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Identification of novel genes selectively expressed in the follicle-associated epithelium from the meta-analysis of transcriptomics data from multiple mouse cell and tissue populations.

Kobayashi A, Donaldson DS, Kanaya T, Fukuda S, Baillie JK, Freeman TC, Ohno H, Williams IR, Mabbott NA - DNA Res. (2012)

Bottom Line: The follicle-associated epithelium (FAE) overlying the Peyer's patches and the microfold cells (M cells) within it are important sites of antigen transcytosis across the intestinal epithelium.This study provides new insight into the FAE transcriptome.Further characterization of the candidate genes identified here will aid the identification of novel regulators of cell function in the FAE.

View Article: PubMed Central - PubMed

Affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Sciences, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK.

ABSTRACT
The follicle-associated epithelium (FAE) overlying the Peyer's patches and the microfold cells (M cells) within it are important sites of antigen transcytosis across the intestinal epithelium. Using a meta-analysis approach, we identified a transcriptional signature that distinguished the FAE from a large collection of mouse cells and tissues. A co-expressed cluster of 21 FAE-specific genes was identified, and the analysis of the transcription factor binding site motifs in their promoter regions indicated that these genes shared an underlying transcriptional programme. This cluster contained known FAE- (Anxa10, Ccl20, Psg18 and Ubd) and M-cell-specific (Gp2) genes, suggesting that the others were novel FAE-specific genes. Some of these novel candidate genes were expressed highly by the FAE and M cells (Calcb, Ces3b, Clca2 and Gjb2), and others only by the FAE (Ascl2, Cftr, Fgf15, Gpr133, Kcna1, Kcnj15, Mycl1, Pgap1 and Rps6kl). We also identified a subset of novel FAE-related genes that were induced in the intestinal epithelium after receptor activator of nuclear factor (NF)-κB ligand stimulation. These included Mfge8 which was specific to FAE enterocytes. This study provides new insight into the FAE transcriptome. Further characterization of the candidate genes identified here will aid the identification of novel regulators of cell function in the FAE.

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IHC analysis of MFG-E8 expression in the SI epithelium. (A) Strong MEG-E8 immunolabelling was detected in the FAE of WT mice with increasing intensity towards the apex (green, left-hand panels, arrow). No MFG-E8 expression was detected throughout the villous epithelium of WT mice. Arrowheads indicate high levels of MFG-E8 in association with mononuclear phagocytes. No MFG-E8 expression was detected in the intestines of Mfge8−/− mice (right-hand panels). SED, subepithelial dome; FO, B cell follicles. (B) Inclusions of MEG-E8 (red) were detected towards the apical surfaces, FAE enterocytes, but not ANXA5+ M cells (green, arrows). (C) MEG-E8 (red) preferentially co-localized to the large LAMP1+ endosomes (green) of FAE enterocytes (arrows). (D) Although occasional CD11c+ cells were detected within the FAE (green; arrow-heads), and some appeared to contain cytoplasmic inclusions of MFG-E8 (arrow), the majority of the MFG-E8 (red) was detected on the apical surfaces of epithelial cells. (E) Many large MFG-E8-containing CD68+ macrophages were detected in the SED (arrowheads), but were rarely detected in the FAE. Dotted lines indicate the border of the FAE.
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DSS022F6: IHC analysis of MFG-E8 expression in the SI epithelium. (A) Strong MEG-E8 immunolabelling was detected in the FAE of WT mice with increasing intensity towards the apex (green, left-hand panels, arrow). No MFG-E8 expression was detected throughout the villous epithelium of WT mice. Arrowheads indicate high levels of MFG-E8 in association with mononuclear phagocytes. No MFG-E8 expression was detected in the intestines of Mfge8−/− mice (right-hand panels). SED, subepithelial dome; FO, B cell follicles. (B) Inclusions of MEG-E8 (red) were detected towards the apical surfaces, FAE enterocytes, but not ANXA5+ M cells (green, arrows). (C) MEG-E8 (red) preferentially co-localized to the large LAMP1+ endosomes (green) of FAE enterocytes (arrows). (D) Although occasional CD11c+ cells were detected within the FAE (green; arrow-heads), and some appeared to contain cytoplasmic inclusions of MFG-E8 (arrow), the majority of the MFG-E8 (red) was detected on the apical surfaces of epithelial cells. (E) Many large MFG-E8-containing CD68+ macrophages were detected in the SED (arrowheads), but were rarely detected in the FAE. Dotted lines indicate the border of the FAE.

Mentions: Among the novel genes that were significantly up-regulated by RANKL treatment in the villous epithelium in vivo, and in small intestinal organoids in vitro, was Mfge8 which encodes MFG-E8 (Supplementary Table S5). IHC was used to confirm the expression of MFG-E8 in the FAE. As anticipated, high levels of MFG-E8 were detected in association with mononuclear phagocytes in the B cell follicles and subepithelial domes (SEDs) of Peyer's patches (Fig. 6A, arrowheads). However, strong MFG-E8 immunolabelling was also detected within epithelial cells within the FAE (Fig. 6A, arrow). No MFG-E8 expression was detected in Mfge8−/− mice (Fig. 6A). Since both the anti-GP2 mAb and anti-MFG-E8 mAb were raised in rats, ANXA5 was used as an alternative marker to detect M cells.20 Our analysis showed that MFG-E8 was strongly expressed within FAE enterocytes but not by M cells, since ANXA5+ cells (Fig. 6B, arrows) lacked MFG-E8. Enterocytes in the FAE, but not the villous epithelium, contain large LAMP1+ endosomes towards their apical surfaces.24 In FAE enterocytes, the MFG-E8 was preferentially co-localized to these large LAMP1+ endosomes (Fig. 6C).Figure 6.


Identification of novel genes selectively expressed in the follicle-associated epithelium from the meta-analysis of transcriptomics data from multiple mouse cell and tissue populations.

Kobayashi A, Donaldson DS, Kanaya T, Fukuda S, Baillie JK, Freeman TC, Ohno H, Williams IR, Mabbott NA - DNA Res. (2012)

IHC analysis of MFG-E8 expression in the SI epithelium. (A) Strong MEG-E8 immunolabelling was detected in the FAE of WT mice with increasing intensity towards the apex (green, left-hand panels, arrow). No MFG-E8 expression was detected throughout the villous epithelium of WT mice. Arrowheads indicate high levels of MFG-E8 in association with mononuclear phagocytes. No MFG-E8 expression was detected in the intestines of Mfge8−/− mice (right-hand panels). SED, subepithelial dome; FO, B cell follicles. (B) Inclusions of MEG-E8 (red) were detected towards the apical surfaces, FAE enterocytes, but not ANXA5+ M cells (green, arrows). (C) MEG-E8 (red) preferentially co-localized to the large LAMP1+ endosomes (green) of FAE enterocytes (arrows). (D) Although occasional CD11c+ cells were detected within the FAE (green; arrow-heads), and some appeared to contain cytoplasmic inclusions of MFG-E8 (arrow), the majority of the MFG-E8 (red) was detected on the apical surfaces of epithelial cells. (E) Many large MFG-E8-containing CD68+ macrophages were detected in the SED (arrowheads), but were rarely detected in the FAE. Dotted lines indicate the border of the FAE.
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Related In: Results  -  Collection

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DSS022F6: IHC analysis of MFG-E8 expression in the SI epithelium. (A) Strong MEG-E8 immunolabelling was detected in the FAE of WT mice with increasing intensity towards the apex (green, left-hand panels, arrow). No MFG-E8 expression was detected throughout the villous epithelium of WT mice. Arrowheads indicate high levels of MFG-E8 in association with mononuclear phagocytes. No MFG-E8 expression was detected in the intestines of Mfge8−/− mice (right-hand panels). SED, subepithelial dome; FO, B cell follicles. (B) Inclusions of MEG-E8 (red) were detected towards the apical surfaces, FAE enterocytes, but not ANXA5+ M cells (green, arrows). (C) MEG-E8 (red) preferentially co-localized to the large LAMP1+ endosomes (green) of FAE enterocytes (arrows). (D) Although occasional CD11c+ cells were detected within the FAE (green; arrow-heads), and some appeared to contain cytoplasmic inclusions of MFG-E8 (arrow), the majority of the MFG-E8 (red) was detected on the apical surfaces of epithelial cells. (E) Many large MFG-E8-containing CD68+ macrophages were detected in the SED (arrowheads), but were rarely detected in the FAE. Dotted lines indicate the border of the FAE.
Mentions: Among the novel genes that were significantly up-regulated by RANKL treatment in the villous epithelium in vivo, and in small intestinal organoids in vitro, was Mfge8 which encodes MFG-E8 (Supplementary Table S5). IHC was used to confirm the expression of MFG-E8 in the FAE. As anticipated, high levels of MFG-E8 were detected in association with mononuclear phagocytes in the B cell follicles and subepithelial domes (SEDs) of Peyer's patches (Fig. 6A, arrowheads). However, strong MFG-E8 immunolabelling was also detected within epithelial cells within the FAE (Fig. 6A, arrow). No MFG-E8 expression was detected in Mfge8−/− mice (Fig. 6A). Since both the anti-GP2 mAb and anti-MFG-E8 mAb were raised in rats, ANXA5 was used as an alternative marker to detect M cells.20 Our analysis showed that MFG-E8 was strongly expressed within FAE enterocytes but not by M cells, since ANXA5+ cells (Fig. 6B, arrows) lacked MFG-E8. Enterocytes in the FAE, but not the villous epithelium, contain large LAMP1+ endosomes towards their apical surfaces.24 In FAE enterocytes, the MFG-E8 was preferentially co-localized to these large LAMP1+ endosomes (Fig. 6C).Figure 6.

Bottom Line: The follicle-associated epithelium (FAE) overlying the Peyer's patches and the microfold cells (M cells) within it are important sites of antigen transcytosis across the intestinal epithelium.This study provides new insight into the FAE transcriptome.Further characterization of the candidate genes identified here will aid the identification of novel regulators of cell function in the FAE.

View Article: PubMed Central - PubMed

Affiliation: The Roslin Institute and Royal (Dick) School of Veterinary Sciences, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK.

ABSTRACT
The follicle-associated epithelium (FAE) overlying the Peyer's patches and the microfold cells (M cells) within it are important sites of antigen transcytosis across the intestinal epithelium. Using a meta-analysis approach, we identified a transcriptional signature that distinguished the FAE from a large collection of mouse cells and tissues. A co-expressed cluster of 21 FAE-specific genes was identified, and the analysis of the transcription factor binding site motifs in their promoter regions indicated that these genes shared an underlying transcriptional programme. This cluster contained known FAE- (Anxa10, Ccl20, Psg18 and Ubd) and M-cell-specific (Gp2) genes, suggesting that the others were novel FAE-specific genes. Some of these novel candidate genes were expressed highly by the FAE and M cells (Calcb, Ces3b, Clca2 and Gjb2), and others only by the FAE (Ascl2, Cftr, Fgf15, Gpr133, Kcna1, Kcnj15, Mycl1, Pgap1 and Rps6kl). We also identified a subset of novel FAE-related genes that were induced in the intestinal epithelium after receptor activator of nuclear factor (NF)-κB ligand stimulation. These included Mfge8 which was specific to FAE enterocytes. This study provides new insight into the FAE transcriptome. Further characterization of the candidate genes identified here will aid the identification of novel regulators of cell function in the FAE.

Show MeSH