New approach for m-cell-specific molecules screening by comprehensive transcriptome analysis

Abstract

A minor population of M cells within the follicle-associated epithelium (FAE) of intestinal Peyer's patches (PPs) serves as a major portal for entry of exogenous antigens. Characterization of the mammalian M cells, including identification of M-cell surface molecules used for bacterial uptake, has been hampered by their relative rarity. In contrast, M cells constitute virtually all of the FAE cells in the avian bursa of Fabricius. We therefore performed comparative gene expression profiling of chicken and murine FAE to identify commonly expressed genes by M cells in both species. The comprehensive transcriptome analysis revealed that 28 genes were commonly up-regulated in FAE from both species. In situ hybridization revealed that annexin A10 (Anxa10) mRNA was scattered in FAE, and co-localized with Ulex europaeus agglutinin-1 binding to M cells. Whole-mount immunostaining also revealed that cellular prion protein (PrP(C)) was expressed on the luminal side of the apical plasma membrane of M cells, and co-localized with grycoprotein 2 that recognizes only M cells in murine PP. Our findings identify new M-cell-specific molecules through using comprehensive transcriptome analysis. These conserved molecules in M cells of mice and chickens may play essential roles in M-cell function and/or differentiation.

Figure 1

Comparative transcriptome analysis. (A) Dissection of bursal FAE and IFE. Upper left, BF was soaked in HBSS containing black ink and observed by stereomicroscopy. FAEs taking up the ink dye are easily distinguished as black spots. Upper right, BF after collecting FAEs. The FAE removal sites appear as white spots. Lower panels show an isolated bursal FAE (left) and bursal IFE (right). (B) Comparison of gene expression profiling between FAE and IFE. Normalized data obtained by microarray analysis and applied filtration on flags are illustrated by a scatter plot of gene expression. The upper and lower lines indicate the threshold levels of 2-fold change. (C) Comparative transcriptome analysis of chicken and murine FAEs. Annotated genes up-regulated 2-fold or more in FAE from both species are shown in a Venn diagram.

Figure 2

Strong expression of Anxa10 and Prnp mRNA in murine PP FAE among various tissues and cells. Immune cell (B, B cell; T, T cell; DC, dendritic cell; NKT, NK T cell; N, natural killer cell; Ne, neutrophil; L, leukocyte) and intestinal epithelium (F, FAE; V, VE) distribution of the expression of Anxa10 and Prnp analyzed with RefDIC. Normalized data of the expression level of each gene are shown as a gradation from green (low) to red (high). With regard to FAE and VE, two different RNA samples were obtained from germ-free mouse (left column) and conventional mouse (right column) conditioned groups, respectively.

Figure 3

Preferential expression of Anxa10 and Prnp mRNA in murine PP FAE compared with VE. Q-PCR analysis was performed for Anxa10 Prnp mRNA expression in murine FAE and VE. The relative expression levels of ‘each gene’ to Gapdh are shown. Values represent the mean ± SD of three samples from different mice. **P < 0.01.

Figure 4

Specific expression of Anxa10 mRNA by murine M cells. (A) ISH analysis of Anxa10 mRNA expression detected using specific antisense probe. Arrows indicate the ISH signals for Anxa10 mRNA. (B) The section after ISH staining (A) was co-staining with UEA-1. Anxa10 signals were co-localized with UEA-1 (arrows). (C) ISH analysis of murine PP FAE using a control probe. Scale bars: 50 µm.

Figure 5

Preferential expression of Cellular prion protein (PrP^C) by murine M cells. Whole-mount specimens of murine PP were stained with a PrP mAb (green), UEA-1 or GP2 (red) and then analyzed by confocal laser-scanning microscopy. (A) The X–Y view from the luminal side stained with PrP^C, UEA-1 and the merged image (PrP^C and UEA-1) is shown. White lines indicate position of FAE in murine PP. (B and D) The X–Y view from the luminal side stained with an isotype-matched control mouse IgG. (C) The X–Y view from the luminal side stained with PrP^C, GP2 and the merged image (PrP^C and GP2) is shown. The X–Z image of FAE sectioning in a vertical direction is displayed below the lower X–Y view (A and C). The upper part of the image is the apical plasma membrane side, and the lower is the basal side. Scale bars: 75 µm.