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Rabbit M cells and dome enterocytes are distinct cell lineages

Laboratoire de Dynamique Moléculaire des Interactions Membranaires, CNRS UMR 5539, cc 107, Université Montpellier II, 34095 Montpellier Cedex 5, France

View larger version (108K): [in a new window]   Fig. 1. EDTA treatment allows the visualization of whole rabbit dome epithelium and its associated crypts. Rabbit epithelium from appendix (A-C) and caecal patch (D,E) were isolated from the underlying tissue by EDTA before fixation according to the Materials and Methods. (A) Side view of an apex-less dome (broken line) seen through its associated surrounding colonic epithelium by trans-illumination; intact crypts are distributed regularly around the dome base (arrowheads). (B) Dome epithelium revealed by pulling down the surrounding colonic epithelium. Arrowheads mark each end of the crevice from which crypts supply, first, M cells and enterocytes to the dome above (broken line), and, second, goblet cells and enterocytes to the colonic epithelium (lower part of the figure, bracket). The colonic epithelium, normally lying in apposition to the dome, is clearly visible under the crypt row (arrowheads) down to the beginning of its apical surface, characterized by long tubular structures opening on the apex (arrows). (C) Tearing away the colonic epithelium left only the FAE with its associated crypts (arrowheads and inset). Note the central apex torn off during isolation treatment. (D) Bottom view (in bright field) of dissociated caecal patch crypts lined up in several rows. (E) DIC side view of caecal patch crypts, with some of them branched.

View larger version (129K): [in a new window]   Fig. 2. Vimentin-positive immature M cells appear in the area of rapidly proliferative cells of crypts in three rabbit GALTs. After 1 hour in vivo incorporation of BrdU, FAE was dissociated, fixed, permeabilized and labeled with anti-vimentin (red) and anti BrdU (green) antibodies. (A) Half dome epithelium from rabbit appendix revealed with DIC; (B) fluorescent labeling of the corresponding field. BrdU labeling is confined to crypts and the neck of crypts. (C,D) Enlarged fields from the delimited area in A, showing vimentin labeling starting around BrdU-labeled cells in crypts (arrow). (E-G) Vimentin-labeled cytoskeleton in the three GALTs studied is progressively organized along the crypt-dome axis starting from fine apico-basal filaments in the crypts (E), here seen from the top of cells, to pockets in dome flanks (G). Isolated FAE from Peyer’s patch (H) and caecal patch (I) shows the first vimentin-expressing cells around BrdU-incorporating cells of crypts (arrows). Insets in D,H,I show vimentin-positive cells co-labeled with BrdU.

View larger version (107K): [in a new window]   Fig. 3. M cell distribution is already regular in crypts of rabbit Peyer’s patches. Cryostat serial cross-sections of crypts in normal ileum villi (A) and the dome region (B-D) were permeabilized and double-labeled for M cells with anti-vimentin (A-D, red) and MAb 58 (C,D, green) or for dividing cells with anti-BrdU (B, green). Arrowheads mark the junction between crypts (delineated by discontinuous lines) and domes or villi (dotted lines). In normal ileum (A), vimentin labels interstitial tissue but is absent from the overall epithelium, including crypts. In FAE (B-D), vimentin labeling surrounds M cell nuclei on the dome side of crypts (B,C) and above the crypt neck (D). In crypts, starting from BrdU-positive cells (arrows in B), vimentin-labeled cells are regularly spaced (B-D). Apical surface of vimentin-positive cells are also labeled with MAb 58 (C), as in the dome flanks (D). A few cells are co-labeled with anti-vimentin and MAb 58 on the villus side (arrows), both in the crypts (C) and above (D).

View larger version (178K): [in a new window]   Fig. 4. The spatial distribution of M cells and dome enterocytes is already established at the base of domes from rabbit appendix and Peyer’s patch. Micro-dissected domes from Peyer’s patch (A-C) and appendix (D) were permeabilized and co-labeled with MAb 58 (green) and MAb 214 (red in A, B, D) or anti-vimentin (red in C). (A) The regular mosaic distribution pattern of M cells and enterocytes, already established at the very base of the dome around a crypt region. (B) A higher magnification reveals marked variations in M cell apical surfaces (thin and open arrows). (C) is the maximal projection of 60 focal planes to localize both the apical surface of M cells and the lateral corresponding pocket: note that M cells with large apical surfaces (open arrows) correspond to large vimentin-delimited pockets, while M cells with compressed apical surfaces (thin arrows) correspond to small pockets. (D) The relative distribution of M cells and enterocytes in appendix reveals a very regular mosaic pattern from the base to the upper part of the dome. A few goblet cells were double labeled in yellow at the lower part of the domes (arrows).