Materials and methods

Tissue

Human fetal tissue was acquired through the Central Laboratory of Human Embryology at the University of Washington, Seattle, with the approval of the Human Subjects Review Board and in accordance with the United States DHEW policies. Human embryonic and fetal skin specimens were obtained from aborted fetuses of 49–163 d EGA. The numbers of the embryos and the fetuses and the ages were as follows: four embryos (49–65 d EGA, two-layered epidermis), five fetuses (66–95 d EGA, fetal stratified three-layered epidermis), five fetuses (96–160 d EGA, four-layered or more epidermis), and two fetuses (>160 d EGA, epidermis showing interfollicular keratinization). Skin specimens were taken from the trunk and scalp and processed for observation. EGA was determined from maternal histories, fetal measurements (crown rump and foot length), and comparative histologic appearance of epidermis (^{Shepard 1975;Holbrook 1979;Mercer et al. 1987}).

Electron microscopy

Skin autopsy and biopsy samples were fixed in one-half strength Karnovsky's fixative or 2% glutaraldehyde solution, postfixed in 1% OsO₄, dehydrated, and embedded in Epon 812 (^{Perry et al. 1987}). All the samples were serially sectioned, sampled every 10–15 m for light microscopy (1 m thick), and thin sectioned for electron microscopy (70 nm thick). The histologic sections were stained by the method of^{Richardson et al. (1960)}. The thin sections were stained with uranyl acetate and lead citrate (^{Reynolds 1963}) and examined in JEOL 1200EXII transmission electron microscope in the transmission mode at 80 kV.

Antibodies

The primary antibodies to CCE proteins used in the present study were rabbit polyclonal anti-human involucrin antibody (Biomedical Technologies Inc., Stoughton, MA), rabbit polyclonal anti-human SPRP 2 antibody, 1B (^{Hohl et al. 1995}), and rabbit polyclonal anti-human SPRP 3 antibody, 4A (^{Hohl et al. 1995}), and rabbit polyclonal anti-human loricrin antibody (^{Mehrel et al. 1990}). Goat polyclonal antibody against human recombinant TGase 1 (major variant) (^{Kim et al. 1992}) whose epitope(s) is located in the central core of TGase 1 (^{Kim et al. 1995}), rabbit polyclonal anti-TGase 2 and rabbit polyclonal anti-TGase 3 antibodies (^{Tarcsa et al. 1997}) were also used. In addition, mouse monoclonal anti-LGP antibody, AE17, a generous gift of Dr. W. Michael O'Guin, was employed (^{O'Guin et al. 1989}).

Immunofluorescent labeling

Six micrometer thick sections of fresh fetal skin cut with a cryostat were used. The sections were incubated in primary antibody solution for 1 h at 37°C. Antibody dilutions were as follows; 1:1 for anti-involucrin, 1:200 for anti-SPRP 2 antibody, 1:500 for anti-SPRP 3 antibody, 1:50 for anti-loricrin antibody, 1:10 for anti-TGase 1 antibody, 1:50 for anti-TGase 2 and anti-TGase 3 antibodies, and 1:2 for AE17. The sections were then incubated in fluorescein isothiocyanate-conjugated to rabbit anti-mouse immunoglobulins, goat anti-rabbit immunoglobulins, or rabbit anti-goat immunoglobulins diluted 1:100 (DAKO, Glostrup, Denmark) for 30 min at room temperature, followed by 10 g per ml propidium iodide to counterstain nuclei (Sigma, St. Louis, MO) for 10 s. The sections were extensively washed with phosphate-buffered saline between incubations. The stained sections were mounted with a coverslip in 50% glycerol mounting medium and observed by epifluorescence microscope.

Post-embedding immunoelectron microscopy using cryofixation and cryosubstitution without chemical fixative

Post-embedding immunoelectron microscopy using cryofixed and cryosubstituted skin specimens was carried out as described previously (^{Shimizu et al. 1989}), with slight modification. Briefly, skin specimens from a fetus at 91 d EGA were cryoprotected with 15% glycerol/phosphate-buffered saline (PBS) at 4°C for 30 min and cryofixed by plunging them into liquid propane cooled to –190°C, followed by cryosubstitution (CS-auto, Reichert-Jung, Wien, Austria) with acetone at –80°C for 120 h. They were then embedded in Lowicryl K11M (Chemische Werke Lowi, Waldkraiburg, Germany) at –60°C. The specimens were polymerized by ultraviolet irradiation at –60°C for 72 h and at room temperature for another 72 h. Ultrathin sections were incubated for 2 h at 37°C with a primary antibody. Dilutions of primary antibodies were 1:4 for anti-loricrin antibody and 1:10 for anti-involucrin antibody. After being washed, each section was placed on a drop of 1 nm gold-labeled goat anti-mouse or rabbit immunoglobulins (Amersham International, Buckinghamshire, UK) diluted 1:40 at room temperature for 2 h, and then washed with distilled water. For easier observation, the 1 nm gold particles were enlarged by incubation with immunogold silver-enhancement solution (Amersham International) at room temperature for 6 min (for observation at high magnification) or 10 min (for observation at low magnification) (^{Shimizu et al. 1992}). The sections were counterstained with saturated uranyl acetate and lead citrate for 6 and 2 min, respectively.

Results

In the two-layered epidermis (49–65 d EGA), TGase 1–3, involucrin and SPRP2/3, but not loricrin, were expressed in the cell periphery of the periderm

Periderm cells of the two-layered epidermis had a flat cell membrane 7–9 nm thick in the early period. Periderm cells showed thickening of their cell membrane thereafter (Figure 2a). No thickening was observed in the cell membrane of basal cells in this stage. Immunoreactivities of involucrin and SPRP2/3 were seen in the periderm cells with some potentiation in the cell periphery, but not in the basal cells in the two-layered epidermis (49–65 d EGA) (Figure 3a,e). Loricrin immunoreactivity was not observed in either the periderm cells or the basal cells (Figure 3i). TGase 1–3 immunoreactivities were seen strongly in the periderm and weakly in the basal cells (Figure 4a,e,i). LGP detected by AE17 antibody was seen only in the periderm cells (Figure 5a).

Figure 2.

Thickening of cell membrane of periderm cells in the developing epidermis. (a) Two-layered stage (49–65 d EGA). (b) Three-layered stage (66–95 d EGA). (c) Four-layered or more stage (96–160 d EGA). (d) The stage of interfollicular keratinization (>160 d EGA). Membrane thickening has already started in periderm cells of the two-layered epidermis (a) and electron-dense, thick cell envelope similar to CCE in adult epidermis is observed in the periphery of periderm cells in the three-layered stage (b) and four-layered or more stage of epidermis (c). After the regression of periderm, CCE is seen in the cell periphery of the cornified cells of the keratinized interfollicular epidermis (d). Scale bar: 0.3 m.

Full figure and legend (72K)

Figure 3.

Expression of CCE precursor proteins in developing human epidermis. The periderm cells (arrowheads) are positive for involucrin and SPRP detected at the stage of embryonic 2-layered epidermis (a, e). The periderm cells (arrowheads) are stained for loricrin after formation of three-layered epidermis (j). Involucrin immunoreactivity is also seen in the upper intermediate cells of the late stage of stratified epidermis (c). Each of the three CCE precursor proteins are expressed in the granular cells and the cornified cells of keratinizing epidermis (d, h, l). Involucrin (a–d), SPRP2 (e, h), SPRP3 (f, g), and loricrin (i–l) immunolocalizations are demonstrated with FITC (green) and nuclei are stained with propidium iodide (red). (a, e, i) Embryonic two-layered epidermis (49–65 d EGA); (b, f, j) fetal stratified three-layered epidermis (66–95 d EGA); (c, g, k) four-layered or more epidermis (96–160 d EGA); (d, h, l) epidermis showing interfollicular keratinization (>160 d EGA). Scale bars: 50 m.

Full figure and legend (98K)

Figure 4.

Expression of TGase in developing human epidermis. Epidermal TGase 1–3 are observed in the periderm cells (arrowheads) of embryonic two-layered epidermis, although the basal cells are negative for the TGase in the stage of development (a, e, i). TGase 1–3 immunoreactivities are detected in all the layers of stratified, three-layered or more epidermis and keratinized epidermis (b–d, f–h, j–l). TGase 1 (a–d), TGase 2 (e–h), and TGase 3 (i–l) immunolocalizations are labeled with FITC (green) and nuclear stain is performed with propidium iodide (red). (a, e, i) Embryonic two-layered epidermis (49–65 d EGA); (b, f, j) fetal stratified three-layered epidermis (66–95 d EGA); (c, g, k) four-layered or more epidermis (96–160 d EGA); (d, h, l) epidermis showing interfollicular keratinization (>160 d EGA). Scale bar: 50 m.

Full figure and legend (100K)

Figure 5.

Expression of LGP in developing human epidermis. LGP detected by AE17 antibody is seen in the late stage of indifferent epithelium. In embryonic epidermis, only the periderm cells (arrowheads) are positive for LGP (a). Both the intermediate cells and the periderm cells are stained for LGP in the stratified, three-layered or more epidermis (b, c). LG-associated protein is observed in the entire epidermis showing keratinization (d). Strong, linear staining by AE17 (arrows) was seen in the subperidermal area (b, c) and between the cornified cells and the granular cells (d). AE17 antibody stain (LGP) are visualized with FITC (green) and nuclear stain is done with propidium iodide (red). (a) Embryonic two-layered epidermis (49–65 d EGA); (b) fetal stratified three-layered epidermis (66–95 d EGA); (c) four-layered or more epidermis (96–160 d EGA); (d) epidermis showing interfollicular keratinization (>160 d EGA). Scale bar: 50 m.

Full figure and legend (79K)

In the three-layered and more epidermis (66–160 d EGA), TGase 1–3, CCE precursor proteins, and LGP were strongly expressed in the periderm cells

Periderm cells of the three-layered, stratified epidermis (66–95 d EGA) had thickened cell membrane (Figure 2b). Involucrin immunoreactivity was observed in both the periderm cells and the intermediate cells in the three-layered, stratified epidermis (66–95 d EGA) (Figure 3b). Loricrin and SPRP2/3 were seen only in the periderm cells (Figure 3f,j). The staining of all the four CCE precursor proteins in the periderm was relatively stronger in the cell periphery. All three TGase were detected in the entire epidermis, although the labeling for TGase 1 and 3 in the basal cells was weaker than that of upper epidermis (Figure 4b,f,j). AE17-positive LGP was observed in the intermediate cells and the periderm cells and sharp, linear staining was seen in the subperidermal area (Figure 5b).

In four-layered or more stratified epidermis (96–160 d EGA), electron-dense, thickened cell membrane about 15 nm in thickness, the same thickness as that of the cornified cells of human adult epidermis, was seen in periderm cells (Figure 2c), although complex blebs and microvilli were prominent in the cell periphery of periderm cells. In this stage of epidermal development, not only the periderm cells but also the upper intermediate cells were positive for involucrin, although loricrin and SPRP2/3 immunoreactivities restricted to the periderm cells (Figure 3c,g,k). Stainings for the three CCE precursor proteins were stronger in the cell periphery of the periderm cells at the light microscopic level. The entire epidermis was immunohistologically positive for TGase 1–3, although the staining for TGase 1 in the basal cells was weak (Figure 4c,g,k). TGase 3 staining in the basal cells was weak in the three-layered epidermis and was increased in four-layered or more epidermis. TGase 1–3 remained in the periderm cells until the regression of periderm cells and the onset of interfollicular keratinization. Also in this stage, LGP was observed in the intermediate cells and the periderm cells, and sharp, linear staining of this protein was seen in the subperidermal area (Figure 5c).

In the epidermis showing interfollicular keratinization (>160 d EGA), all the TGase, CCE precursor proteins, and LGP were expressed in the granular and cornified layers

The periderm disappeared and now the outermost keratinocytes of the interfollicular epidermis showed keratinization (>160 d EGA). The superficial keratinized cells had thickened cell membrane, approximately 15 nm in thickness, similar to CCE in cornified cells in adult epidermis (Figure 2d). Involucrin, SPRP2/3, and loricrin were expressed in the granular and cornified layers with a linear peripheral localization in this stage (Figure 3d,h,l). Loricrin immunoreactivity was weak in the cornified cells. The anti-TGase 1–3 antibodies stained the entire epidermis, and TGase 1 and 3 showed diffuse cytoplasmic staining, although the staining was stronger in the cell periphery (Figure 4d,h,l). LGP was seen in the spinous, granular and cornified cells (Figure 5d). Strong linear staining of this protein was observed between the cornified cells and the granular cells.

Immunoelectron microscopy confirmed the deposition of CCE precursor proteins to the cell membrane of periderm cells

Immunoelectron microscopic observation of the fetal skin in the stage of four-layered or more epidermis revealed that the major CCE precursor proteins, involucrin (Figure 6) and loricrin, were restrictively localized in the CCE both in the flat surface and in the bleb of periderm cells.

Figure 6.

Ultrastructural localization of involucrin in the periphery of periderm cells in developing human epidermis. Immunoelectron microscopy reveals the deposition of involucrin to the cell membrane of the periderm of three-layered epidermis (b, c), although the deposition is sparse compared with that seen in adult skin (a). (a) Normal adult human skin; (b) flat surface of the periderm of three-layered epidermis; (c) bleb surface of the periderm of three-layered epidermis. Scale bar: 200 nm.

Full figure and legend (115K)

Discussion

In the present study, major CCE precursor proteins, involucrin, SPRP2/3, and loricrin, were expressed in the periderm cells ontogenically during human skin development. In addition, TGase 1–3 were expressed in the periderm from the stage of two-layered epidermis and in the underlying epidermis from the stage of three-layered epidermis. LPG was also detected from the stage of two-layered epidermis in the periderm and, from the stage of three-layered epidermis, in the underlying intermediate cell layer of epidermis. These sequential expression patterns were associated with the thickening of cell membrane of periderm cells revealed by electron microscopy. These findings indicated that CCE is being formed in the periderm cells from the stage of two-layered epidermis by coordinated expressions of CCE precursor proteins, TGase 1–3 and LGP in the developing human skin.

The formation of a structurally mature CCE occurs in several steps (^{Eckert et al. 1993;Reichert et al. 1993;Steinert & Marekov 1995;Ishida-Yamamoto et al. 1997a;Jarnik et al. 1998}). Initially, in response to an increase in the calcium concentration in the cytoplasm of keratinocytes, epidermal TGase start cross-linking a membrane-bound protein with a cytosolic precursor protein. During the first stage, a scaffolding is assembled from soluble precursors such as involucrin. In the second stage, other precursors, including SPRP and loricrin, are added to this scaffold. Finally, lipids are covalently bound to the outer surface of the protein cell envelope to form the complete water barrier. Our findings in the present study clearly demonstrate that CCE precursor proteins, epidermal TGase and LGP are expressed at precise times during epidermal development corresponding to distinct morphologic events.

The periderm cells undergo a remarkable series of changes in morphology beginning at the time of the embryonic–fetal transition and continuing until interfollicular epidermal keratinization. The changes that occur in the later stages of peridermal morphogenesis mimic some of the events of keratinization: the cells lose most of their few organelles, the nucleus is rarely evident, and the cells fill increasingly with loosely organized intermediate filaments. Thus, it has been described as 'an anomalously keratinizing keratinized layer' (^{Wolf 1967a}). In addition, these changes are similar to those that occur in the first intermediate cells that undergo partial keratinization. It is likely that initial cornification of the periderm cells followed by keratinization of the epidermis protects the fetus from immediate environmental alteration as the amniotic fluid changes before birth. In this context, CCE formation in periderm cells may have an important implication. Morphologically, a dense band beneath the plasma membrane (structurally equivalent to CCE) is seen before 90 d EGA (^{Holbrook & Odland 1975}). By 135 d EGA, all periderm cells demonstrated thickened cell membrane as we showed in the present study, and the periderm cells flatten and 'regress' just prior to keratinization. According to our results, the order of CCE precursor protein expression in periderm cells and, later, in the intermediate or granular cells during epidermal development was consistent with the suggested order of deposition of the precursor proteins in CCE formation in the adult epidermis, i.e., involucrin first, followed by SPRP, and finally loricrin (^{Steinert & Marekov 1995}). During this developmental process, TGase were always expressed in the site of CCE formation. LGP was detected in the periderm or intermediate cells at almost the same time as the expression of involucrin and SPRP2/3. Finally, in the regression stage of the periderm cells (96–160 d EGA), all three TGase expressed in the periderm cells and all CCE precursor proteins are abundant in the periderm and showed membranous localization in the cell periphery. In addition, LGP was mainly localized linearly between the granular cells and the periderm or cornified cells. Thus, these results indicate that the process of CCE formation during periderm regression and epidermal keratinization are similar if not identical (Figure 7). This fact implies that periderm regression in the fetal skin is a similar event to the keratinization of mature epidermis.

Figure 7.

Model of structure of CCE in the human fetal periderm cells. The innermost (cytoplasmic surface) consists of loricrin and SPRP. These proteins are deposited over a scaffold of unknown structure that consist of involucrin, cystatin , and probably other yet unknown proteins. Keratin filaments bound together by filaggrin are not associated with peridermal CCE. The outer surface (intercellular surface) is coated with a lipid envelope derived from lamellar granules.

Full figure and legend (33K)

Recently, abnormal CCE has been reported to be the cause of several congenital skin diseases of cornification, including lamellar ichthyosis (^{Huber et al. 1995;Russell et al. 1995}), Vohwinkel's syndrome (^{Maestrini et al. 1996;Korge et al. 1997}), autosomal dominant erythrokeratoderma (^{Ishida-Yamamoto et al. 1997b}), and mutilating palmoplantar keratoderma (^{Akiyama et al. 1998}). In this context, our findings are not only important for understanding the developmental mechanism of cornified cells, but also provide an important clue to clarifying the pathomechanism of congenital cornification disorders, especially, severe ichthyosis which have their onset in the fetal period.

In a previous report on ontogenic expression of TGase 1 in developing human fetal skin (^{Polakowska et al. 1994}), the periderm cells were positive for TGase 1 from about 65 d EGA and the underlying epidermal cell layers became positive for TGase 1 only after the disappearance of the periderm. The discrepancy in the expression pattern of TGase 1 between the previous report and our observation is thought to be due to the difference in nature of anti-TGase 1 antibody used in each study. Anti-TGase 1 antibody, B.C1, was used in the report of^{Polakowska et al. (1994)}. Immunologic and protein chemical analyses, however, revealed that the major epitope of several batches of the commercially available form of the B.C1 antibody resides on SPRP2 (^{Kim et al. 1995}). Thus, we employed a newly developed anti-TGase 1 antibody (^{Kim et al. 1995}) whose epitope(s) is located in the central core of TGase 1. Using this antibody, TGase 1 expression is detected in the entire epidermis of mature skin (^{Kim et al. 1995;Akiyama et al. 1997}). From our results, TGase 2 was expressed in the entire epidermis throughout epidermal development. On the other hand, expression of TGase 1 and 3 in the periderm cells preceded their expression in the other epidermal layers. Our findings suggest that TGase, especially TGase 2, work not only in CCE formation but also in other processes of the epidermal morphogenesis.

In conclusion, our ultrastructural and immunohistologic studies confirmed that periderm cells indeed form CCE by coordinated expressions of CCE precursor proteins, TGase, and LGP and periderm regression was suggested to be a similar process in terms of CCE formation to the subsequent epidermal keratinization.

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Acknowledgments

We thank Dr. W. Michael O'Guin for his generous gift of AE17 antibody; Dr. Soo-Youl Kim for providing us the anti-TGase antibodies; Dr. Akemi Ishida-Yamamoto for her generous help and Ms. Megumi Sato, Ms. Yuriko Kanzaki, Ms. Yu Umebayashi, Ms. Shuko Nonaka, Ms. Marcia L. Usui, and Mr. Robert A. Underwood for their fine technical assistance on this project. This work was supported in part by Grant-in-Aid for Encouragement of Young Scientists (nos 08770700 and 9770668) to M.A. from the Ministry of Education, Science, Sports and Culture of Japan and by grants to L.T.S. (HD-17664 and AR-21557) from the National Institutes of Health, U.S.A and support from the George F. Odland Endowment Funds.