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Expression of Differentiation Markers During Fetal Skin Development in Humans: Immunohistochemical Studies on the Precursor Proteins Forming the Cornified Cell Envelope

1999; Elsevier BV; Volume: 112; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.1999.00602.x

1523-1747

Seung‐Chul Lee, Jee-Bum Lee, Jung-Pio Kook, Jae-Jung Seo, Young Pio Kim, Kwang Il Nam, Sung-Sik Park,

Wound Healing and Treatments

The cornified cell envelope is formed during the terminal differentiation of epidermis through cross-linking of specific proteins by transglutaminases. The specific arrangement of individual protein in the cornified cell envelope and participation of individual protein in the cornified cell envelope at different regions of skin, i.e., palm, foreskin, lips, etc. are not clearly understood. In order to understand the pattern and expression schedule of each individual precursor protein during the differentiation and formation of cornified cell envelope, the expression of precursor proteins in developing human fetal skins from the first to the third trimester were examined by immunohistochemical studies. Involucrin was found in the periderm and intermediate layer from 14 wk estimated gestational age, while loricrin and small proline-rich protein 1 were found in the periderm from 16 wk estimated gestational age. Filaggrin and trichohyalin that are absent in the adult cornified cell envelope were found in the granular and horny layers from 24 wk estimated gestational age. The precursor proteins except trichohyalin did not change their patterns after the onset of initial expression during development. Trichohyalin was transiently expressed in the granular and horny layers of the epidermis from 24 wk estimated gestational age with peak expression at 27 wk estimated gestational age, but was not detected in adult skin. In hair follicles, trichohyalin expression was stable without change from 20 wk estimated gestational age. These findings suggest that fetal skin may have different sets of barriers from the second trimester; the immature cornified cell envelope is formed in the early second trimester and the mature cornified cell envelope is formed in the late second or early third trimester when filaggrin and trichohyalin appear. The cornified cell envelope is formed during the terminal differentiation of epidermis through cross-linking of specific proteins by transglutaminases. The specific arrangement of individual protein in the cornified cell envelope and participation of individual protein in the cornified cell envelope at different regions of skin, i.e., palm, foreskin, lips, etc. are not clearly understood. In order to understand the pattern and expression schedule of each individual precursor protein during the differentiation and formation of cornified cell envelope, the expression of precursor proteins in developing human fetal skins from the first to the third trimester were examined by immunohistochemical studies. Involucrin was found in the periderm and intermediate layer from 14 wk estimated gestational age, while loricrin and small proline-rich protein 1 were found in the periderm from 16 wk estimated gestational age. Filaggrin and trichohyalin that are absent in the adult cornified cell envelope were found in the granular and horny layers from 24 wk estimated gestational age. The precursor proteins except trichohyalin did not change their patterns after the onset of initial expression during development. Trichohyalin was transiently expressed in the granular and horny layers of the epidermis from 24 wk estimated gestational age with peak expression at 27 wk estimated gestational age, but was not detected in adult skin. In hair follicles, trichohyalin expression was stable without change from 20 wk estimated gestational age. These findings suggest that fetal skin may have different sets of barriers from the second trimester; the immature cornified cell envelope is formed in the early second trimester and the mature cornified cell envelope is formed in the late second or early third trimester when filaggrin and trichohyalin appear. cornified cell envelope estimated gestational age small proline-rich protein The cornified cell envelope (CE), an essential epidermal integument, is thought both to serve a barrier function for organisms, and to help maintain the structural integrity of the epidermis (Hohl, 1990Hohl D. Cornified cell envelope.Dermatologica. 1990; 180: 201-211Crossref PubMed Scopus (164) Google Scholar;Reichert et al., 1993Reichert U. Michel S. Schmidt R. The cornified cell envelope: a key structure of terminally differentiating keratinocytes.in: Darmon M. Blumenberg M. Molecular Biology of the Skin. Academic Press Inc, London1993: 107-150Crossref Google Scholar). It is formed during the terminal differentiation process as several precursor proteins are cross-linked by transglutaminases (TGase) (Steven and Steinert, 1994Steven A.C. Steinert P.M. The protein composition of cornified cell envelopes.J Cell Sci. 1994; 107: 693-700Crossref PubMed Google Scholar;Steinert, 1995Steinert P.M. A model for the hierarchical structure of the human epidermal cornified cell envelope.Cell Death Differ. 1995; 2: 33-40PubMed Google Scholar). The CE is composed of a numerous precursor proteins, such as involucrin (Rice and Green, 1977Rice R.H. Green H. The cornified envelope of terminally differentiated human epidermal keratinocytes consists of crosslinked protein.Cell. 1977; 11: 417-422Abstract Full Text PDF PubMed Scopus (396) Google Scholar;Eckert et al., 1993Eckert R.L. Yaffe M.B. Crish J.F. Murthy S. Rorke E.A. Welter J.F. Involucrin—structure and role in envelope assembly.J Invest Dermatol. 1993; 100: 613-617Abstract Full Text PDF PubMed Google Scholar), loricrin (Mehrel et al., 1990Mehrel T. Hohl D. Rothnagel J.A. et al.Identification of a major keratinocyte cell envelope protein, loricrin.Cell. 1990; 61: 1103-1112Abstract Full Text PDF PubMed Scopus (360) Google Scholar;Hohl et al., 1991Hohl D. Mehrel T. Lichti U. Turner M.L. Roop D.R. Steinert P.M. Characterization of human loricrin. Structure and function of a new class of epidermal cell envelope proteins.J Biol Chem. 1991; 266: 6626-6636Abstract Full Text PDF PubMed Google Scholar), keratolinin/cystatin A (Zettergren et al., 1984Zettergren J.G. Peterson L.L. Wuepper K.D. Keratolinin: the soluble substrate of epidermal transglutaminase from human and bovine tissue.Proc Natl Acad Sci USA. 1984; 81: 238-242Crossref PubMed Scopus (77) Google Scholar;Takahashi et al., 1992Takahashi M. Tezuka T. Katunuma N. Phosphorylated cystatin alpha is a natural substrate of epidermal transglutaminase for formation of skin cornified envelope.FEBS Lett. 1992; 308: 79-82Abstract Full Text PDF PubMed Scopus (69) Google Scholar), small proline rich-protein (SPRR) (Kartasova et al., 1988Kartasova T. van Muijen G.N. van Pelt-Heerschap H. van de Putte P. Novel protein in human epidermal keratinocytes: regulation of expression during differentiation.Mol Cell Biol. 1988; 8: 2204-2210Crossref PubMed Scopus (67) Google Scholar, Kartasova et al., 1996Kartasova T. Darwiche N. Kohno Y. et al.Sequence and expression patterns of mouse SPR1: correlation of expression with epithelial function.J Invest Dermatol. 1996; 106: 294-304Crossref PubMed Scopus (59) Google Scholar), and SKALP/elafin (Wiedow et al., 1990Wiedow O. Schroder J.-M. Gregory H. Young J.A. Christophers E. Elafin: an elastase-specific inhibitor of human skin. Purification, characterization, and complete amino acid sequence.J Biol Chem. 1990; 265: 14791-14795Abstract Full Text PDF PubMed Google Scholar;Nara et al., 1994Nara K. Ito S. Ito T. Suzuki Y. Ghoneim M.A. Tachibana S. Hirose S. Elastase inhibitor elafin is a new type of proteinase inhibitor which has a transglutaminase-mediated anchoring sequence termed 'cementoin'.J Biochem. 1994; 115: 441-448PubMed Google Scholar). All of these precursor proteins are good substrates for calcium-dependent TGase, which catalyzes the formation of ε(γ-glutamyl)lysine isopeptide bond (Goldsmith et al., 1974Goldsmith L.A. Baden H.P. Roth S.I. Colman R. Lee L. Fleming B. Vertebral epidermal transamidases.Biochim Biophys Acta. 1974; 351: 113-125Crossref PubMed Scopus (29) Google Scholar;Steinert and Idler, 1979Steinert P.M. Idler W.W. Postsynthetic modifications of mammalian epidermal keratin.Biochemistry. 1979; 18: 5664-5669Crossref PubMed Scopus (64) Google Scholar). Therefore, it is understandable that the proper expression of enzymes and their substrates is an essential regulatory process in forming the CE to serve a barrier function. Expression of precursor proteins, markers of terminal differentiation, was determined by various factors, including calcium, retinoic acid, and cultural conditions, such as exposed or submerged cultures (Yuspa et al., 1982Yuspa S.H. Ben T. Steinert P. Retinoic acid induces transglutaminase activity but inhibits cornification of cultured epidermal cells.J Biol Chem. 1982; 257: 9906-9908Abstract Full Text PDF PubMed Google Scholar;Lichti et al., 1985Lichti U. Ben T. Yuspa S.H. Retinoic acid induces transglutaminase in mouse epidermal cells is distinct from epidermal transglutaminase.J Biol Chem. 1985; 260: 1422-1426Abstract Full Text PDF PubMed Google Scholar;De Nagae et al., 1987De Nagae S. Lichti U. Luca L.M. Yuspa S.H. Effect of retinoic acid on cornified envelope formation: difference between spontaneous envelope formation in vivo or in vitro and expression of envelope competence.J Invest Dermatol. 1987; 89: 51-58Crossref Scopus (40) Google Scholar). The normal epidermis proceeds in multiple stages to form the mature CE, but cultured cells are not able to complete all stages, causing different forms of the CE in vitro (De Nagae et al., 1987De Nagae S. Lichti U. Luca L.M. Yuspa S.H. Effect of retinoic acid on cornified envelope formation: difference between spontaneous envelope formation in vivo or in vitro and expression of envelope competence.J Invest Dermatol. 1987; 89: 51-58Crossref Scopus (40) Google Scholar). Similarly, prenatal skin is exposed to the aqueous condition of the placenta instead of air, suggesting that different sets of differentiation are possible in developing fetal skin. In addition, the periderm is found as a simple epithelium of fetal skin with an unique structure. It functions in the outermost layer until the second trimester, in contrast with the stratifying squamous epithelium in mature skin (Holbrook, 1979Holbrook K.A. Human epidermal embryogenesis.Int J Dermatol. 1979; 18: 329-356PubMed Google Scholar, Holbrook, 1991Holbrook K.A. Structure and function of the developing human skin.in: Goldsmith L.A. Physiology, Biochemistry, and Molecular Biology of the Skin. Oxford University Press, New York1991: 63-110Google Scholar). To date, however, there are only a few reports on the sequential changes in the markers of terminal differentiation during fetal skin development in humans. Involucrin was expressed in the periderm and intermediate layer, while loricrin was detected in the intermediate cells of human skin from the first trimester (Holbrook et al., 1991Holbrook K.A. Underwood R.A. Dale B.A. Thacher S.M. Wuepper K.D. Banks-Schlegel S. Cornified cell envelope (CCE) in human fetal skin: involucrin, keratolinin, loricrin, and transglutaminase expression and activity.J Invest Dermatol. 1991; 96: 542Google Scholar). SPRR1 expression was observed from the early second trimester in the periderm (Kartasova et al., 1988Kartasova T. van Muijen G.N. van Pelt-Heerschap H. van de Putte P. Novel protein in human epidermal keratinocytes: regulation of expression during differentiation.Mol Cell Biol. 1988; 8: 2204-2210Crossref PubMed Scopus (67) Google Scholar). In the late second or early third trimester, filaggrin expression was detected in the granular layer of the epidermis, whereas keratohyalin granules appear with the onset of epidermal keratinization (Holbrook, 1991Holbrook K.A. Structure and function of the developing human skin.in: Goldsmith L.A. Physiology, Biochemistry, and Molecular Biology of the Skin. Oxford University Press, New York1991: 63-110Google Scholar). Trichohyalin is a new putative precursor of the CE which acts as a matrix protein to bind with keratin intermediate filaments (Lee et al., 1993Lee S.-C. Kim I.-G. Marekov L.N. O'Keefe E.J. Parry D.A.D. Steinert P.M. The structure of human trichohyalin: potential roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and intermediate filament-associated (cross-linking) protein.J Biol Chem. 1993; 268: 12164-12176Abstract Full Text PDF PubMed Google Scholar). Unfortunately, few studies have been reported on the relative relationship among the CE precursors in terms of their sequential orders and localization during fetal skin development. Also, trichohyalin expression in the epidermis and hair follicles has not been reported in fetal skin. Under the assumption that transient barrier structures exist in fetal skin with different sets of differentiation process, we have evaluated the expression of the markers of terminal differentiation from the first to the third trimester, consecutively, in human fetuses. From this study, fetal skin is noted to be similar to postnatal mature skin in the sequential expression of precursor proteins of the CE. Also, trichohyalin was detected in the epidermis transiently as a component of the CE, whereas it was stably expressed in hair follicles, suggesting that a different regulatory mechanism is implicated in trichohyalin expression between the epidermis and hair follicles. Human fetal samples were obtained by dilation and curettage (D&C) or by an artificial termination of pregnancy. Consent forms were approved by the Committee for Human Experimental Study of Chonnam University Medical School, and all practices were performed under the rules for human sampling. The estimated gestational age (EGA) of fetal samples ranged from 10 wk to 32 wk, and the ages were determined by mothers' histories of last menstruation. Results on immunohistochemical experiments were repeated at least three times with different samples of the same EGA. A monoclonal antibody to anti-human filaggrin and a polyclonal antibody to anti-human involucrin were purchased from Biomedical Technologies (Stoughton, MA). The anti-filaggrin antibody was extracted from mice with partially purified filaggrin from the newborn human epidermis (Lynley and Dale, 1983Lynley A.M. Dale B.A. The characterization of human epidermal filaggrin, a histidine-rich, keratin filament-aggregating protein.Biochim Biophys Acta. 1983; 744: 28-35Crossref PubMed Scopus (109) Google Scholar). The anti-involucrin anti-serum was obtained from rabbits with purified cultured human epidermal cells (Murphy et al., 1984Murphy G.F. Flynn T.C. Rice R.H. Pinkus G.S. Involucrin expression in normal and neoplastic human skin: a marker for keratinocyte differentiation.J Invest Dermatol. 1984; 82: 453-457Abstract Full Text PDF PubMed Scopus (174) Google Scholar). A polyclonal anti-human loricrin antibody was raised against the carboxyl-terminal peptide of human loricrin in rabbits (Hohl et al., 1991Hohl D. Mehrel T. Lichti U. Turner M.L. Roop D.R. Steinert P.M. Characterization of human loricrin. Structure and function of a new class of epidermal cell envelope proteins.J Biol Chem. 1991; 266: 6626-6636Abstract Full Text PDF PubMed Google Scholar). A polyclonal anti-SPRR1 antibody was raised against the carboxyl-terminal peptide of human SPRR1 in rabbits (Kartasova et al., 1988Kartasova T. van Muijen G.N. van Pelt-Heerschap H. van de Putte P. Novel protein in human epidermal keratinocytes: regulation of expression during differentiation.Mol Cell Biol. 1988; 8: 2204-2210Crossref PubMed Scopus (67) Google Scholar). A polyclonal anti-trichohyalin antibody was obtained from rabbits with recombinant human trichohyalin of 1250–1849 residues (domain 8) located in the carboxy-terminus (Tarcsa et al., 1996Tarcsa E. Marekov L.N. Mei G. Melino G. Lee S.-C. Steinert P.M. Protein unfolding by peptidylarginine deiminase.J Biol Chem. 1996; 271: 30709-30716Crossref PubMed Scopus (280) Google Scholar). Specimens from the skin of thigh were fixed in 10% formalin solution for the immunohistochemical labeling with corresponding antibodies. After the fixation, the specimens were rinsed in absolute alcohol, rehydrated to 70% ethanol, and were processed for paraffin embedding. Paraffin sections, 5 μm in thickness, were deparaffinized by placing them through two changes of xylene. The sections were preincubated with 3% hydrogen peroxide in distilled water for 10 min to block endogenous peroxidase activity. The slides were then washed in Tris-buffered saline (pH 7.2). The sections were incubated with primary antibodies as follows: 1:10 titer at room temperature for 30 min with anti-involucrin; 1:200 titer at room temperature for 2 h with anti-loricrin; 1:200 titer at room temperature for 30 min with anti-filaggrin; 1:100 titer at room temperature for 2 h with anti-SPRR1; and 1:50 titer at room temperature for 30 min with anti-trichohyalin antibody. After incubation with the primary antibodies, the samples were rinsed with Tris-buffered saline and re-incubated with a biotinylated secondary antibody (anti-rabbit, mouse immunoglobulins) from LSAB kit (DAKO, CA) for 15 min at room temperature. After washing, the sections were incubated for 15 min with the streptavidin conjugated with horseradish peroxidase. They were incubated for 5 min with 3,3′-diaminobenzidine tetrahydrochloride or 3-amino-9-ethyl carbazole as the chromogenic substrate, and were counterstained with Meyer's hematoxylin. As a negative control, sections were incubated with nonimmunized sera instead of primary antibodies. In immunohistochemical staining for trichohyalin, the sections were boiled for 5 min at 121°C, 15 lb per in2 in an autoclave prior to incubating with the anti-trichohyalin antibody to restore its antigenicity. All sections were examined under the light microscope. The expression levels of differentiation markers were graded with a scale ranging from 0 to 3+ for their intensity accordingly. Involucrin was expressed from 14 wk EGA in the periderm and intermediate layer (Figure 1b), and it was observed in the granular and upper spinous layers of mature skin (Figure 1d). Loricrin and SPRR1 were expressed from 16 wk EGA in the periderm (Figure 1f,j), and they were observed in the granular layer of mature skin (Figure 1h,l). SPRR1 expression was also observed focally in the upper spinous layer of mature skin (Figure 1l). Filaggrin expression was observed from 24 wk EGA in the granular and horny layers of the entire epidermis (Figure 1o,p), whereas it was observed focally from 22 wk EGA in the perifollicular epidermis (Figure 1n). These precursor proteins did not demonstrate changes in their expression patterns after their initial onset of fetal skin development (Table 1). In experiments to exclude the possibility of nonspecific background staining, especially in the early stages, samples of 10 and 12 wk EGA were negatively stained when they were incubated with preimmune sera instead of primary antibodies (Figure 2).Table 1Immunoreactivities of precursor proteins of the cornified cell envelope during fetal skin development in humanImmunoreactivityPeriderm (granular)aThe epidermis consisted of the periderm, intermediate, and basal layers until 22 wk EGA, but they were replaced with the granular, spinous, and basal layers from 24 wk EGA as the periderm disappeared.Intermediate layer (spinous) bSPRR1 expression was focally positive in the upper spinous layer of the mature epidermis.Basal layer (basal) cFilaggrin expression was focally positive from 22 wk EGA in the perifollicular epidermis, while it was positive from 24 wk EGA in the entire epidermis.Precursor proteinsNo. of samples141620222432141620222432141620222432Involucrin5++++++++++++++++++++++++++++++––––––Loricrin5–++++++++++––––––––––––SPRR13–++++++++++––––+ bSPRR1 expression was focally positive in the upper spinous layer of the mature epidermis.+ bSPRR1 expression was focally positive in the upper spinous layer of the mature epidermis.––––––Filaggrin4–––– cFilaggrin expression was focally positive from 22 wk EGA in the perifollicular epidermis, while it was positive from 24 wk EGA in the entire epidermis.++++––––––––––––Trichohyalin3––– dTrichohyalin expression was positive from 24 wk EGA in the epidermis, but it was positive from 20 wk EGA in hair follicles.–+++––––––––––––a The epidermis consisted of the periderm, intermediate, and basal layers until 22 wk EGA, but they were replaced with the granular, spinous, and basal layers from 24 wk EGA as the periderm disappeared.b SPRR1 expression was focally positive in the upper spinous layer of the mature epidermis.c Filaggrin expression was focally positive from 22 wk EGA in the perifollicular epidermis, while it was positive from 24 wk EGA in the entire epidermis.d Trichohyalin expression was positive from 24 wk EGA in the epidermis, but it was positive from 20 wk EGA in hair follicles. Open table in a new tab Figure 2Samples treated with preimmune sera show no background staining. Fetal skin samples of 10 wk EGA (a) and 12 wk EGA (b) were stained negatively, when they were incubated with preimmune sera instead of specific primary antibodies. Scale bar: 50 μm.View Large Image Figure ViewerDownload (PPT) Trichohyalin was transiently expressed in the granular and horny layers of the epidermis from 24 wk EGA (Figure 3c), with a peak expression at 27 wk EGA (Figure 3d). Trichohyalin expression diminished after 32 wk EGA (Figure 3e), and it was not detected in adult skin (Figure 3f). In contrast, trichohyalin expression was stable in hair follicles without any changes since it started expression from 20 wk EGA in the inner root sheath and medulla (Figure 3b) (Table 1). This study was carried out to establish a better understanding of the functional aspects of fetal skin by evaluating certain markers of terminal differentiation, namely the precursor proteins of the CE.Holbrook, 1979Holbrook K.A. Human epidermal embryogenesis.Int J Dermatol. 1979; 18: 329-356PubMed Google Scholar suggested that the periderm has a barrier function until the underlying epidermis keratinizes with the formation of the epidermal CE from the late second or early third trimester. During the first trimester, the amniotic environment supplies nutrition to the fetus for growth, so the periderm is not a barrier in this early stage (Holbrook, 1979Holbrook K.A. Human epidermal embryogenesis.Int J Dermatol. 1979; 18: 329-356PubMed Google Scholar). The periderm was also reported to contain many cytoplasmic vesicles to exchange fluid or other materials over 12–16 wk EGA. Later it undergoes a transformation into a layer of flattened squames with aggregates of keratin filaments (Hoyes, 1968Hoyes A.D. Electron microscopy of the surface layer (periderm) of human foetal skin.J Anat. 1968; 103: 321-336PubMed Google Scholar;Breathnach, 1971Breathnach A.S. Embryology of human skin: a review of ultrastructural studies.J Invest Dermatol. 1971; 57: 133-143Abstract Full Text PDF PubMed Scopus (60) Google Scholar). From this study, precursor proteins, except filaggrin and trichohyalin, were identified in the periderm stages of the second trimester, suggesting that the periderm differentiates into a functional barrier as keratinization proceeds during this stage. The sequential order of their expression in fetal skin was similar to that of their incorporation into the CE in postnatal skin. Involucrin starts expression in the earliest stage followed by loricrin and SPRR. Filaggrin and trichohyalin are expressed in the last stage, causing the formation of the mature CE. An existing model for the hierarchical order of CE assembly predicts that involucrin and cystatin A act as a scaffold in the early stage of assembly, then loricrin and SPRR are deposited, and finally filaggrin is incorporated in the very late stage (Steinert, 1995Steinert P.M. A model for the hierarchical structure of the human epidermal cornified cell envelope.Cell Death Differ. 1995; 2: 33-40PubMed Google Scholar). Comparing our results with the previous reports, it appears that there are discrepancies in the expression of some precursor proteins in human skin. Our study showed that involucrin was present from 14 wk EGA in the periderm and intermediate layer, whereas it was reported to be found from 12 wk EGA (Watt et al., 1989Watt F.M. Keeble S. Fisher C. Hudson D.L. Codd J. Salisburt J.R. Onset of expression of peanut lectin-binding glycoproteins is correlated with stratification of keratinocytes during human epidermal development in vivo and in vitro.J Cell Sci. 1989; 94: 355-359Google Scholar) or from 8 to 9 wk EGA (Holbrook et al., 1991Holbrook K.A. Underwood R.A. Dale B.A. Thacher S.M. Wuepper K.D. Banks-Schlegel S. Cornified cell envelope (CCE) in human fetal skin: involucrin, keratolinin, loricrin, and transglutaminase expression and activity.J Invest Dermatol. 1991; 96: 542Google Scholar). Loricrin expression was reported to be positive in intermediate cells (Bickenbach et al., 1995Bickenbach J.R. Greer J.M. Bundman D.S. Rothnagel J.A. Roop D.R. Loricrin expression is coordinated with other epidermal proteins and the appearance of lipid lamellar granules in development.J Invest Dermatol. 1995; 104: 405-410Crossref PubMed Scopus (91) Google Scholar) from 7 to 8 wk EGA (Holbrook et al., 1991Holbrook K.A. Underwood R.A. Dale B.A. Thacher S.M. Wuepper K.D. Banks-Schlegel S. Cornified cell envelope (CCE) in human fetal skin: involucrin, keratolinin, loricrin, and transglutaminase expression and activity.J Invest Dermatol. 1991; 96: 542Google Scholar). The finding of loricrin expression only in the intermediate layer is exceptional, because other markers were found in the periderm. In fact, expression patterns of marker proteins in terms of their localization did not change markedly after initial onset of expression (Table 1). From this study, loricrin was found in the periderm of developing skin from 16 wk EGA, and the pattern did not change in mature skin, to be detected only in the granular layer. In SPRR1 expression, our result is largely consistent with the previous reports which show that it is localized in the periderm of mice (Kartasova et al., 1996Kartasova T. Darwiche N. Kohno Y. et al.Sequence and expression patterns of mouse SPR1: correlation of expression with epithelial function.J Invest Dermatol. 1996; 106: 294-304Crossref PubMed Scopus (59) Google Scholar) and humans from 15 wk old (Kartasova et al., 1988Kartasova T. van Muijen G.N. van Pelt-Heerschap H. van de Putte P. Novel protein in human epidermal keratinocytes: regulation of expression during differentiation.Mol Cell Biol. 1988; 8: 2204-2210Crossref PubMed Scopus (67) Google Scholar). SPRR1 expression was also found in developing hair follicles (Figure 1k), supporting the previous report that it was present in the outer layers of follicular infundibulum and isthmus (Koizumi et al., 1996Koizumi H. Kartasova T. Tanaka H. Ohkawara A. Kuroki T. Differentiation-associated localization of small proline-rich protein in normal and diseased human skin.Br J Dermatol. 1996; 134: 686-692Crossref PubMed Scopus (33) Google Scholar). The initial expression of filaggrin was observed from 24 wk EGA, whereas the epidermal CE forms as fetal skin reaches maturation, supporting the previous report (Dale et al., 1985Dale B.A. Holbrook K.A. Kimball J.R. Hoff M. Sun T.-T. Expression of epidermal keratins and filaggrin during human fetal skin development.J Cell Biol. 1985; 101: 1257-1269Crossref PubMed Scopus (216) Google Scholar). It is known that the mature epidermis starts keratinization from this stage when keratohyalin granules appear, while the periderm sloughs away (Holbrook, 1991Holbrook K.A. Structure and function of the developing human skin.in: Goldsmith L.A. Physiology, Biochemistry, and Molecular Biology of the Skin. Oxford University Press, New York1991: 63-110Google Scholar). The finding of focal staining of filaggrin in the perifollicular epidermis from 22 wk EGA, however, suggests that the maturation of the follicular epidermis precedes that of the interfollicular epidermis with a time-gap in keratinization of fetal skin. Together, the marker proteins of terminal differentiation did not change markedly in their expression patterns during fetal skin development. The same stage in the initial expression of loricrin and SPRR implies the presence of a functional relationship between them during terminal differentiation. From the analysis of peptide sequences, SPRR is believed to function as cross-bridging protein between and among the much larger amounts of loricrin (Steinert, 1995Steinert P.M. A model for the hierarchical structure of the human epidermal cornified cell envelope.Cell Death Differ. 1995; 2: 33-40PubMed Google Scholar). SPRR expression, however, is affected by many factors since its expression starts in fetal skin. SPRR1 is strongly expressed in the periderm of a 16 d old fetus in mouse, whereas the expression was not detectable in a newborn mouse (Kartasova et al., 1996Kartasova T. Darwiche N. Kohno Y. et al.Sequence and expression patterns of mouse SPR1: correlation of expression with epithelial function.J Invest Dermatol. 1996; 106: 294-304Crossref PubMed Scopus (59) Google Scholar). The age-associated decrease of SPRR1 was also reported in an mRNA expression study of human skin (Yaar et al., 1995Yaar M. Eller M.S. Bhawan J. Harkness D.D. Gilchrest B.A. In vivo and in vitro SPRR1 gene expression in normal and malignant keratinocytes.Exp Cell Res. 1995; 217: 217-226Crossref PubMed Scopus (35) Google Scholar). Further studies should be performed on factors to regulate SPRR expression during terminal differentiation, including age, location of skin, and various exogenous stimuli in normal and pathologic conditions (Kartasova et al., 1988Kartasova T. van Muijen G.N. van Pelt-Heerschap H. van de Putte P. Novel protein in human epidermal keratinocytes: regulation of expression during differentiation.Mol Cell Biol. 1988; 8: 2204-2210Crossref PubMed Scopus (67) Google Scholar;Yaar et al., 1995Yaar M. Eller M.S. Bhawan J. Harkness D.D. Gilchrest B.A. In vivo and in vitro SPRR1 gene expression in normal and malignant keratinocytes.Exp Cell Res. 1995; 217: 217-226Crossref PubMed Scopus (35) Google Scholar;Koizumi et al., 1996Koizumi H. Kartasova T. Tanaka H. Ohkawara A. Kuroki T. Differentiation-associated localization of small proline-rich protein in normal and diseased human skin.Br J Dermatol. 1996; 134: 686-692Crossref PubMed Scopus (33) Google Scholar). Trichohyalin is present mainly in the inner root sheath and medullary cells of hair follicles, as well as in the nail matrix, tongue, hard palate, and epidermis of humans (O'Guin and Manabe, 1991O'Guin W.M. Manabe M. The role of trichohyalin in hair follicle differentiation and its expression in non-follicular epithelia.Ann N Y Acad Sci. 1991; 642: 51-63Crossref PubMed Scopus (23) Google Scholar;O'Keefe et al., 1993O'Keefe E.J. Hamilton E.H. Lee S.-C. Steinert P.M. Trichohyalin: a structural protein of hair, tongue, nail, and epidermis.J Invest Dermatol. 1993; 101: 65S-71SAbstract Full Text PDF PubMed Scopus (54) Google Scholar). In the epidermis, it is transiently expressed only in neonatal foreskin, and is not detected in adult skin. But, analysis of the human trichohyalin gene suggests that trichohyalin is a precursor protein of the epidermal CE, because of its similarity to involucrin; both consist largely of peptide repeats that possess similar periodicity in charged residues to form a single-stranded helical rod as a scaffold proteins (Lee et al., 1993Lee S.-C. Kim I.-G. Marekov L.N. O'Keefe E.J. Parry D.A.D. Steinert P.M. The structure of human trichohyalin: potential roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and intermediate filament-associated (cross-linking) protein.J Biol Chem. 1993; 268: 12164-12176Abstract Full Text PDF PubMed Google Scholar). Trichohyalin is also similar to filaggrin in gene structure: both are composed of three exons and intervening introns; both have two EF-hands at the amino terminus, enabling them to bind with calcium; and both are regarded as an intermediate filament associated protein to bind with keratin filaments (Lee et al., 1993Lee S.-C. Kim I.-G. Marekov L.N. O'Keefe E.J. Parry D.A.D. Steinert P.M. The structure of human trichohyalin: potential roles as a functional EF-hand-like calcium-binding protein, a cornified cell envelope precursor, and intermediate filament-associated (cross-linking) protein.J Biol Chem. 1993; 268: 12164-12176Abstract Full Text PDF PubMed Google Scholar). From this study, trichohyalin and filaggrin have an intimate functional relationship as precursor proteins, in that both are expressed in the granular and horny layers at the same stage of 24 wk EGA. In fact, filaggrin and trichohyalin are reported to be found as hybrid granules in the normal foreskin, and pathologic conditions of psoriasis and molluscum contagiosum (Manabe and O'Guin, 1994Manabe M. O'Guin W.M. Existence of trichohyalin-keratohyalin hybrid granules: co-localization of two major intermediate filament-associated proteins in non-follicular epithelia.Differentiation. 1994; 58: 65-75Crossref PubMed Scopus (47) Google Scholar;Manabe et al., 1996Manabe M. Yaguchi H. Butt K.I. O'Guin W.M. Sun T.T. Ogawa H. Expression of keratohyalin-trichohyalin hybrid granules in molluscum contagiosum.Int J Dermatol. 1996; 35: 106-108Crossref PubMed Scopus (13) Google Scholar;Ishida-Yamamoto et al., 1997Ishida-Yamamoto A. Hashimoto Y. Manabe M. O'Guin W.M. Dale B.A. Iizuka H. Distinctive expression of filaggrin and trichohyalin during various pathways of epithelial differentiation.Br J Dermatol. 1997; 137: 9-16Crossref PubMed Scopus (26) Google Scholar). Further studies should be done to confirm the role of filaggrin as a component of the CE, because it may be a contaminant of the CE during the preparation of them as an intermediate filament associated protein (Steinert and Marekov, 1995Steinert P.M. Marekov L.N. The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope.J Biol Chem. 1995; 270: 17702-17711Crossref PubMed Scopus (464) Google Scholar). Trichohyalin expression, however, was unstable in the epidermis to be found only in the late stage of fetal skin development, suggesting that trichohyalin is a minor component in forming the epidermal CE. On the other hand, trichohyalin expression in hair follicles was stable without changes of expression pattern from 20 wk EGA, implying that a different mechanism exists to control trichohyalin expression between the epidermis and hair follicles. In contrast with the stratified epithelium of the mature epidermis, the periderm is a single cell layer, which may lead to a different mechanism in forming a barrier structure. For example, cultured human keratinocytes produce the fragile CE that differs markedly with the rigid CE of keratinocytes in vivo in amino acid composition primarily due to the absence of loricrin (Mehrel et al., 1990Mehrel T. Hohl D. Rothnagel J.A. et al.Identification of a major keratinocyte cell envelope protein, loricrin.Cell. 1990; 61: 1103-1112Abstract Full Text PDF PubMed Scopus (360) Google Scholar). Even in normal skin, there was intra- and inter-individual variations in peptide composition of the CE (Legrain et al., 1991Legrain V. Michel S. Ortonne J.P. Reichert U. Intra- and inter-individual variations of cornified envelope peptide composition in normal and psoriatic skin.Arch Dermatol Res. 1991; 283: 512-515Crossref Scopus (7) Google Scholar). Therefore, it is suggested that the periderm uses different sets of precursor proteins in forming a barrier structure by assembling the available precursors. A similar idea has been presented as a 'dust-bin hypothesis': the composition of the CE might be determined by available substrate proteins at the moment when TGase mediate cross-linking (Michel et al., 1987Michel S. Schmidt R. Robinson S. Shroot B. Reichert U. Identification and subcellular distribution of cornified envelope precursors proteins in the transformed human keratinocyte line SV-K14.J Invest Dermatol. 1987; 88: 301-305Abstract Full Text PDF PubMed Google Scholar;Hohl, 1990Hohl D. Cornified cell envelope.Dermatologica. 1990; 180: 201-211Crossref PubMed Scopus (164) Google Scholar;Reichert et al., 1993Reichert U. Michel S. Schmidt R. The cornified cell envelope: a key structure of terminally differentiating keratinocytes.in: Darmon M. Blumenberg M. Molecular Biology of the Skin. Academic Press Inc, London1993: 107-150Crossref Google Scholar). In this study, there was a drastic change in expression patterns of the precursor proteins during fetal development: (i) they were not identified in the first trimester; (ii) several precursor proteins are expressed from the early second trimester; and (iii) filaggrin and trichohyalin are expressed from the late second or early third trimester as a final stage. These results suggest that the immature CE is formed in the early second trimester, whereas the mature CE is formed in the late second or early third trimester in adult skin. Therefore, the periderm is not a functional barrier in the first trimester in that no identifiable precursors of the CE are present at this stage, but it becomes a functional barrier as the epidermis differentiates in the second trimester, supporting the previous report (Holbrook, 1991Holbrook K.A. Structure and function of the developing human skin.in: Goldsmith L.A. Physiology, Biochemistry, and Molecular Biology of the Skin. Oxford University Press, New York1991: 63-110Google Scholar). Our morphologic findings are consistent with previous biochemical results which show that an osmotic gradient develops between amniotic fluid and fetal and maternal plasmas from the second trimester (Breathnach, 1971Breathnach A.S. Embryology of human skin: a review of ultrastructural studies.J Invest Dermatol. 1971; 57: 133-143Abstract Full Text PDF PubMed Scopus (60) Google Scholar;Benzie et al., 1974Benzie R.J. Doran T.A. Harkins J.L. Owen V.M. Porter C.J. Composition of the amniotic fluid and maternal serum in pregnancy.Am J Obstet Gynecol. 1974; 119: 798-810Abstract Full Text PDF PubMed Scopus (72) Google Scholar). In summary, the precursor proteins of the CE initiates their expression from the early second trimester in a sequential order of involucrin, loricrin and SPRR1, and filaggrin and trichohyalin, which is similar to the sequential order of deposits in the CE of the postnatal mature epidermis. Therefore, fetal skin is a good model to investigate the dynamic process of terminal differentiation of skin. The antibodies to loricrin and trichohyalin were generous gifts from Dr. Steinert PM (N.I.H., MD). The antibody to SPRR1 was a generous gift from Dr. Kartasova T (N.I.H., MD). We are gratefully indebted to Dr. Soo-Il Chung for critically reading the manuscript, and to Mrs. In-Sook Choi for her skillful technical assistance. This work was supported in part by a grant from the Chonnam University Hospital Research Institute of Clinical Medicine (1995).