ARTICLE |
Correspondence to: Ken Arita, Dept. of Dermatology, Hokkaido U. Graduate School of Medicine, North 15 West 7, Kita-ku, Sapporo 060-8638, Japan. E-mail: ariken@med.hokudai.ac.jp
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Summary |
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Gap junctions are intercellular channels composed of connexin subunits that mediate cellcell communication. The functions of gap junctions are believed to be associated with cell proliferation and differentiation and to be important in maintaining tissue homeostasis. We therefore investigated the expression of connexins (Cx)26 and 43, the two major connexins in human epidermis, and examined the formation of gap junctions during human fetal epidermal development. By immunofluorescence, Cx26 expression was observed between 49 and 96 days' estimated gestational age (EGA) but was not present from 108 days' EGA onwards. Conversely, Cx43 expression was observed from 88 days' EGA onwards. Using electron microscopy, the typical structure of gap junctions was observed from 120 days' EGA. The number of gap junctions increased over time and they were more common in the upper layers, within the periderm and intermediate keratinocyte layers rather than the basal layer. Immunoelectron microscopy revealed Cx43 labeling on the gap junction structures after 105 days' EGA. Formation of gap junctions increased as skin developed, suggesting that gap junctions may play an important role in fetal skin development. Furthermore, the changing patterns of connexin expression suggest that Cx26 is important for early fetal epidermal development. (J Histochem Cytochem 50:14931500, 2002)
Key Words: connexin 26, connexin 43, electron microscopy, epidermis, fetal skin, gap junction, morphogenesis, periderm
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Introduction |
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THE GAP JUNCTION is an important intercellular channel that is located at areas of closely associated plasma membranes of adjacent cells. Gap junctions consist of members of the connexin (Cx) proteins. Each Cx subunit has four transmembrane sequences and a cluster of six Cx molecules forms the hexameric connexon unit, which penetrates the cell membrane to form a tube-like structure. Opposing connexons of adjacent cells connect with each other to form an intercellular channel through which ions and low molecular weight (<1000 D) second messengers, such as Ca2++, cyclic nucleotides, and inositol triphosphate can permeate. Gap junctions thus allow cellcell communication, which is believed to regulate tissue homeostasis (
Although the Cx family has more than 20 members in vertebrates, the major Cxs of the human epidermis are Cx26 and Cx43 (
It has been hypothesized that gap junctions play an important role in skin development, especially during epidermal morphogenesis. In the mouse and rat, the formation of gap junctions and the changing patterns of Cx expression have been studied in detail (
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Materials and Methods |
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Tissue
Human embryonic and fetal skin specimens were obtained from several institutes with the approval of the appropriate ethical boards and in accordance with their policies. The ages and the autopsy sites of fetuses included in the present study were as follows: 49 days' EGA, trunk; 57 days' EGA, trunk; 88 days' EGA, scalp; 96 days' EGA, trunk; 108 days' EGA, scalp; 135 days' EGA, scalp; 163 days' EGA, scalp (all above for immunofluorescence); 102 days' EGA, trunk; 108 days' EGA, scalp; 120 days' EGA, scalp; 152 days' EGA, scalp (for transmission electron microscopy); 42 days' EGA, digit and hand; 105 days' EGA, digit and arm; 154 days' EGA, digit and arm [for immunoelectron microscopy (
Antibodies
The primary antibodies used in the present study were rabbit polyclonal anti-Cx26 antibody (Zymed Laboratories; San Francisco, CA) and mouse anti-Cx43 monoclonal antibody, clone 4E6.2 (Chemicon International; Temecula, CA). The secondary linker antibodies used in immunoelectron microscopy were rabbit anti-mouse immunoglobulins (DAKO; Glostrup, Denmark) and mouse anti-rabbit immunoglobulin (Jackson Immunoresearch Laboratories; West Grove, PA).
Immunofluorescent Labeling
Fetal skin was snap-frozen in isopentane and 6-µm-thick sections were cut with a cryostat. The sections were washed with 0.01 M PBS for 10 min and then incubated in primary antibody solution for 1 hr at 37C. Antibody dilutions were 1:10 for anti-Cx26 antibody and 1:100 for anti-Cx43 antibody. The sections were then incubated in fluorescein (FITC)-conjugated goat anti-mouse immunoglobulins (Jackson Immunoresearch) or in FITC conjugated to goat anti-rabbit immunoglobulins (Jackson Immunoresearch) for 30 min at room temperature, followed by 10 µg/ml propidium iodide as a nuclear counterstain (Sigma Chemical; St Louis, MO) for 10 sec. The sections were extensively washed with 0.01 M PBS between incubations. The stained sections were mounted with a glycerol-based mounting medium (Permafluor; Shandon, Pittsburgh, PA) and stored in the refrigerator. Photos were taken with an Olympus IX70 confocal laser scanning microscope. Immunostaining was detected as green (FITC) and nuclear stain was observed as red (propidium iodide). Overlap of both FITC and propidium iodide was demonstrated as a yellowish color. Image collection was performed by software Fluoview version 2.0 (Olympus America; Melville, NY). As negative control, non-immunize normal rabbit serum or mouse myeloma supernatant instead of primary antibodies was used.
Quantitative Analysis of Cx Expression in Human Fetal Epidermis
Single-channel FITC immunofluorescence images of fetal epidermis were digitized as TIFF images (using Photoshop v6.0; Adobe, CA) and subsequently analyzed quantitatively with the MCID/M2 software image analysis program (Imaging Research; Ontario, Canada). Briefly, rectangular areas containing linear epidermal basal cells up to the periderm or stratum corneum (10 cell widths on average) were selected. Image analysis was then performed to determine the overall image intensity at various levels from the base of the rectangle. Propidium iodide (PI) nuclear counterstain that was removed using Photoshop from the original image was used to determine the boundary of various layers on the MCID image analysis program.
Electron Microscopy
Fetal skin was fixed in half-strength Karnovsky's fixative or 2% glutaraldehyde solution, postfixed in 1% OsO4, dehydrated, and embedded in Epon 812 (
Cryofixed and Cryosubstituted Postembedding Immunoelectron Microscopy Without Chemical Fixatives
Postembedding immunoelectron microscopy using cryofixed and cryosubstituted skin specimens was carried out with slight modifications to the previously described method (. After being washed, each section was incubated for 1 hr at 37C with a secondary linker antibody. After another wash, each section was placed on a drop of 5-nm gold-labeled goat anti-mouse or rabbit IgG (British Biocell International; Cardiff, UK) diluted 1:200 at room temperature for 1 hr, and was then washed with distilled water. The sections were counterstained with 15% uranyl acetate for 2 min.
Quantitative Analysis of Gap Junctions in Fetal Epidermal Keratinocytes
Quantitative analysis of morphologically apparent gap junctions was performed within the periderm, intermediate, and basal cells. Gap junctions were counted using fetal specimens of ultrathin sections for standard high-power transmission electron microscopy. We defined a gap junction on a standard electron micrograph as a plasma membrane area in close apposition to another cell plasma membrane that exhibited a central electron-dense line between each plasma membrane lipid bilayer of adjacent cells. Gap junction counts were done on at least 26 cells in each specimen. In addition, the number of immunogold-labeled gap junctions was counted on sections stained for immunoelectron microscopy. We calculated the numbers of gap junctions per epidermal cell to evaluate the number of gap junctions in the three epidermal cell populations, i.e., basal cells, intermediate cells, and periderm cells.
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Results |
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Cx26 Is Expressed in Early Fetal Stages, Whereas Cx43 Is Expressed in Later Stages of Epidermal Development
Cx26 was expressed in the basal cells and periderm at 49 days' EGA (corresponding to the two-layered stage) (Fig 1a), but this expression gradually became restricted to the periderm and became weaker during fetal skin development (Fig 1b). From 108 days' EGA (four or more layered stage), expression of Cx26 was not detected in the epidermis (Fig 1c).
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Conversely, the expression of Cx43 was not detected at 49 days' EGA nor at 57 days' EGA (Fig 1d), but from 88 days' EGA (four or more layered stage), expression of the Cx43 molecule was detected in the intermediate and periderm layers, although the expression was very weak in the basal layer (Fig 1e). At 163 days' EGA (the stage of interfollicular keratinization), expression of Cx43 was apparent in all the intermediate cells. Again, weak expression was seen in the basal cells and no expression was detected in the horny layer (Fig 1f). Periderm had been already been lost at this stage.
Quantitative analysis for Cx26 immunofluorescence revealed high FITC levels at the intermediate cellperiderm interface site at 96 days' EGA (Fig 2a). However, intense FITC levels for Cx43 were noted at the intermediate cell layer at the same time point (Fig 2b).
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Formation of Ultrastructurally Mature Intact Gap Junctions Is Observed After the Four or More Layered Stage of Epidermal Development
Morphologically apparent gap junction structures were not observed in the epidermis at 108 days' EGA or younger. The intact gap junction structures were observed from 120 days' EGA (four or more layered stage) (Fig 3a and Fig 3b). In the epidermis at 120 days' EGA, most gap junctions were located on the sites of peridermperiderm or peridermintermediate cell attachment and a small number of gap junctions were seen on the intermediate cellintermediate cell attachment region. There were no obvious gap junctions between the basal cells. At this stage of epidermal development, the numbers of ultrastructurally apparent gap junctions per periderm cell and intermediate cell were 1.8 (gap junction counts/number of periderm cells examined) and 0.15 (gap junction counts/number of intermediate cells examined), respectively (Fig 5a).
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At 152 days' EGA, most gap junctions were located between intermediate cell attachment sites and fewer numbers of gap junctions were found on any of the borders of the basal cell attachment sites. At this stage of epidermal development, the periderm had already senescenced and become detached. The numbers of apparent gap junctions per intermediate cell and basal cell were 1.0 and 0.13, respectively (Fig 5a).
Mature Intact Gap Junction Structures in Intermediate Cells of Fetal Epidermis Contained Cx43
In the epidermis at 42 days' EGA, there were no obvious gap junction structures but sparse immunogold labeling for Cx26 was observed on the cell membrane, forming small clusters of gold particles between both basal cells and periderm cells (data not shown). Immunogold labeling for Cx43 was not seen.
At 105 days' EGA, immunogold labeling for Cx43 was observed at immature gap junction-like structures that comprised closely apposed cell membranes of the intermediate cells (Fig 4a). The number of labeled immature gap junction-like structures per intermediate cell was 0.051 (Fig 5b). No Cx43 labeling was seen in the basal cells or the periderm. Cx26 immunogold labeling was not detected in any part of the epidermis at this stage of development.
At 154 days' EGA, immunogold labeling for Cx43 was observed on the mature intact gap junctions on the intermediate cells (Fig 4b), and the number of labeled gap junctions per intermediate cell was 0.17 (Fig 5b). At this stage of epidermal development, no Cx43 labeling was seen in the basal cells, and the periderm had already been lost. Cx26 immunogold labeling was not detected in any layer of the epidermis.
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Discussion |
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In the developing human fetal epidermis before interfollicular keratinization, three major populations of epidermal cells are identified: the basal cells, the intermediate cells, and the periderm. Before 36 days' EGA, there is only a single-layered epithelium. Between 36 and 55 days' EGA, two layers, the basal cell layer and the periderm, are observed in the primitive epidermis. Between 55 and 75 days' EGA, the intermediate cell layer appears between the basal cell layer and the periderm and, as the fetal skin develops, the intermediate cell layer forms into two and then three or more layers. At about 160 days' EGA, the periderm is lost and interfollicular keratinization begins (
Cellcell junction proteins are identifiable from the early stage of human epidermal development. For example, desmosomal proteins, the desmoplakin, desmocollin and desmoglein, and adherens junction components E-cadherin, -catenin, and vinculin were reported to be expressed in the epidermis at 56 days' EGA (
By immunofluorescence, Cx26 was expressed at 49 days' EGA within the basal cells and periderm. However, Cx26 expression gradually disappeared and was not observed at 108 days' EGA. Cx43 expression was observed from 88 days' EGA. Switching of the expression pattern of the Cx family subunits was also observed during mouse embryonic development (
At 42 days' EGA, we failed to observe mature gap junction structures, but the presence of clusters of gold particles at the cell membrane, forming immature-looking gap junction-like structures, was observed by immunoelectron microscopy. In this stage of epidermal development, Cx26 was expressed, but Cx26 molecules might only form immature gap junctions. From the four or more layered stage of epidermal development (105 days' EGA), Cx43 staining was observed at the gap junctional structures by immunoelectron microscopy. This finding, together with the results of immunofluorescence data and electron microscopic observations, further confirms that, from the four or more layered stage, gap junctions mainly comprise Cx43. Previous studies from another group showed that the gap junction protein Cx43 was expressed on a few locations in the cell membrane of basal cells at 56 days' EGA (
Quantitative study revealed that the number of gap junctions increased according to the maturation of the fetal epidermis. This was also highlighted by the differentiation process, in which gap junctions were more frequently observed in the intermediate cell layer than in the basal cell layer (Fig 6).
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The number of gap junctions per cell was counted on ultrathin sections approximately 70 nm thick. The diameter of the keratinocyte is about 2040 µm according to semithin section measurement. Therefore, the total number of gap junctions per single whole cell is estimated to be 300500-fold of our data. The number of Cx43-labeled gap junctions detected in immunoelectron microscopy was about 1/6 for the data of transmission electron microscopy, as expected from the limitations of the postembedding immunoelectron microscopic technique. This is caused by immunogold particles binding only to the antigens that are exposed at the very surface of the section. The increase in Cx43-labeled gap junctions on epidermal maturation was also demonstrated by this technique. Thus, our results were confirmed not only at the ultrastructural level but also at the protein expression level. From the quantitative analysis, we can conclude that gap junctions are formed more frequently in the intermediate cells than in basal cells in accordance with keratinocyte differentiation. In contrast, in developing rat epidermis, the gap junctions were observed with the highest frequency in the basal layer at 20 days of gestation, corresponding to the period of interfollicular keratinization in rat epidermis (
In our study, many gap junctions were observed in the periderm. The existence of gap junctions in the periderm layer was also shown in rat fetal epidermis (
In conclusion, our results clearly demonstrate that Cx26 is expressed in the early stages of fetal epidermal development and Cx43 in the later stages. In addition, gap junctions are more frequently assembled as keratinocytes mature from the basal cell layer to upper epidermal layers. These facts add further evidence that the gap junctions are essential structures involved in human epidermal cell proliferation, differentiation, and morphogenesis, and suggest that Cx26 plays a crucial role in early fetal epidermal morphogenesis.
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Acknowledgments |
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Supported in part by grants-in-aid for Scientific Research (nos. 13357008 and 12470175) to HS, and by no. 12670839 to MA from the Japan Society for the Promotion of Science.
We thank Prof John E. Olerud, Ms Marcia L. Usui, Mr Robert A. Underwood, Dr Lynne T. Smith, Megumi Sato, and Hideki Nakamura for their generous help in this project.
Received for publication April 23, 2002; accepted May 29, 2002.
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