negative bacteria with high affinity via the lipid A domain of LPS, blocking the endotoxin activity.40
Thus, in the FAE, the expression of MFG-E8 by enterocytes and ANXA5 by M cells may each play important anti-inflammatory roles to protect against LPS-mediated endotoxemia.
The promoter analysis of the cluster 65 genes identified over-representation of 11 candidate TFBS motifs supporting the conclusion that the genes in this cluster shared an underlying transcriptional programme. Many of these motifs bind transcription factors that control epithelial cell and enterocyte differentiation and homeostasis. The nuclear transcription factor Y subunit α can regulate the differentiation of a human epithelial colorectal adenocarcinoma cell line.44
Similarly, hepatocyte nuclear factor 1α maintains enterocyte terminal differentiation and cell fate commitment in the intestinal epithelium.45
Two high mobility group box transcription factors were also identified. Sox9 is expressed in intestinal epithelium stem/progenitor cells and in Paneth cells and can regulate homeostasis in the intestinal epithelium.46
Sox17 can promote the growth and differentiation of respiratory epithelial cells.47,48
Motifs for basic helix-loop-helix (bHLH) transcription factors were also identified by the promoter analysis. Furthermore, genes encoding two bHLH transcription factors, L-myc (encoded by Mycl1
) and achaete scute-like 2 (encoded by Ascl2
), were co-expressed in cluster 65. These transcription factors are important regulators of cell fate in the intestine49
implying a similar role in the FAE.
NF-κB functions as a dimeric transcription factor and plays a central role in regulating gene expression during intestinal inflammation. The identification of enriched binding motifs for NF-κB transcription factor family members is consistent with the role for RANK–RANKL signalling in controlling M cell differentiation.1
RelA has been shown to be required for the homeostatic regulation of cell death and division in the intestinal epithelium under steady-state conditions and for protection during acute inflammation.50
Similarly, in the large intestine, Rel regulates the innate inflammatory response to the gut microflora.51
The chronological comparison of the effects of RANKL stimulation on the expression of the cluster 65 genes revealed that although stromal cells beneath the FAE express high levels of RANKL, not all of the FAE-related genes identified in this study are directly induced by RANKL stimulation. Furthermore, although Ccl20
were rapidly up-regulated following RANKL stimulation, Gp2
were induced at later times. The promoter analysis indicated that Ccl20
all contained NF-κB family TFBS motifs, whereas they were absent in Gp2
. These data indicate that although RANKL stimulation is important for the induction of M cell differentiation from enterocytes, it does not directly regulate the expression of genes such as Gp2
which are associated with terminally differentiated M cells. High levels of the E26 transformation-specific family transcription factor transcription factor Spi-B (encoded by Spib
) were induced in the villous epithelium after RANKL stimulation. Recent data show that Spi-B is an important transcription factor which acts downstream of RANKL–RANK signalling to control the subsequent terminal differentiation of functionally mature M cells.5,6
Data in this study show that gene expression signatures specifically associated with the FAE and the M cells within it can be readily identified from the meta-analysis of a large collection of publicly available gene expression data. Little is known of the genes that specifically regulate the differentiation and function of cells within the FAE. Further characterization of the candidate genes identified in the current study will aid the identification of novel regulators of FAE and M cell function and homeostasis.