1. Volume 129, Issue 3, Pages 902-912 (September 2005)
Epithelial Barrier Function In Vivo Is Sustained Despite Gaps in Epithelial Layers 
Alastair J.M. Watson, Shaoyou Chu, Leah Sieck, Oleg Gerasimenko, Tim Bullen, Fiona Campbell, Michael McKenna, Tracy Rose, Marshall H. Montrose 
Gastroenterology 
Volume 129, Issue 3, Pages (September 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
Intestinal epithelium is a discontinuous monolayer in the living animal. (A) Autofluorescence of intestinal epithelial cytosol. (B) Confocal reflectance of intestinal epithelial cells. (C) Overlay of images A and B. (D) Nuclear fluorescence with the Hoechst DNA stain. (E) Cytosolic BCECF fluorescence. (F) Confocal reflectance. (G) Overlay of images D, E, and F. Images from transgenic YC3.0 calcium cameleon mice. (H) Nuclear fluorescence. (I) Cytosolic CFP fluorescence. (J) Confocal reflectance. (K) Overlay of images H, I, and J. (L) Nuclear fluorescence with Hoechst DNA stain. (M) Membrane DiI fluorescence. (N) Confocal reflectance. (O) Overlay of images L, M, and N. Bars = 20 μm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
Frequency and appearance of epithelial cell gaps. (A) En face and associated orthogonal views of villus from cameleon transgenic mouse (green; fluorescent protein) additionally stained with HOESCHT (blue) as in Figure 1. En face gap shown in crosshairs is confirmed by associated orthogonal views optically slicing thru epithelial layer. Orthogonal views along green and red axes are presented in the green and red boxes. (B) Percentage of epithelial cell positions that lack cells, as a function of distance from the villus tip. Serial optical sections were taken at 1-μm intervals 0–70 μm from the villus tip. Cell positions were visualized by nuclear staining and confocal reflectance. Gaps were defined as a region ∼10 μm in diameter in the x, y, and z dimension with coincident loss of nucleus (Hoechst stain) and cytosol (assayed by cytosolic BCECF, autofluorescence, or CFP). Thirty-two villi were counted from 10 mice. (C) En face view of villus cells of cameleon transgenic mouse imaged as in Figure 1 demonstrates variability in diameters and angularity of the boundary of cell-free zones (yellow arrows) vs epithelial cells (green: fluorescent protein; red: reflectance). (D) Formalin-fixed sections of mouse tissue. Histologic stain for mucins positively distinguishes goblet cells (red arrows) from gaps (yellow arrow) when villus viewed en face.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
Features at basal pole of gaps and shedding cells. A–D are images from cameleon transgenic mice showing CFP fluorescence (A,C) or CFP fluorescence in green overlaid with confocal reflectance in red (B,D, respectively). Yellow arrow, apical pole of cell; yellow arrowhead, basal pole. (A and B) Example of gap with cytoplasmic extensions across basal pole. (C and D) Example of gap without cytoplasm at basal pole. (E and F) Formalin-fixed sections of mouse tissue immunostained for ZO-1 (brown peroxidase) and counterstained with hematoxylin. (E) Red arrow indicates location of ZO-1 staining at base of shedding cell. (F) Red arrow shows absence of ZO-1 staining at base of shedding cell.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
Restricted permeation of luminal Lucifer Yellow into epithelial layer. Lucifer Yellow (100 μmol/L) was added to the fluid bathing the mucosal surface to image all compartments accessible to luminal fluids. Images were compensated for limited bleed over of Hoechst fluorescence into the Lucifer Yellow channel. (A and E) Lucifer Yellow fluorescence in intervillus space does not permeate the epithelial layer or enter most cell-free gaps (arrow). Limited permeation of some gaps was observed (arrowheads; see text). (B and F) Confocal reflectance of 800-nm light. (C and G) Nuclear fluorescence of Hoechst stained DNA used to define gaps (arrow/arrowhead). (D and H) Overlay images. Bars = 20 μm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
Permeation of gaps from the serosal fluid compartment. Fluorescent dextran (10,000 mw, conjugated to Alexa Fluor 647) was injected intravenously into transgenic YC3.0 calcium cameleon mice to image all compartments accessible to serosal fluids. Panels A, C, D, and E show dextran fluorescence alone, overlaid as red channel with CFP fluorescence (green channel) in panels B, F, G, and H, respectively. Asterisks indicate location of gap in epithelium. (A and B) Arrow indicates apical pole and arrowhead the basal pole of epithelium. Dextran permeates into lateral intercellular spaces and perimeter surrounding basal portion of gap. (C and F) En face views of villus epithelium near apical boundary of cells. Dextran fluorescence is excluded from the region surrounding gaps, although it can be readily detected in lateral space between adjacent cells. D, G and E, H are 2 other representative gaps to reinforce conclusions from C, F.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
Biogenesis of gaps: time-lapse studies of cell shedding. (A) Time-lapse images of a Hoechst stained cell being shed (arrow). (B) Upper graph of internuclear distance between 3 shed cells and their immobile neighbors. Lower graph of nuclear fluorescence intensity in same cells over same time course. Panels C and D are fixed sections of mouse tissue. (C) Cells being shed from the murine villus tip stained by H&E. (D) Stained for mucins (arrow in inset shows goblet cell). (E) Fixed section of human small intestine stained with H&E. Bars = 20 μm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

8. Figure 7
Caspase activation is occasionally seen during cell shedding. Time course studies of tissues stained with Hoechst and exposed to 10 μmol/L PhiPhiLux in the luminal fluid. A” PhiPhiLux fluorescence; A’ nuclear fluorescence of Hoechst stained DNA; A overlay of images A” and A’. Nuclei with condensed chromatin (A’, arrowhead) are in the lumen, surrounded by brighter PhiPhiLux (A”). (B and C) Overlay images collected 2 and 5 minutes later. The arrow indicates a cell being shed but remaining PhiPhiLux negative (A–C). (D–G) Time course of shedding for 1 cell (at arrow) with PhiPhiLux-positive cytoplasm.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions