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Correspondence to: Fiona M. Watt, Keratinocyte Laboratory, Imperial Cancer Research Fund, 44 Lincolns Inn Fields, London WC2A 3PX, UK.
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Summary |
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The basal layer of the epidermis contains two types of proliferating keratinocyte: stem cells, with high proliferative potential, and transit amplifying cells, which are destined to undergo terminal differentiation after a few rounds of division. It has been shown previously that two- to three-fold differences in the average staining intensity of fluorescein-conjugated antibodies to ß1 integrin subunits reflect profound differences in the proliferative potential of keratinocytes, with integrin-bright populations being enriched for stem cells. In the search for additional stem cell markers, we have stained sections of normal human epidermis with antibodies to proteins involved in intercellular adhesion and quantitated the fluorescence of individual cell-cell borders. In the basal layer, patches of brightly labeled cells were detected with antibodies to E-cadherin, ß-catenin, and -catenin, but not with antibodies to P-cadherin,
-catenin, or with pan-desmocollin and pan-desmoglein antibodies. In the body sites examined, palm and foreskin, integrin-bright regions were strongly labeled for
-catenin and weakly labeled for E-cadherin and ß-catenin. Our data suggest that there are gradients of both cell-cell and cell-extracellular matrix adhesiveness within the epidermal basal layer and that the levels of E-cadherin and of ß-and
-catenin may provide markers for the stem cell compartment, stem cells expressing relatively higher levels of
-catenin and lower levels of E-cadherin and ß-catenin than other basal keratinocytes. (J Histochem Cytochem 45:867-874, 1997)
Key Words: keratinocyte, cadherin, catenin, integrin, desmocollin, desmoglein
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Introduction |
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The basal layer of the epidermis, which is attached to an underlying basement membrane, contains both proliferating keratinocytes and keratinocytes that have withdrawn from the cell cycle and are committed to undergo terminal differentiation (
We recently showed that two- to threefold differences in cell surface levels of the 2ß1,
3ß1, and
5ß1 integrins reflect profound differences in the proliferative potential of human epidermal keratinocytes (
Variation in integrin levels can be detected by confocal microscopy of epidermis labeled with FITC-conjugated anti-integrin antibodies. Isolation of integrin-bright cells directly from the epidermis confirms that they are enriched in stem cells (
The proportion of integrin-bright basal cells is 25-50%, depending on body site (
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Materials and Methods |
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Immunofluorescence Staining
One specimen of adult human palm skin was obtained postmortem and neonatal foreskins were obtained from routine circumcisions. Skin samples were embedded in OCT compound (Miles; Elkhart, IN), snap-frozen in liquid nitrogen, and stored at -70C until required. Frozen sections (5µm) were prepared with a cryomicrotome (Reichart-Jung; Leitz, Milton Keynes, UK) and collected on silane-coated slides.
Sections were fixed for 10 min in 3.7% formaldehyde, 0.1% Triton X-100 in PBS containing 1 mM calcium and 1 mM magnesium (PBSABC). Fixed sections were blocked for 1 hr with PBSABC containing 0.2% bovine serum albumin and 0.01% Triton X-100. Each primary antibody was applied to sections and incubated for 1 hr in a moist chamber at room temperature, then washed three times in PBSABC. The relevant secondary antibodies were applied for 30 min and then sections were extensively washed before mounting with Gelvatol (Monsanto; St. Louis, MO). Double labeling was performed exclusively with primary antibodies from different species and involved sequential addition of first primary antibody, first secondary antibody, second primary antibody, and second secondary antibody.
The following primary antibodies were used. Mouse monoclonals HECD-1 to E-cadherin and NCC-CAD-299 to P-cadherin were kindly provided by M. Takeichi and S. Hirohashi (3ß1;
-catenin, ß-catenin, and
-catenin (VB1, VB2, and VB3, respectively) were described by
Quantitation of Fluorescence
Tissue sections were analyzed with a confocal microscope (MRC 600; BioRad, Richmond, CA) as described previously (
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Results |
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Quantitation of Indirect Immunofluorescence
In our previous studies of integrin levels within the epidermis, we stained sections with anti-integrin antibodies that had been directly conjugated to fluorescein isothiocyanate (FITC), thereby obviating the amplification of signal provided by a secondary antibody (3 integrin subunit and the same antibody unconjugated and detected with an FITC-conjugated secondary antibody (p=0.32). On scalp epidermis the ratio of fluorescence in the bright and dull regions of the basal layer was 2.37 ± 0.2 (p<0.0002) by indirect labeling and 2.1 ± 0.1 (p<0.0001) by direct labeling.
Range of Antibodies Tested
Frozen sections of foreskin epidermis were stained with antibodies to proteins that are known to mediate intercellular adhesion of keratinocytes. We examined E-cadherin, which is expressed in all the living layers of the epidermis, P-cadherin, which is confined to the basal layer (-, ß-, and
-catenin (plakoglobin), cytoplasmic proteins that regulate cadherin function (reviewed by
-catenin is associated both with the classical cadherins and with desmosomes (
Foreskin Epidermis
Quantitation of fluorescence at individual intercellular borders of basal keratinocytes is shown in Figure 1 and Figure 2. Figure 1 shows the micrographs that provided the data for Figure 2. In each panel of Figure 1, the cell-cell border marked with an asterisk corresponds to cell-cell border number 1 in the corresponding panel of Figure 2. Subsequent cell-cell borders are numbered from left to right along the basal layer. Note that in these measurements it is the relative rather than the absolute fluorescence values that are important. Absolute values varied according to antibody labeling conditions and the basal cell-cell border with maximal fluorescence in any given section was assigned a pixel intensity of 255 units.
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When fluorescence at individual intercellular borders of basal keratinocytes was quantitated, all antibodies tested showed at least a two-fold variation in staining intensity, but in the case of E-cadherin, ß-catenin, and -catenin the range was greater (4-25 fold; Figure 2, and data not shown). Furthermore, in sections labeled with antibodies to E-cadherin, ß-catenin, and
-catenin, groups of bright cells were found (>8 contiguous bright borders; Figure 1A, Figure 1C, Figure 1D, Figure 2A, 2C, and 2D), whereas other antibodies tended to give strong staining of individual cells or pairs of cells (e.g., Figure 1B and Figure 2B). Cells at the tips of the dermal papillae (horizontal lines in Figure 2) tended to have the highest fluorescence labeling for
-catenin (Figure 1D and Figure 2D) and the lowest fluorescence labeling for E-cadherin (Figure 1A and Figure 2A) and ß-catenin (Figure 1C and Figure 2C).
To examine whether the variation in fluorescence at individual cell-cell borders was statistically significant, the fluorescence intensity of the 8-15 cell-cell borders at the tips of individual dermal papillae or rete ridges was averaged. At least 24 dermal papillae and 24 rete ridges were analyzed per antibody (Table 1). The ratio of fluorescence intensity of cells at the tips of the dermal papillae and rete ridges was significantly different in sections labeled with antibodies to E-cadherin, ß-catenin, or -catenin, but not in sections labeled with
-catenin antibodies.
Palm Skin
Whereas in foreskin the integrin-bright patches lie at the tips of the dermal papillae, in palm they lie at the tips of the rete ridges (-catenin-bright patches were at the tips of the rete ridges (data not shown). The variation in fluorescence could be visualized more easily when the lowest 50% of pixel intensity was electronically subtracted (Figure 3B cf.3A, Figure 3D cf.3C).
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Integrin/Catenin Double-label Immunofluorescence
We also compared the size and position of the patches defined by integrin staining with those of the patches defined by catenin labeling. Because the number of cells per rete ridge varies, we did not compare the average number of bright cells per patch but investigated the variation of fluorescence intensity of all the basal cells in a single rete ridge by double-label immunofluorescence. Double labeling was performed on nine sections, using either ß1 integrin and ß-catenin antibodies or ß1 integrin and -catenin antibodies. In the example of epidermis shown in Figure 4, the cluster of integrin-bright cells at the tip of the dermal papilla extends from cell border 19 to 32, whereas the
-catenin-bright patch is between borders 13 and 30. From these observations we conclude that there is approximate coincidence of the patches defined with integrin and catenin antibodies.
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Discussion |
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In a search for further markers of epidermal stem and transit amplifying cells, we stained sections of foreskin epidermis with antibodies to a range of cell adhesion molecules. The overall staining patterns were as reported previously. E-cadherin was found in all the living cell layers, whereas P-cadherin was confined to the basal layer (Nicholson et al. 1991; -, ß-, and
-catenin stained all the living cell layers, consistent with the presence of E-cadherin in those layers (
In previous studies of integrin levels we used FITC-conjugated primary antibodies and we measured the average fluorescence of groups of cells at defined locations within the epidermal basal layer (
Of the antibodies tested, only three showed patchy staining within the basal layer (see Figure 5). In foreskin, the tips of the dermal papillae, where the ß1 integrin-bright cells lie, showed low levels of E-cadherin and ß-catenin and high levels of -catenin. In palm, the ß1-,
-catenin-bright, E-cadherin-, ß-catenin-dull cells were at the tips of the rete ridges. The ratio of the average fluorescence intensity of bright and dull cells ranged from 1.6 to 2.1 (Table 1), which is comparable to the ratio of 2.1 previously reported for the
2ß1 and
3ß1 integrins (
-catenin and ß1 integrins showed broad coincidence of bright cells.
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The finding of heterogeneity in the levels of E-cadherin, ß-catenin, and -catenin is of interest because of evidence that cadherins play a major role in epithelial morphogenesis (
2ß1,
3ß1, and E-cadherin. The complementary distribution of ß-catenin- and
-catenin-bright cells in the basal layer is interesting, given that the cytoplasmic domains of cadherins form complexes that contain either ß-catenin or
-catenin in addition to
-catenin (
-catenin not only binds to the cytoplasmic domain of cadherins but also is a component of desmosomes. Furthermore, ß-catenin is believed to have signaling functions that are independent of its role in cell adhesion (
In conclusion, our observations suggest that there are gradients of cell-cell and cell-matrix adhesiveness within the basal layer (see Figure 5; see also
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Footnotes |
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1 Present address: Laboratoire de Dermatologie Moléculaire, Institut Univesitaire de Recherche Clinque, Montpéllier, France.
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Acknowledgments |
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J-PM was supported by fellowships from the European Community (Human Capital and Motility) and from INSERM (Bourse Formation Postdoctorale). We gratefully acknowledge additional funding from Bristol-Myers Squibb.
We are grateful to M. Takeichi, A.I. Magee, and S. Hirohashi for generous gifts of antibodies, to P.H. Jones for interesting discussions, and to P. Jordan for help with confocal microscopy. We thank W. Senior for typing the manuscript.
Received for publication July 17, 1996; accepted December 2, 1996.
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