Portal de Periódicos da CAPES

Isolation and Characterization of a Putative Keratin-Associated Protein Gene Expressed in Embryonic Skin of Mice

1998; Elsevier BV; Volume: 111; Issue: 1 Linguagem: Inglês

10.1046/j.1523-1747.1998.00241.x

1523-1747

Mikiro Takaishi, Yoshimi Takata, Toshio Kuroki, Nam‐ho Huh,

Wnt/β-catenin signaling in development and cancer

Embryonic mouse skin undergoes substantial morphologic changes from 13 days post-coitus (dpc) to 16 dpc, i.e., from simple layers of epithelial cells and periderm at 13.5 dpc to almost fully differentiated stratified epithelium with the rudiments of hair follicles at 16.5 dpc. Using RNA differential display, we isolated a gene involved in the development of mouse epidermis. This gene, tentatively designated as 4C32, encodes 197 amino acids containing six direct repeats of 10 amino acids with the CQ motif. The repetitive structure with the CQ motif is seen in most keratin-associated protein families, which are known to be specifically expressed in hair follicles. 4C32 is expressed in the outermost layer of the embryonic epidermis at 15.5 and 16.5 dpc, and abruptly disappears at 17.5 dpc, suggesting that 4C32 is expressed in the periderm. The periderm is a superficial layer of embryonic epidermis, and is known to disappear at 17 dpc in mouse embryos. The 4C32 transcripts were also detected in the developing and matured tongue tissues and in the tail scale, but not at any stage in hair follicles. Embryonic mouse skin undergoes substantial morphologic changes from 13 days post-coitus (dpc) to 16 dpc, i.e., from simple layers of epithelial cells and periderm at 13.5 dpc to almost fully differentiated stratified epithelium with the rudiments of hair follicles at 16.5 dpc. Using RNA differential display, we isolated a gene involved in the development of mouse epidermis. This gene, tentatively designated as 4C32, encodes 197 amino acids containing six direct repeats of 10 amino acids with the CQ motif. The repetitive structure with the CQ motif is seen in most keratin-associated protein families, which are known to be specifically expressed in hair follicles. 4C32 is expressed in the outermost layer of the embryonic epidermis at 15.5 and 16.5 dpc, and abruptly disappears at 17.5 dpc, suggesting that 4C32 is expressed in the periderm. The periderm is a superficial layer of embryonic epidermis, and is known to disappear at 17 dpc in mouse embryos. The 4C32 transcripts were also detected in the developing and matured tongue tissues and in the tail scale, but not at any stage in hair follicles. days post-coitus keratin-associated protein Growth and differentiation of cells are strictly regulated, keeping tissue architecture in vivo. In skin, epidermal keratinocytes, among others, proliferate in the basal layer contacting directly to the dermal tissue via the basement membrane, progressively differentiate in supra basal spinous, granular, and horny layers, and finally slough off the uppermost horny layer. Such an elaborate control is mediated by diffusible agents as well as direct contact between cells or to matrix. Alteration of genes encoding regulatory or structural proteins leads to several disorders with abnormal differentiation and/or growth of cells, including epidermolytic hyperkeratosis (Cheng et al., 1992Cheng J. Syder A.J. Yu Q.C. Letai A. Paller A.S. Fuchs E. The genetic basis of epidermolytic hyperkeratosis: a disorder of differentiation-specific epidermal keratin genes.Cell. 1992; 70: 811-819Abstract Full Text PDF PubMed Scopus (274) Google Scholar;Chipev et al., 1992Chipev C.C. Korge B.P. Markova N. Bale S.J. DiGiovanna J.J. Compton J.G. Steinert P.M. A leucine Cheng J,. proline mutation in the H1 subdomain of keratin 1 causes epidermolytic hyperkeratosis.Cell. 1992; 70: 821-828Abstract Full Text PDF PubMed Scopus (244) Google Scholar;Compton et al., 1992Compton J.G. DiGiovanna J.J. Santucci S.K. et al.Linkage of epidermolytic hyperkeratosis to the type II keratin gene cluster on chromosome 12q.Nature Genet. 1992; 1: 301-305Crossref PubMed Scopus (87) Google Scholar), epidermolysis bullosa simplex (Bonifas et al., 1991Bonifas J.M. Rothman A.L. Epstein E.H. Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities.Science. 1991; 254: 1202-1205Crossref PubMed Scopus (330) Google Scholar;Coulombe et al., 1991Coulombe P.A. Hutton M.E. Letai A. Hebert A. Paller A.S. Fuchs E. Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses.Cell. 1991; 66: 1301-1311Abstract Full Text PDF PubMed Scopus (507) Google Scholar;Lane et al., 1992Lane E.B. Rugg E.L. Navsaria H. Leigh I.M. Heagerty A.H. Ishida-yamamoto A. Eady R.A. A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering.Nature. 1992; 356: 244-246Crossref PubMed Scopus (322) Google Scholar), ichthiotic disorders (Michels and Juhlin, 1990Michels S. Juhlin L. Cornified envelopes in congenital disorders of keratinization.Br J Dermatol. 1990; 122: 15-21Crossref Scopus (15) Google Scholar;Resing and Dale, 1991Resing K.A. Dale B.A. Proteins of keratohyalin.in: Goldsmith L.A. Physiology, Biochemistry, and Molecular Biology of the Skin. Oxford University of Press, New York1991: 148-167Google Scholar), and basal cell nevus syndrome (Hahn et al., 1996Hahn H. Wicking C. Zaphiropoulous P.G. et al.Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome.Cell. 1996; 85: 841-851Abstract Full Text Full Text PDF PubMed Scopus (1611) Google Scholar;Unden et al., 1997Unden A.B. Zaphiropoulos P.G. Bruce K. Toftgard R. Stahle-backdahl M. Human patched (PTCH) mRNA is overexpressed consistently in tumor cells of both familial and sporadic basal cell carcinoma.Cancer Res. 1997; 57: 2336-2340PubMed Google Scholar). Although considerable progress has thus far been made in identifying genes involved in skin diseases, a vast majority of genes involved in the control of growth and differentiation of epidermal cells remain unknown. One possible systematic approach may be to screen genes whose expression levels are controlled during development and differentiation. In this respect, we noted that embryonic mouse skin undergoes considerable morphologic changes from 13 days post-coitus (dpc) to 16 dpc (Sengel, 1976Sengel P. Morphogenesis of Skin. Cambridge University Press, New York1976Google Scholar;Kashiwagi et al., 1997Kashiwagi M. Kuroki T. Huh N. Specific inhibition of hair follicle formation by epidermal growth factor in an organ culture of developing mouse skin.Dev Biol. 1997; 189: 22-32Crossref PubMed Scopus (39) Google Scholar). At 13.5 dpc, the epidermis is composed of simple layers of epithelial cells and periderm. Periderm is a transitory embryonic tissue, and is present in the flank and foot-sole regions from 8 dpc to 17 dpc. From 14.5 dpc, the epidermis becomes stratified and progressively differentiates to form a keratinized layer by 16.5 dpc. Among approaches to isolate differentially expressed genes, we employed RNA differential display (Liang and Pardee, 1992Liang P. Pardee A.B. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction.Science. 1992; 257: 967-971Crossref PubMed Scopus (4631) Google Scholar), by which gene expression among tissues of different origin or during the process of development or differentiation can be compared. In this report, we describe a development-related gene by the use of RNA differential display applied to embryonic mouse epidermis from 12.5 to 16.5 dpc. Dorsal skin tissue was isolated from ICR mouse embryos (Nippon SLC, Shizuoka, Japan) of different gestational days under dissecting microscopy. When necessary, the epidermis was separated from the dermis by incubating in 10 mM ethylenediamine tetraacetic acid in phosphate-buffered saline for 0.5–2 h at 37°C. Total RNA was extracted by acid guanidinium thiocyanate-phenol-chloroform extraction (Chomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62289) Google Scholar) using 1 ml of the denaturing solution for 100 mg tissue. RNA differential display was performed using RNAmap kit (GenHunter, Brookline, MA) according to the manufacturer's protocol. Briefly, total RNA samples from dorsal epidermis of mouse embryos were reverse-transcribed using 5′-TTTTTTTTTTTTMC-3′ (T12MC: M = A or C or G) as a primer. The cDNA were amplified by polymerase chain reaction (PCR) using T12MC and 5′-GGTACTCCAC-3′ (AP4) as primers, four dNTP including [35S]dATP (Amersham, Arlington Heights, IL) and rTaq polymerase (Takara, Shiga, Japan). The PCR products were then separated on 6% denaturing polyacrylamide gel, transferred onto 3 MM filter paper (Whatman, Maidstone, U.K.), and autoradiographed. Differentially expressed DNA fragments were cut out, extracted with water, and re-amplified under similar conditions to the first PCR except for using cold dATP. After confirming the sizes by agarose gel electrophoresis, the amplified fragments were subcloned into pCRII vector (Original TA Cloning Kit; Invitrogen, NV Leek, The Netherlands) and at least five independent clones per original DNA fragment were sequenced by automated sequencer (Pharmacia, Uppsala, Sweden). The resulting sequences were used to search for homology with genes registered in GenBank by BLAST. Northern analysis was performed according to conventional conditions (Ausubel et al., 1994Ausubel F. Brent R. Kingston R. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates & Wiley-Interscience, New York1994Google Scholar). Briefly, 20 μg of total RNA was electrophoresed on 1.5% agarose/formalin gel, transferred onto Hybond-N (Amersham). The filters were finally washed twice with 0.2×sodium citrate/chloride buffer, 0.1% sodium dodecyl sulfate, at 43°C for 15 min, and the signals were visualized by exposing to X-ray films. The 4C32 probe was obtained from the RNA differential display. Probes for cytokeratin K10, K14, and loricrin were obtained by reverse transcriptase PCR. A probe of glyceraldehyde-3-phosphate dehydrogenase, a constitutively expressed gene, was provided by Dr. K. Nose (Showa University, Tokyo) and used as a control for applied amounts of RNA. A cDNA library was prepared from 16.5 dpc embryonic mouse skin. Poly(A) + RNA was enriched by Oligotex-dT30 (Takara, Shiga, Japan), reverse-transcribed using random hexamers, and integrated into λZAP II (Stratagene, La Jolla, CA) under the recommended conditions by the manufacturer. After screening ≈5 × 105 independent plaques of the cDNA library with32P-labeled probe, positive clones were transformed to pBluescript by in vivo excision, and sequenced as above. A pLITMUS 28 vector (New England Biolabs, Beverly, MA) containing an ≈350 bp fragment of the 3′-untranslated region of 4C32 was linealized either with AflII or with SpeI, and anti-sense or sense RNA probe was synthesized with T7 RNA polymerase (Gibco BRL, Rockville, MD). The ribo-probes were labeled with digoxigenin using DIG-UTP (Boehringer, Mannheim, Germany). Fragments of cytokeratin K10, K14, and loricrin obtained by reverse transcriptase PCR were subcloned into pLITMUS 28 vector, and RNA probes were prepared as above. In situ hybridization was performed as described previously (Nomura et al., 1988Nomura S. Wills A.J. Edwards D.R. Heath J.K. Hogen B.L.M. Developmental expression of 2ar (osteopontin) and SPARC (osteonectin) RNA as revealed by in situ hybridization.J Cell Biol. 1988; 106: 441-450Crossref PubMed Scopus (445) Google Scholar). Mouse embryos at 16.5 dpc were fixed in 4% paraformaldehyde/phosphate-buffered saline at 4°C for 36 h and processed to paraffin sections. The tissue sections were hybridized with the probes, and specific signals were visualized as alkaline phosphatase activity conjugated to anti-digoxigenin antibody (Boehringer). Among several thousand DNA fragments amplified by PCR using 20 combinations of the primers, we identified 20 and 18 cDNA fragments whose band intensity increased and decreased, respectively, during the progression from 12.5 dpc to 16.5 dpc. Of these clones, the six and two cDNA fragments were confirmed by northern analysis to be expressed in increasing and decreasing manners with development, respectively. One of the clones with increasing expression, tentatively named as 4C32 (Figure 1), was used for cDNA cloning. About 5 × 105 independent plaques of the cDNA library prepared from embryonic ICR mouse skin were screened with the 4C32 fragment as a probe. Among more than 100 of positive plaques, 10 clones were isolated after three rounds of purification for further characterization. The nucleotide sequence of 4C32 cDNA is shown in Figure 2(a). The size of the transcript is estimated to be ≈900 bp plus poly(A) + tail. Only one reasonably long open reading frame of 594 nucleotides, encoding 197 amino acids, was identified. The putative initiation codon is shown in bold, whose surrounding nucleotide sequence principally meets Kozak's rule (Kozak, 1987Kozak M. An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs.Nucl Acid Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4103) Google Scholar). Furthermore, we have isolated a genomic clone of 4C32, and found a TATA box at 79 bp upstream and an in-frame stop codon at 87 bp upstream from the putative initiation codon (data not shown). The amino acid sequence of 4C32 shows moderate homology with some of the keratin associated proteins in hair follicles, such as ultra-high sulfur keratins (Wood et al., 1990Wood L. Mills M. Hatzenbuhler N. Vogeli G. Serine-rich ultra high sulfur protein gene expression in murine hair and skin during the hair cycle.J Biol Chem. 1990; 265: 21375-21380Abstract Full Text PDF PubMed Google Scholar), cysteine-rich hair keratin-associated protein (Powell et al., 1995Powell B.C. Arthur J. Nesci A. Characterization of a gene encoding a cysteine-rich keratin associated protein synthesized late in rabbit hair follicle differentiation.Differentiation. 1995; 58: 227-232Crossref PubMed Scopus (20) Google Scholar), and high sulfur matrix proteins (Rogers et al., 1994Rogers G.R. Hickford J.G. Bickerstaffe R. Polymorphism in two genes for B2 high sulfur proteins of wool.Anim Genet. 1994; 25: 407-415Crossref PubMed Scopus (57) Google Scholar). A more salient feature of 4C32 is the presence of repeated sequence. As underlined in Figure 2(a), 4C32 has six tandem repeats of 10 amino acids in the N-terminal side. Among the six repeats, the amino acid sequence in the middle four repetitive units is the best conserved, the consensus being CQES/TCIEPIR/S (Figure 2b). The repeated sequence is commonly seen in hair follicle-specific sheep high-sulfur matrix proteins (Rogers et al., 1994Rogers G.R. Hickford J.G. Bickerstaffe R. Polymorphism in two genes for B2 high sulfur proteins of wool.Anim Genet. 1994; 25: 407-415Crossref PubMed Scopus (57) Google Scholar) and mouse hacle-1 gene (Huh et al., 1994Huh N. Kashiwagi M. Konishi C. Hashimoto Y. Kohno Y. Nomura S. Kuroki T. Isolation and characterization of a novel hair follicle-specific gene, hacl-1.J Invest Dermatol. 1994; 102: 716-720Abstract Full Text PDF PubMed Google Scholar). From the result of RNA display, 4C32 was expected to be expressed from 15.5 dpc. As shown in Figure 3, the transcript of 4C32 was detected by northern analysis in the epidermis of 15.5 dpc and became more abundant at 16.5 dpc, but not in the epidermis of 13.5 and 14.5 dpc, nor in dermis at any stage. The size of the transcript was estimated to be a little shorter than 1 kb. Loricrin, a major component of cornified envelope (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), was expressed in a very similar manner to 4C32. Cytokeratin K10 transcripts were first detected on 14.5 dpc and its level increased progressively, whereas the expression of cytokeratin K14 remained unchanged throughout the period. In the matured epidermis, cytokeratin K14 is expressed in the basal layer (Fuchs, 1995Fuchs E. Keratins and the skin.Annu Rev Cell Dev Biol. 1995; 11: 123-153Crossref PubMed Google Scholar), K10 in supra basal layers (Fuchs, 1995Fuchs E. Keratins and the skin.Annu Rev Cell Dev Biol. 1995; 11: 123-153Crossref PubMed Google Scholar), and loricrin in the granular and horny layers (Hohl et al., 1993Hohl D. Ruf Olano B. de Viragh P.A. Huber M. Detrisac C.J. Schnyder U.W. Roop D.R. Expression patterns of loricrin in various species and tissues.Differentiation. 1993; 54: 25-34Crossref PubMed Scopus (62) Google Scholar). To examine the expression of 4C32 in the later developmental stages, we performed northern analysis using RNA from skin at different ages (Figure 4). 4C32 was detected only in the RNA from 16.5 dpc skin, but not in those from 17.5 dpc, 18.5 dpc, 1 d, 3 d, and 1 wk old mouse skin (Figure 4a). On the contrary, loricrin was expressed continuously during this period (Figure 4b). Apparent reduction of the band intensity of loricrin relative to that of glyceraldehyde-3-phosphate dehydrogenase at the later stages possibly reflects the change of cell populations associated with tissue remodeling during the development. The location of 4C32 expression in whole embryo at 16.5 dpc was examined by in situ hybridization using the 3′-untranslated region of 4C32 cDNA as a probe. As shown in Figure 5(a), the expression of 4C32 was observed in the uppermost layer of the embryonic epidermis. Figure 6 shows the transitional zone from skin to oral mucosa of the lower lip at 16.5 dpc. 4C32 is expressed in the outermost layer of skin, but not in the oral mucosa (Figure 6b). Loricrin (Figure 6c) and cytokeratin K10 (Figure 6d) are also expressed only in the skin region, contrary to cytokeratin K13 whose transcripts were detected in oral mucosa (Figure 6f). Cytokeratin K14 is expressed in both of the regions, although only in the basal layer in the skin, but in all the layers in the oral epithelium (Figure 5e). In accordance with the result by northern blotting, the transcripts of 4C32 were not detected in adult epidermis, nor in hair follicles (data not shown).Figure 64C32 is expressed in skin, but not in oral mucosa. Serial sections were made from the lower lip of 16.5 dpc ICR mouse embryo, and stained with hematoxylin and eosin (a) or hybridized with anti-sense RNA probe of 4C32 (b), loricrin (c), cytokeratin K10 (d), K14 (e), and K13 (f). Hybridization with corresponding sense probes was performed in parallel, giving consistently negative signals. Scale bar, 100 μm.View Large Image Figure ViewerDownload (PPT) 4C32 was expressed in the dorsal epithelium of the tongue of 16.5 dpc embryo (Figure 5b). In contrast to the epidermis, the region with positive 4C32 expression in the tongue is not continuous, but intermitted and dotted on the surface. This is more clearly seen in the adult tongue (Figure 5c), where the 4C32 transcript was detected asymmetrically in filiform papillae with stronger signals on the surface at the posterior side. This asymmetrical expression of 4C32 was also observed in the scale epithelium of the adult tail (Figure 5d), with positive signals only at the anterior slope. 4C32 was not expressed in any internal organs, including the esophagus and forestomach (data not shown). For a better understanding of skin biology and the development of fundamental measures against skin diseases, it is essential to elucidate underlying biologic phenomena with molecular terms, including the identification and characterization of the genes involved. Systematic screening of genes involved in the regulation of cell growth/differentiation is promising for this goal. This study was aimed at isolating genes whose expression levels change during the morphogenic process of mouse embryonic skin by the use of RNA differential display. After a partial screening, we identified eight as yet unknown genes, one of which, 4C32, is described in this paper. 4C32 showed moderate amino acid sequence homology with some keratin-associated proteins (KAP), which are best studied in sheep and comprise two big families rich in either cysteine or glycine/tyrosine (Powell et al., 1991Powell B.C. Nesci A. Rogers G.E. Regulation of keratin gene expression in hair follicle differentiation.Ann NY Acad Sci USA. 1991; 642: 1-20Crossref PubMed Scopus (73) Google Scholar;Fratini et al., 1993Fratini A. Powell B.C. Rogers G.E. Hair follicle differentiation: sequence and expression of a gene encoding a glycine/tyrosine-rich protein.J Biol Chem. 1993; 268: 4511-4518Abstract Full Text PDF PubMed Google Scholar;Rogers and Powell, 1993Rogers G.R. Powell B.C. Organization and expression of hair follicle genes.J Invest Dermotol. 1993; 101: 50S-55SAbstract Full Text PDF PubMed Google Scholar). Several corresponding family members were identified in humans (MacKinnon et al., 1990MacKinnon P.J. Powell B.C. Rogers G.E. Structure and expression of genes for a class of cysteine-rich proteins of the cuticle layers of differentiating wool and hair follicles.J Cell Biol. 1990; 111: 2587-2600Crossref PubMed Scopus (70) Google Scholar;Zhumabaeva et al., 1992Zhumabaeva B.D. Gening L.V. Gazaryan K.G. Cloning and structural characterization of human hair sulfur-rich keratin genes.Mol Biol. 1992; 26: 550-555Google Scholar) and in mice (McNab et al., 1989McNab A.R. Wood L. Theriault N. Gierman T. Vogeli G. An ultra-high sulfur keratin gene is expressed specifically during hair growth.J Invest Dermatol. 1989; 92: 263-266Abstract Full Text PDF PubMed Google Scholar;Wood et al., 1990Wood L. Mills M. Hatzenbuhler N. Vogeli G. Serine-rich ultra high sulfur protein gene expression in murine hair and skin during the hair cycle.J Biol Chem. 1990; 265: 21375-21380Abstract Full Text PDF PubMed Google Scholar). We previously reported an additional member of the KAP family, which is expressed specifically in the hair cortex of mouse (Huh et al., 1994Huh N. Kashiwagi M. Konishi C. Hashimoto Y. Kohno Y. Nomura S. Kuroki T. Isolation and characterization of a novel hair follicle-specific gene, hacl-1.J Invest Dermatol. 1994; 102: 716-720Abstract Full Text PDF PubMed Google Scholar). 4C32 has six direct repeats of 10 amino acids containing the CQ motif conserved perfectly among the repetitive units (Figure 2b). Most of the reported KAP have a varying number of the characteristic 10 amino acid repeats containing the CQ motif. For example, sheep high-sulfur matrix protein B2A, B2B, B2C, and B2D have five, four, three, and six repeats, respectively (Powell et al., 1983Powell B.C. Sleigh M.J. Ward K.A. Rogers G.E. Mammalian keratin gene families: organisation of genes coding for the B2 high-sulphur proteins of sheep wool.Nucl Acids Res. 1983; 11: 5327-5346Crossref PubMed Scopus (60) Google Scholar;Rogers et al., 1994Rogers G.R. Hickford J.G. Bickerstaffe R. Polymorphism in two genes for B2 high sulfur proteins of wool.Anim Genet. 1994; 25: 407-415Crossref PubMed Scopus (57) Google Scholar). The sequence homology and the common characteristic repeats between 4C32 and the KAP indicate that 4C32 belongs to the KAP family. These KAP are expressed exclusively in the hair follicle, with some exceptional concomitant expression in the epidermis (Wood et al., 1990Wood L. Mills M. Hatzenbuhler N. Vogeli G. Serine-rich ultra high sulfur protein gene expression in murine hair and skin during the hair cycle.J Biol Chem. 1990; 265: 21375-21380Abstract Full Text PDF PubMed Google Scholar). KAP are considered to be involved in forming the rigid structure of the hair shaft with hair-specific keratins and trichohyalin (Rogers and Powell, 1993Rogers G.R. Powell B.C. Organization and expression of hair follicle genes.J Invest Dermotol. 1993; 101: 50S-55SAbstract Full Text PDF PubMed Google Scholar). The transcripts of 4C32 were not found in the hair follicles, nor in the matured epidermis, but were detected in the epidermis at 16.5 dpc, in the tongue, and the scales of the tail (Figures 4, 5). This suggests that KAP-like proteins, such as 4C32, are necessary also in some tissues other than hair follicles. 4C32 may contribute to form a rigid cytoskelton and keep cell shapes by interacting with cytokeratins. In this respect, it is noteworthy that mouse small proline-rich proteins, a component of the cornified envelope of epidermal keratinocytes (Steinert and Marekov, 1995Steinert P. Marekov L.N. Thr proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and SPRs are isopeptide cross-linked components of the human epidermal cornified envelope.J Biol Chem. 1995; 270: 17702-17711Crossref PubMed Scopus (464) Google Scholar), have 13 or seven tandem repeats of eight amino acids with the CQP motif (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). This structural feature of small proline-rich protein is conserved among different species, e.g., pig, rabbit, monkey, and humans (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 tandem amino acid repeats with the CQ motif may contribute to form protein–protein cross-links including disulfide bond and Nε-(γ-glutamyl)lysine isodipeptide bond catalyzed by transglutaminase. In the epidermis, the transcripts of 4C32 were detected only in the outermost layer at 16.5 dpc (Figure 5a), and completely disappeared from 17.5 dpc (Figure 4). This suggests that 4C32 is expressed in periderm, which appears from the early embryonic development of 8.5 dpc and is eliminated at 17.5 dpc (Sengel, 1976Sengel P. Morphogenesis of Skin. Cambridge University Press, New York1976Google Scholar). Though not fully characterized, the periderm cells are considered to cease proliferation at 14.5 dpc and to undergo some type of terminal differentiation (Sengel, 1976Sengel P. Morphogenesis of Skin. Cambridge University Press, New York1976Google Scholar). Expression of 4C32 is possibly induced in accordance with the differentiation of periderm. An intriguing question remains to be answered, i.e., what kind of common molecular processes the periderm, the scale of tail, and the tongue share. The hair-specific KAP comprise a large gene family. Considering the possibility that 4C32 may comprise its own subfamily, we performed genomic southern analysis using either the coding region or the 3′-untranslated region of 4C32 as a probe. Multiple bands were detected with the coding region probe, but not with the more specific 3′-probe (data not shown), suggesting the presence of a possible subfamily. Actually, we have already isolated several distinct cDNA clones from the cDNA library prepared from 16.5 dpc embryonic mouse skin, and some genomic clones of mouse. Using a mixture of the cDNA clones, we identified several human genomic clones as well. Characterization and functional analysis of these clones will reveal the involvement of KAP-like proteins in the differentiation of various tissues, not only in mice but also in humans. This work was supported in part by Grand-in-Aid from the Ministry of Education, Science and Culture of Japan, and by a Research Grand from the Mitsubishi Foundation and Shiseido Foundation for Skin Aging Research.