The type I keratin 17 (K17) shows a peculiar localization in human epithelial appendages including hair follicles, which undergo a growth cycle throughout adult life. Additionally K17 is induced, along with K6 and K16, early after acute injury to human skin. To gain further insights into its potential function(s), we cloned the mouse K17 gene and investigated its expression during skin development. Synthesis of K17 protein first occurs in a subset of epithelial cells within the single-layered, undifferentiated ectoderm of embryonic day 10.5 mouse fetuses. In the ensuing 48 h, K17-expressing cells give rise to placodes, the precursors of ectoderm-derived appendages (hair, glands, and tooth), and to periderm. During early development, there is a spatial correspondence in the distribution of K17 and that of lymphoid-enhancer factor (lef-1), a DNA-bending protein involved in inductive epithelial–mesenchymal interactions. We demonstrate that ectopic lef-1 expression induces K17 protein in the skin of adult transgenic mice. The pattern of K17 gene expression during development has direct implications for the morphogenesis of skin epithelia, and points to the existence of a molecular relationship between development and wound repair.

In spite of a recent revolution in the discovery of novel genes, the most reliable criterium to define the epithelial nature of a cell remains keratin gene expression. The pattern of keratin protein synthesis provides a useful tool for studying progression through complex programs of epithelial differentiation and cycles of epithelial self-renewal (Moll et al., 1982; O'Guin et al., 1990; Byrne et al., 1994). Keratins belong the superfamily of intermediate filament (IF)1 proteins. There are in excess of 35 keratin genes, and these can be partitioned into two distinct sequence types, I and II, based their genomic structure and nucleotide sequence homology (Fuchs and Weber, 1994). Most keratin genes, including the type II K1–K8 and the type I K9–K20, are expressed in soft epithelia (Moll et al., 1982; O'Guin et al., 1990). In addition, a group of seven type I (Ha1–Ha7) and six type II (Hb1–Hb6) keratin genes are expressed in hard epithelia such as hair and nail (Rogers et al., 1997; Winter et al., 1997). Epithelial cells coordinately regulate the expression of at least one type I and one type II gene, reflecting the requirement for both types of keratin proteins in a 1:1 molar ratio to sustain the assembly of 10-nm filaments (Coulombe, 1993). Many type I and type II keratin genes are de facto regulated in a pairwise fashion and in an epithelial tissue type- and differentiation-specific manner, even though any combination of type I and type II proteins can produce a filamentous polymer in vitro (Hatzfeld and Franke, 1985). The notions of sequence diversity, pairwise regulation, and differentiation-specific expression of keratin genes have largely been conserved throughout evolution, suggesting the existence of a functional link between keratin proteins and the diversity of structure and function of epithelial tissues.

Our laboratory is interested in the regulation and function of three keratin genes, the type II K6 and the type I K16 and K17, which cannot be readily associated with a well-defined epithelial context (O'Guin et al., 1990). These genes show an analogous pattern of expression in human skin that includes a constitutive expression in the hair follicle, sporadic expression in the thick epidermis of palm and sole skin, but no expression in interfollicular epidermis (Moll et al., 1983; Troyanovsky et al., 1989). K17 is also expressed in the myoepithelium surrounding secretory epithelial cells in many glandular tissues (Troyanovsky et al., 1989). Moreover, K6, K16, and K17 are induced under conditions associated with enhanced proliferation and abnormal differentiation, such as viral infection, inflammation, psoriasis, carcinoma, and after acute injury to various stratified epithelia (Weiss et al., 1984; Stoler et al., 1988; Jiang et al., 1994; Leigh et al., 1995; Paladini et al., 1996; McGowan and Coulombe, 1998). Although its functional significance has not yet been established, the accumulation of K6, K16, and K17 occurs at the expense of the keratins normally expressed under basal conditions and correlates with major changes in epithelial cytoarchitecture (Paladini et al., 1996; Coulombe, 1997; McGowan and Coulombe, 1998).

In comparison with the other wound-induced keratins, K6 and K16, relatively little is known about the significance of K17 gene expression and the properties and function(s) of K17 protein. To gain some insight into this question we wanted to examine K17 expression in a context potentially related to wound repair, epidermal development. Whereas one functional human 17 gene and two pseudogenes have been characterized (Troyanovsky et al., 1992), only a partial cDNA is available for mouse K17 (Knapp et al., 1987). To facilitate our studies we cloned the full-length gene for mouse K17 and examined its expression by producing a high-titer monospecific antiserum. We discovered that K17 is expressed at an early stage of skin morphogenesis according to a complex spatial pattern, and that the transcription lymphoid-enhancer factor (lef-1) is likely to play a role in its regulation. Our data led us to propose a revised model for skin morphogenesis, and suggest the existence of a molecular relationship between development and the wound repair response in adult skin epithelia.

Materials And Methods

Materials

Materials were obtained from the following sources: 129SvJ mouse genomic library packaged into Lambda Fix II and pBluescript vector from Stratagene (La Jolla, CA); restriction endonucleases and DNA modifying enzymes from New England Biolabs (Beverly, MA); Superscript II reverse transcriptase from Life Technologies (Grand Island, NY); Nytran membranes from Schleicher & Schuell (Keene, NH); Hybond-N filters from Amersham Corp. (Arlington Heights, IL); phage and plasmid DNA purification kits from QIAGEN Corp. (Santa Clarita, CA); LA-Taq DNA polymerase from Panvera Corp. (Madison, WI); alkaline-phosphatase antibody detection kit from Bio-Rad Laboratories (Hercules, CA). All other chemicals were reagent grade and were typically obtained from Sigma Chemical Co. (St. Louis, MO).

Animal Protocols

All studies involving animals were reviewed by the Johns Hopkins University Animal Use and Care Committee (Baltimore, MD). For studies involving experimental injury adult mice were anesthetized with avertin and their backs were epilated with Nair cream (Carter-Wallace Inc., New York, NY). A surgical area was disinfected and full thickness skin wounds were made with a 4-mm punch (AcuPunch; Acuderm Inc., Ft. Lauderdale, FL). The injured area was cleaned and covered with anti-bacterial ointment until the time of killing. For developmental studies, mouse embryos were isolated from timed pregnancies of B6C3F1/J mice (Jackson Laboratory, Bar Harbor, ME). Individual matings were initiated at 6:00 pm and monitored the next morning at 8:00 am. At this time the males were removed and the females were examined for the appearance of a vaginal plug. If copulatory plugs were detected, the date was recorded and the pregnancy was timed using 12 noon as embryonic day (e) = 0.5 d (Kaufman, 1992). On days 8, 9, 10, 12, 14, 16, and 18 at 12:00 noon, the embryos were isolated and fixed in either Bouin's fixative for subsequent embedding in paraffin or frozen in OCT Compound (Tissue-Tek; Sakura Finetek, Torrance, CA) for preparation of cryosections. Additional tissues from lef-1 overexpressing and lef-1–null mice were obtained from E. Fuchs (University of Chicago, Chicago, IL) (Zhou et al., 1995) and R. Grosschedl (University of California, San Francisco, CA) (van Genderen et al., 1994), respectively.

RNA Isolation

RNA samples were obtained from control and experimentally wounded mouse back skin using guanidine thioisocyanate (GTC)-phenol extraction method (Bessho et al., 1993). The wounded tissue samples were extracted 72 h after experimental injury to enhance the abundance of wound-induced keratin gene mRNAs.

Immunological Analyses

For immunoblot analyses, known quantities of recombinant human keratins were electrophoresed, transferred to nitrocellulose, and the blots were incubated with primary antisera diluted in blocking buffer (Tris-buffered saline with 0.5% Tween 20 and 5% powdered milk). Bound primary antibodies were revealed by alkaline phosphatase–conjugated secondary antibodies as recommended by the manufacturer (Bio-Rad Laboratories). The primary antisera for K17 and K6 were prepared as described below. Immunohistochemical analyses were performed on 5-μm sections prepared from either paraffin-embedded or fresh-frozen tissues. The sections were first reacted with the primary antisera and then revealed with either a peroxidase-based reaction (Kirkegaard and Perry Laboratories, Gaithersburg, MD) or by indirect immunofluorescence (Jackson Immunological Reagents, West Grove, PA). In addition to the antisera mentioned above, other primary antisera used in these studies were: a guinea pig anti-K8/18 purchased from ARP laboratories (Belmont, MA); a mouse monoclonal anti-K10 (clone K8.60) from Sigma Chemical Co.; a mouse monoclonal anti-K14 (LL001) donated by I. Leigh (London Hospital Medical College, London, UK) (Purkis et al., 1990); and a rabbit anti–lef-1 donated by R. Grosschedl (van Genderen et al., 1994). Double-immunofluorescence staining using primary antibodies from the same species (rabbit anti-K17 and rabbit anti–lef-1) was performed as described (Lewis-Carl et al., 1993).

Screening the Mouse Genomic Library

Approximately 1.5 d × 106 phage clones were screened with [α-32P]dCTP radiolabeled probes. The initial round of screening was performed using two probes. The first probe was derived from the rod portion of the published human keratin 16 cDNA (Paladini et al., 1995). This domain is highly conserved among the type I keratin sequences, particularly K14, K16, and K17. The second probe was generated by reverse transcription (RT)-PCR using RNA isolated from mouse back skin 72 h after experimental wounding as described (Takahashi et al., 1998). The oligonucleotides were based on the partial mouse K17 cDNA sequence published by Knapp and colleagues (Knapp et al., 1987) and amplified a 700-bp fragment containing the COOH terminus and 3′ untranslated regions of this mRNA. The strategy was to use the first probe as a means of identifying all the type I keratin genes contained in the library and then apply the K17-specific probe on a duplicate set of filters to identify the K17 containing phages. Hybridization and washes were carried out as suggested by the manufacturer (Stratagene). Positive colonies were isolated by repeated plaque purification as described (Sambrook et al., 1989). Phage DNA was extracted and analyzed by restriction enzyme digestion and Southern blotting. DNA fragments expected to contain expressed regions of the K17 gene were subcloned in pBluescript KS+ and sequenced. DNA sequencing and reconstruction of mouse keratin 17 gene locus was performed with the assistance of the Johns Hopkins University DNA Sequencing Core Facility (Baltimore, MD).

Mouse K17 cDNA Clone

A cDNA clone containing the entire coding sequence of K17 was obtained via RT-PCR with oligonucleotides derived from the SvJ129 genomic sequence using RNA from experimentally wounded back skin of a SvJ129 mouse. The primers used were: forward primer, 5′-ACGTGGATCCCGCTGCCACCATGACCACCA-3′; and reverse primer, 5′-GCTGTAGCAGGAGGGTGATGCCGGAGCGGA-3′.

The PCR conditions applied were 94°C for 1 min (denaturation); 55°C for 1 min (annealing), and 72°C for 2 min (elongation) for 25 cycles. The 1.3-kb product obtained (as expected) was subcloned in pGEM-T (Promega Corp., Madison, WI) and subjected to DNA sequence analysis as described above. Sense and anti-sense probes corresponding to the 3′-untranslated region-specific probe were also generated and used by Carroll et al. (1997) for in situ analysis of K17 mRNA expression.

Antibody Production and Characterization

Synthetic peptides (see below) were conjugated to maleimide-activated keyhole limpet hemocyanin carrier as described by the manufacturer (Pierce Chemical Co., Rockford, IL) and used for the production of monospecic anti-keratin polyclonal antisera in rabbits according to standard procedures (Covance Research Products, Denver, PA). A 12-mer peptide, NH2-C-S-S-R-E-Q-V-H-Q-T-T-R-COOH, was used for the production of an antiserum to K17 (anti-K17), and a 9-mer peptide, NH2-C-S-S-T-I-K-Y-T-T-COOH, was used for the production of an antiserum that recognizes all K6 isoforms (anti-K6gen; Takahashi et al., 1998). Bleeds were tested with the immunoblot procedures described above using recombinant keratin proteins prepared as described (Takahashi et al., 1994; Wawersik et al., 1997).

Results

Isolation and Characterization of Genomic and cDNA Clones for Mouse K17

We screened 106 clones from a 129 SvJ mouse genomic library (average insert size: 15–20 kb). An initial pool of 31 phage clones was established in which two clones (designated 16-9 and 16-61) were found to hybridize strongly with a K17-specific probe. Further analyses on these two clones revealed that they contain overlapping inserts that included the 3′ portion of a type I keratin gene whose coding and 3′ noncoding sequences were identical to the previously reported partial K17 cDNA (Knapp et al., 1987). Genomic walking yielded a third phage, designated 16-8, which contained the 5′ portion of this gene. DNA sequence analyses revealed the presence of a 925 nucleotide overlap between the genomic inserts contained in phages 16-8 and 16-9. The contiguity of these two inserts in 129 SvJ mouse genomic DNA was confirmed by a PCR experiment (data not shown).

The nucleotide sequence of all exons and intron–exon junctions, 5′ upstream and 3′ noncoding sequences was determined for the candidate K17 genomic clone (see Fig. 2). The boundaries of the functional elements in the gene isolated were established via comparison with the previously cloned human K17 gene (Troyanovsky et al., 1992) and a near full-length mouse K17 cDNA clone (see below). The candidate mouse K17 gene contains eight exons interrupted by seven introns (1–7). The sequence of the exon– intron boundaries (Fig. 2) conforms to the consensus splicing signals (Shapiro and Senapathy, 1987). All introns occur within the protein coding sequence (Figs. 1 and 2) at positions that are perfectly conserved in all type I keratin genes, including human K17 (Troyanovsky et al., 1992).

Oligonucleotide primers spanning the putative translation start codon (forward) and 3′ non-translated region (reverse) in the candidate K17 gene were used to obtain an ∼1.3-kb cDNA clone by RT-PCR. The nucleotide sequence of the cDNA clone is identical to the open reading frame contained within exons 1–8 in the candidate K17 genomic sequence and to the previously reported mouse partial K17 cDNA (data not shown) (Fig. 2). Together with the distribution of the corresponding keratin protein observed via immunohistochemistry (see below), this led us to conclude that the gene and cDNA that we have isolated encode mouse keratin 17.

An alignment of the cDNA and genomic sequences is shown diagramatically in Fig. 1. As for other keratin genes there is no obvious correspondence between the placement of introns and the predicted protein domains. The 5′ leader, the entire head domain and the 1A subdomain of the rod are encoded by exon 1, the largest of the eight exons (Table I). Exons 2–6 code for nearly all the remaining portions of the postulated α-helical rod domain including linkers, the exception being the last residue of subdomain 2B at the COOH terminus. The very short exon 7 codes for this last residue and the first eight amino acids of the nonhelical tail domain. Finally, the remainder of the tail domain (31 amino acids) and the entire 3′ nontranslated region are encoded by exon 8.

Inspection of the 5′ upstream sequence from our cloned sequence revealed the presence of a TATAA motif located 95 bases upstream of a potential translation start site with the consensus sequence defined by Kozak (1987) (Fig. 2). Inspection of the proximal 5′ upstream sequence revealed the presence of consensus binding sites for several known transcription factors (Fig. 2). These include AP-2, which has been shown to concentrate at sites of future hair follicle formation during epidermal development (Byrne et al., 1994), and several NF-IL-6(C/EBP-β) and GAS sites, which are involved in the upregulation gene expression during inflammation and viral infection as previously discussed for the human K17 gene (Proby et al., 1993; Jiang et al., 1994; Troyanovsky and Leube, 1994; Bonnekoh et al., 1995; Vogel et al., 1995; Komine et al., 1996). A detailed analysis of the complete K17 promoter sequence will be reported elsewhere.

The Human and Mouse K17 Gene and Predicted Protein Sequences Are Highly Conserved

Table I compares various functional elements from the mouse (this study) and human K17 gene (Troyanovsky et al., 1992). At ∼4.8 kb in total length, the mouse gene is slightly shorter than its human orthologue, due primarily to shorter introns 1 and 2. Whereas exon 8 is shorter in mouse at the level of the 3′ noncoding portion, exons 1–7 are of identical size between mouse and human (Table I). As a result, the K17 protein is predicted to be of similar size in human and mouse (see below). As illustrated in Fig. 1, the mouse K17 gene is flanked by a K17-like gene on its 5′ side and the K16 gene on the 3′ side. A characterization of the latter will be reported elsewhere (McGowan, K.M., K.M. Hess, and P.A. Coulombe, manuscript in preparation). Of note, Troyanovsky et al. (1992) showed that the functional human K17 gene is flanked by a K17 pseudogene on its 5′ side and the functional K16 gene on its 3′ side, all of which reside within the cluster of type I keratin genes on chromosome 17q. It appears likely that these keratin genes reside on chromosome 11q in mouse, since it is syntenic with human chromosome 17q and contains a cluster of type I genes (Nadeau et al., 1989).

An alignment of the predicted amino acid sequences of mouse and human K17, as well as human K14 and K16, is shown on Fig. 3. The mouse sequence (433 amino acids) is longer than human K17 by one amino acid (Table I) due to the insertion of a Pro residue at position 400 within the tail domain (Fig. 3). The primary sequence of the head, rod, and tail domains of mouse and human K17 show 88% (73 out of 83 residues), 96% (302 of 312 residues), and 97% identity (37 of 38 residues), respectively. Most of the amino acid substitutions between mouse and human K17 are conservative in nature, and four of them render mouse K17 identical to human K14 (see residues marked by # in Fig. 4). This exceptionally high degree of sequence relatedness between mouse and human K17 suggests, when interpreted in light of their highly conserved regulation (see below), that a K17 ancestral gene was present before the divergence of these species during evolution, and that the function(s) of the protein is conserved.

As pointed out for the human sequences (Troyanovsky et al., 1992), mouse K17 is more related to K14 (73% identity) than to K16 (66% identity), due primarily to a better conservation of the tail domains in K17 and K14 (Fig. 3). In addition, the central rod domain of mouse K17 is more related to that of human K14 (88% identity) than to human K16 (81% identity). We recently showed that unlike K16, human K14 and K17 are capable of forming very stable heterotetramers with a variety of type II keratin partners, including K5, K6, and K8 (Wawersik et al., 1997).

Expression of K17 in Intact and Wounded Adult Mouse Trunk Skin

The commercially available antiserum against K17 (monoclonal; Sigma Chemical Co.) has a low titer and does not react with mouse K17 (our unpublished data). This prompted us to produce a polyclonal antiserum in rabbits using a hapten-conjugated 11-mer peptide that is perfectly conserved at the COOH terminus of human and mouse K17, and which is distinct from the related K14 sequence (Fig. 3). When used for Western blotting at a high dilution (1:10,000), the rabbit antiserum does not react with K14 and K16, but reacts strongly with purified recombinant human K17 (Fig. 4,A). In total insoluble proteins extracted from mouse trunk skin, the rabbit antiserum recognizes a single antigen with an apparent Mr of 48 kD (Fig. 4,B) which comigrates with human K17 under standard gel buffer conditions (Fig. 4). This reactivity is not seen with the preimmune serum from the same rabbit, and can be successfully competed out with the free peptide used in the production of the antiserum (data not shown). These data establish that we produced a high-titer antiserum that is monospecific for K17, and confirms that the mouse and human K17 proteins do not differ significantly in their apparent Mr by conventional gel electrophoresis.

When applied on sections of adult mouse trunk skin (Fig. 5), the K17 antiserum reacts strongly with hair follicles (Fig. 5, A and B) and sweat glands (data not shown), but not with normal interfollicular epidermis. In hair follicles, the entire outer root sheat (ORS) is immunostained, whether the follicle is actively growing (anagen phase; Fig. 5, A and B) or resting (data not shown). Most, if not all, ORS keratinocytes are positively stained for K17, unlike K6, whose protein distribution is restricted to the innermost ORS layers (Fig. 5, compare panels C, D, and E). Although the difference in K6 and K17 distribution is best seen in the context of larger hair follicles, such as those presented in Fig. 5, it applies for all hair types (see Fig. 11, showing longitudinally sectioned pelage hair). Similar findings were obtained in human skin samples (data not shown).

Close examination of the hair bulb area revealed the consistent presence of faint immunostaining for K17 in the matrix cells and stronger staining in the early differentiating hair epithelial cells (trichocytes) (Fig. 5, F–H). The signal is lost in the upper segments of the hair (data not shown), owing probably to epitope masking. Such immunostaining for K17 occurs in all types of hair follicles, and its specificity is supported by control experiments involving the use of preimmune serum or the addition of the peptide immunogen to the anti-K17 serum (data not shown). Reexamination of in situ hybridization data (Carroll et al., 1997), which made use of a probe corresponding to the 3′ portion of the K17 mRNA, revealed the presence of K17 transcripts in the early differentiating trichocytes of vibrissae (Fig. 5 I) and pelage hair follicles (data not shown). A similar outcome was observed in a previous report by Panteleyev et al. (1997), but these authors attributed their results to the presence of melanin granules. We have observed this same pattern of immunostaining using samples obtained from unpigmented mice. Furthermore, immunoreactivity for K17 can be observed in Western blots analysis of protein extracts prepared from mouse hair clippings (the lack of K14 in such extracts confirms the absence of ORS contamination; data not shown). Finally, we can detect reporter transgene activity in trichocytes in the context of studies involving the 5′ upstream sequence of the mouse K17 gene (McGowan, K.M., and P.A. Coulombe, unpublished observations). Our ability to detect K17 protein in trichocytes, an occurrence that has never been reported before, may be primarily the result of the high titer and specificity displayed by our polyclonal antiserum.

We previously showed, using the commercially available monoclonal antibody (Sigma Chemical Co.), that K17 is induced at the wound edge starting 12 h after acute injury to human epidermis (Paladini et al., 1996). Likewise, Knapp et al. (1987) showed that a mouse type I keratin mRNA, which later on was identified as K17 (see Troyanovsky et al., 1992; Schweizer, 1993), is induced in skin wounds. Application of our novel antiserum to sections of adult mouse wounded skin showed that K17 is already induced by 8 h after acute injury (Fig. 5, E and F). In older wounds, K17 synthesis extends distally from the cut edge of the wound and is particularly strong in the migrating epidermal tissue, where it occurs in all epidermal layers (Fig. 5, G and H; 48 h after injury). The epidermal staining for K17 eventually disappears after wound closure (data not shown). These data establish that, as was found to be the case for K6 and K16 in human skin (Paladini et al., 1996), K17 is an early marker of the process of keratinocyte activation after injury and is expressed in the migrating epithelium.

Onset of K17 Expression Occurs Early in Developing Mouse Skin

Until ∼e9.5 d, the single-layered ectoderm covering the surface of the embryo expresses the keratins 8 and 18 typical of simple epithelia (Jackson et al., 1981). The earliest molecular indices of epidermal gene expression, starting at ∼e9.5 d, occur in the form of the transcription factor AP2 and its target genes K5 and K14. Byrne et al. (1994) showed that onset of expression of these genes takes place according to a complex spatiotemporal pattern that does not correlate with the initial stratification of the ectoderm. In agreement with previous reports (Jackson et al., 1981; Thorey et al., 1993), we find that e9.5-d ectoderm stains uniformly with an antiserum directed against K8/K18 but sporadically with antisera directed against K14 (data not shown). At e10.5 d, expression of K14 has generalized to a significant portion of the surface ectoderm. These data establish the validity of our antibody tools. We performed triple-staining studies that include the DNA stain Hoechst to analyze the context in which K17 expression is initiated in the early stages of skin morphogenesis. These studies made use of saggital sections of whole e12.5-d embryos, as these feature areas with single layered ectoderm (e.g., upper dorsum), two-layered ectoderm (e.g., lower dorsum), and stratified epithelium (e.g., ventral surface).

We find that induction of K17 expression takes place in cells that express both K8–K18 and K14. The induction is independent of stratification of the epithelium, as it is seen in single-layered ectoderm (Fig. 6, A–F), in the bottom layer of two-layered epithelium (Fig. 6, G and H), in the top layer of the stratified epithelium on the ventral surface of the embryo (Fig. 6, I and J). In favorable sections, the filamentous nature of the K17 staining can clearly be seen at higher magnification (Fig. 6 K). As previously reported (Greer and Roop, 1991; Byrne et al., 1994), no expression of K10 can be detected in sections prepared from e12.5-d embryos (data not shown). These data establish that K17 protein synthesis occurs shortly after the onset of K14 expression, before the onset of epithelial differentiation-specific gene expression.

Onset of K17 Expression Reflects the Adoption of a Nonepidermal Cell Fate in Embryonic Skin

As mentioned above, we observe very patchy expression of the K17 gene in the ectoderm of the upper dorsum region of e10.5-d embryos (Fig. 7,A). Examination of sections prepared from several embryos suggest a periodicity in the distribution of K17, in that clusters of one to three positively stained cells occur at regularly spaced intervals along the dorsal surface of the embryo. This staining pattern is more firmly established at e12.5 d (Fig. 7,B). Between e12.5 and e14.5 d, the two-layer ectoderm further stratifies to give rise to embryonic epidermis over most of the embryo surface, whereas the first signs of hair follicle induction occur in the form of an invagination of small groups of epithelial cells into the underlying mesenchyme. We find that the placodes and the proximal epithelial tissue are both strongly immunostained for K17 (Fig. 7, C, D, and E show various stages of placode formation in e14.5-d epidermis). Continued downward expansion of the placodes gives rise to primary hair germs, starting at e14.5 d and well established by e16.5 d (Mann, 1962; Hardy and Vielkind, 1996). The strong expression of K17 persists in the primary hair germs at e14.5 d (Fig. 7,F) and e16.5 d (Fig. 7 G), and examination of sections from several embryos shows that the majority of the epithelial cells in primary hair germs are K17 positive. Collectively, our data suggest that the precursors of pelage and vibrissae hair follicles arise from small clusters of K17–positive ectoderm that first appear in the undifferentiated ectoderm of e10.5 and e12.5 mouse embryos.

Another ectodermal cell type that is uniformly and consistently immunopositive when stained with anti-K17 is the periderm. As is the case of K6 (see below), the periderm staining begins at e14.5 d (Fig. 7, C–F) and extends (Fig. 7 G, e16.5-d sample) until this layer is lost from the surface of the embryos shortly before birth (Fisher, 1994).

The embryonic epidermis is mostly negative for K17, with the exception of a transient expression in the basal layer starting at e14.5 (Fig. 7,F) and peaking at e16.5 d (Fig. 7,G). This finding is rather intriguing, as differentiation specific gene expression is already ongoing in the embryonic epidermis during this period (data not shown) (Dale et al., 1985; Greer and Roop, 1991; Byrne et al., 1994). This expression has already begun to recede towards developing hair follicles by e18.5 d and is further restricted to the neck of hair follicles openings in dorsal trunk skin of newborn mice (Fig. 7 H).

K17 Expression Is Partially Uncoupled from That of K6 in Embryonic Skin

We examined the expression of K6 in our collection of embryo specimens using a newly produced rabbit polyclonal antiserum (refer to Materials and Methods). This type II keratin is coregulated with K17 in wound edge epidermis after injury to human and mouse adult skin (Paladini et al., 1996; Takahashi et al., 1997, 1998), and is presumed to be the type II pairing partner for K17 in several stratified epithelia (e.g., O'Guin et al., 1990). In e12.5-d embryos, a strong staining for K6 protein occurs in the oral mucosa whereas the surface ectoderm is negative (data not shown). In e14.5-d embryos, K6 immunoreactivity occurs the innermost aspect of the developing vibrissae follicles (Fig. 8,A) and in the periderm layer, which is uniformly stained (Fig. 8, A and B). Similarly to K17, the periderm staining for K6 persists (Fig. 8,C) until the loss of this layer before birth (data not shown). In contrast to K17, however, we detect K6 immunoreactivity at a relatively late stage of pelage hair follicle development (e16.5 d), where it is restricted to a subset of epithelial cells (Fig. 8 C). This pattern is maintained in e18.5-d embryos and newborn mouse skin (data not shown). Consistent with previous reports situating the onset of hair-specific keratin gene expression at e14.5–e15.5 d (Zhou et al., 1995), onset of K6 expression appears to coincide with the beginning of differentiation in developing hair follicles. These data establish that with the notable exception of the entire periderm and a small subset of hair epithelial cells (see Discussion), K17 expression is largely uncoupled from that of K6 in developing skin.

Expression of K17 Coincides with That of the Transcription Factor lef-1 during Skin Development

The epithelial placodes that serve as precursors of hair follicles and other appendages form directly above condensates of specialized mesenchymal cells with inductive properties (Sakakura et al., 1976; Kollar et al., 1983; Hardy, 1992; Jowett et al., 1993). Lef-1, a protein that belongs to the HMG box family of transcription factors (Clevers and Grosscheld, 1996), is known to play a critical role in the epithelial–mesenchymal interactions that underlie appendageal morphogenesis. Lef-1 is expressed in the cell condensates that form at sites of future hair follicle formation (e.g., Zhou et al., 1995; Kratochwil, 1996), and mice harboring a null mutation in their lef-1 gene show a significantly reduced number of hair follicles, a failure to form teeth and mammary glands, and die shortly after birth (van Genderen et al., 1994). Given this, we decided to relate the expression of K17 to that of lef-1 in our collection of mouse embryo sections.

Immunostainings for lef-1 performed on frozen sections prepared from e12.5-, e14.5-, and e16.5-d mouse embryos confirmed the elegant whole mount in situ hybridization findings reported by Zhou et al. (1995). The lef-1 protein staining is predominantly nuclear, as expected for a transcription factor, and is initially manifested in and around epithelial placodes (Fig. 9, A and B; e14.5-d embryo dorsum). The presence of lef-1 in both the epithelial cells forming the placode and in proximal mesenchymal cells is consistent with its role in epithelial–mesenchymal interactions (Kratochwil et al., 1996). These sites of epithelial– mesenchymal interactions, as identified by lef-1 protein stainings, also display a strong signal for K17 (Fig. 9, A and B). A survey of samples obtained from dual-staining experiments of e14.5-d embryos show that the spatial correspondence between the signals for K17 and lef-1 proteins is striking. This is well illustrated in the context of double-staining experiments (hair placode shown in Fig. 9, C and D) and staining of consecutive frozen sections (vibrissae shown in Fig. 9, E and F). As hair placodes evolve into primary hair germs, e.g., in e16.5-d embryos, the spatial correspondence between the two signals persists only at the tip region near the mesenchyme (data not shown). In addition, the basal layer of e16.5-d interfollicular epidermis is clearly positive for both K17 and lef-1 proteins (Fig. 9, B and C). In contrast to K17, however, we could not detect a convincing signal for lef-1 in the periderm layer at any stage of its formation. In newborn skin, the signal for lef-1 is mostly restricted to the actively growing (bulb) segment of the follicles, as previously reported (Zhou et al., 1995), whereas K17 is distributed throughout the outer root sheath and occurs in proximal epidermis as well (refer to Fig. 7 H). Thus, it would appear that lef-1 protein is not synthesized in differentiating epithelial cells of embryonic mouse skin.

Keratin 17 is expressed in several mature epithelia whose development is known to depend on epithelial– mesenchymal interactions mediated by lef-1. These include vibrissae, salivary glands, tooth, and thymus (Fig. 10, A–D, respectively). To further probe the relationship between K17 and lef-1 expression, we examined these tissues in e14.5-d mouse embryos. Again, we detected K17 immunoreactivity at an early step in the morphogenesis of these tissues (Fig. 10, A′–D′). Collectively, these findings establish that the K17 gene is expressed in a number of developing tissues whose formation partly depend upon lef-1.

Ectopic Expression of lef-1 Correlates with K17 Induction in the Epidermis of Adult Transgenic Mice

The intriguing correspondence between the timing and location of lef-1 protein synthesis, a transcription factor allegedly involved in the regulation of hair-specific keratin genes, and K17, a component of the pilosebaceous apparatus, raises the possibility that the former may contribute to regulate the latter during early skin morphogenesis. In support of this possibility, inspection of the 5′ upstream sequence of the mouse K17 gene reveals the presence, in three distinct locations, of a nucleotide sequence motif, 5′-CTTTGWW-3′, which fits perfectly the consensus DNA binding site for lef-1 (Powell et al., 1991; Clevers and Grosscheld, 1996). A perfect consensus binding site for lef-1 occurs in the third intron of the human K17 gene (GenBank/EMBL/DDBJ accession number Z19574) (Troyanovsky et al., 1992). Such lef-1 consensus binding sites occur in the 5′ upstream regulatory region of all the hair follicle-specific genes examined so far, and can mediate the transactivation of a reporter gene in a lef-1–dependent manner in an heterologous expression assay ex vivo (Zhou et al., 1995). Based on these findings we examined the expression of K17 in two mouse models in which lef-1 expression has been genetically manipulated: mice that ectopically overexpress lef-1 in epidermis via a keratin 14 gene promoter (Zhou et al., 1995), and mice harboring a null mutation for lef-1 (van Genderen et al., 1994).

Transgenic mice that express lef-1 under the control of the K14 gene promoter show the presence of lef-1 in the basal layer of epidermis, oral mucosa, and a few other stratified epithelia. These mice feature an abnormal patterning of hair follicles over their trunk, and consistent with the postulated role for lef-1 in appendageal morphogenesis (van Genderen et al., 1994), they show ectopic appendages in the oral mucosa (Zhou et al., 1995). Paraffin-embedded skin samples from 11-d-old transgenic pups and controls were provided by E. Fuchs. The epidermis of these mice display a normal morphology and distribution of key markers of terminal differentiation, such as K10 and filaggrin (data not shown) (Zhou et al., 1995). Additionally, the interfollicular epidermis does not stain positively for K6 (Fig. 11,B), a marker for abnormal proliferation that is coregulated with K17 after various acute challenges to the skin (Takahashi et al., 1997; 1998). Yet, unlike the situation in control (Fig. 11,A), a significant fraction of basal cells in interfollicular epidermis express K17 in lef-1 transgenic tissue (Fig. 11, C and D). The specificity of this induction and the absence of any other detectable changes in the properties of progenitor basal keratinocytes in the mature epidermis of [K14 promoter] lef-1 mice supports the possibility that lef-1 may be involved in aspects of K17 expression at an early stage of appendageal morphogenesis.

Transgenic mice that harbor a null mutation in the lef-1 gene feature a significantly reduced number of hair follicles that are reportedly shorter and delayed in their maturation (van Genderen et al., 1994). We found that the hair follicle profiles present show positive immunostaining for K17 in the ORS, however these samples lacked hair shafts (data not shown). Whether these follicles form as a result of functional redundancy with other related transcription factors in lef-1–null mice, or whether lef-1 preferentially impacts on the formation of a specific type of hair follicle, remains unclear at present. From this we conclude that lef-1 expression is not essential for the morphogenesis of all pelage hairs or for the expression of K17 in the ORS.

Discussion

K17 and the Onset of Epithelial Differentiation during Skin Development

Essential to understanding the mechanisms that underlie the developmental choices that occur during mammalian organogenesis is the identification of the stages at which commitment to various cell lineages and subsequent differentiation occur. The embryonic epidermis represents an excellent system for such studies as it differentiates and expands from a single-layered embryonic ectoderm to form a stratified squamous epithelium. Concomitantly, cells from within this tissue undergo separate programs of differentiation to form epidermally derived appendages like hair, nail, teeth, and glands. The monitoring of epidermal and hair-specific keratin gene expression has significantly contributed to our current understanding of these processes (Dale et al., 1985; Kopan and Fuchs, 1989; Greer and Roop, 1991; Byrne et al., 1994). We surmised that given their constitutive expression in various appendages and their rapid induction after acute injury, the nonepidermal keratin genes K6 and K17 may provide additional insights into skin development. We found that expression of K17 is initiated in the undifferentiated ectoderm at the single- or two-layer stage, starting in e10.5-d mouse embryos. The spatiotemporal pattern of K6 expression is different, and coincides with the onset of expression of several differentiation-specific markers at a later time (see below). Our data point to the intriguing possibility that onset of K17 expression in the embryonic ectoderm reflects a commitment towards distinct epithelial lineages, and prompt us to propose a revised model for skin development as shown in Fig. 12.

In this model, the morphogenesis of the major types of skin epithelia is segmented into four distinct yet logically interconnected phases, which can be described as follows:

Phase 1. Commitment to Form a Complex Epithelium.

Up to ∼e9.5 d, the single-layered ectoderm covering the embryo is undifferentiated and expresses the keratins characteristic of simple epithelia, K8, and K18 (Jackson et al., 1981; Thorey et al., 1993). We propose that the onset of transcription factor AP-2 expression, soon followed by that of its target genes K5 and K14 (Byrne et al., 1994), reflects a commitment towards the formation of a complex epithelium. Of note, K5 and K14 (along with variable levels of K15) are the only keratins that are expressed in all the complex (soft) epithelia in the adult, where they typically occur in the least differentiated progenitor cells (Moll et al., 1982; O'Guin et al., 1990).

Phase 2. Definition of Major Epithelial Lineages.

We propose that onset of K17 expression in a subpopulation of the K5–K14- and K8–K18-expressing embryonic ectodermal cells, which occurs in the e10.5 –e12.5-d time window, signifies a commitment to form a nonepidermal tissue such as a hair follicle, a gland, or the periderm. This commitment is the result of a set of specific molecular instructions (e.g., lef-1–mediated epithelio–mesenchymal interactions) that do not take place in epidermal precursors, which represents the default pathway (Kopan and Fuchs, 1989). We have demonstrated that onset of K17 expression may occur within the single-layer embryonic ectoderm, or in the lower or upper layer of the two-layer ectoderm (depending on the region of the embryo), in cells that are mitotically active (this study; Byrne et al., 1994). These data extend the work of Byrne and colleagues (1994), who observed that the initial stratification of the ectoderm does not correspond to a polarization or differentiation event. Moreover, our temporal assignment for this phase is in complete agreement with the results obtained by Sanes and coworkers (1986), who used retrovirally marked cells to determine the genealogy of the various cell types in several mouse tissues. Their study showed that commitment to peridermal and follicular cell fates occurs at ∼e11 d, but that before this all ectodermal cells maintain pluripotentiality.

Phase 3. Cellular Expansion.

For a period of ∼48 hours that extends from the onset of K17 gene expression to that of differentiation-specific genes, there is a significant increase in epithelial cell number without molecular indices of terminal differentiation (see below). We propose that this period corresponds to an expansion of relatively undifferentiated cell pools within each of the major epithelial lineages. Obviously, additional studies are required to examine gene expression during this poorly-defined phase.

Phase 4. Onset of Lineage-specific Epithelial Differentiation.

Several differentiation-related genes are first expressed starting at e13.5–e15.5 d, such as K1–K10 (this study; Byrne et al., 1994), filaggrin, and loricrin (Byrne et al., 1994; Greer and Roop, 1991) in epidermis, and hard keratin genes (Byrne et al., 1994) as well as K6 (this study) in hair follicles. In all these instances, expression of the marker occurs in a subset of cells within the maturing epithelium, reflecting a polarization in the developing tissue. Consequently, this phase represents the last one in our model for the morphogenesis of skin epithelia, as it corresponds to the onset of terminal differentiation.

Although the four steps described occur sequentially for any individual element (e.g., a single hair follicle), it must be realized, as alluded to in the results section, that they are not synchronized over the entire skin tissue, since development proceeds according to spatial and temporal gradients.

K17 Is a Candidate Target Gene for Regulation by Lef-1

Considerable progress has been made towards identifying the mechanisms and molecules that mediate the reciprocal, inductive interactions between mesenchymal and epithelial tissues that play an integral role in hair follicle morphogenesis. One such molecule is the lymphoid enhancer factor, lef-1 (van Genderen et al., 1994; Zhou et al., 1995; Clevers and Grosschedl, 1996). Lef-1 is a transcription factor capable of bending DNA, a property that is thought to influence gene transcription by facilitating the association of other transcription factors (for review see Clevers and Grosschedl, 1996). Targeted inactivation of the lef-1 gene produced major alterations in epithelial tissues that share a dependency upon epithelial–mesenchymal interactions for proper morphogenesis (van Genderen et al., 1994). The spatiotemporal pattern of K17 expression during development suggest strongly that this keratin gene may be regulated by signals generated in the context of such epithelio–mesenchymal interactions, possibly lef-1.

We presented several lines of evidence that support this notion. First, we have shown a temporal and spatial correspondence between K17 and lef-1 protein synthesis in hair placodes. Second, we have presented data depicting K17 protein synthesis during the formation of other lef-1 target organs. Third, we provide convincing evidence that K17 is expressed in precursor matrix epithelial cells and early differentiating trichocytes of fully formed hair follicles. These cells give rise to the structures of the hair, and the programs of gene expression associated with these include several hard keratin genes postulated to be directly regulated by lef-1. To our knowledge, K17 represents the first instance of a keratin gene expressed in both hard and soft epithelial tissues. Fourth, there are three consensus binding sites for lef-1 in the mouse K17 gene, and at least one in the human K17 gene (the full 5′ upstream sequence of this gene is not yet available). These consensus sites are identical to those found in hair keratin genes (Powell et al., 1991, 1992), where they have been found to mediate a direct transactivation (Zhou et al., 1995). Fifth and finally, we found that K17 protein is synthesized in the epidermal basal layer of transgenic mice that ectopically express lef-1 under the control of the K14 promoter.

The connection between K17 and lef-1 expression is important when one considers the delay between the expression of lef-1, which begins at ∼e11 d (Zhou et al., 1995; Kratochwil et al., 1996), and the subsequent induction of hair keratin genes at ∼e14.5–15.5 d. The potential relationship between lef-1 expression and K17 upregulation could provide a critical molecular link between the expression of this transcription factor and the actual commitment to undergo appendageal organogenesis. The location of binding sites for lef-1 is different in the K17 and the hair-specific keratin genes (data not shown). This may represent an important determinant for the manner with which lef-1, being an architectural transcription factor, impacts on the expression of these keratin genes during development. Studies are in progress to test whether lef-1 can directly transactivate the K17 gene promoter.

K17, Wound Healing, and Epidermal Development

Together with our previous work (Paladini et al., 1996; Takahashi et al., 1997, 1998), the studies reported here establish that K6, K16, and K17 proteins are all induced in epidermis at the proximal edge of the wound within hours after acute injury to mouse and human skin. The K17 specific antisera used for this study allows us to modify our early studies, in that it appears that the induction of K6, K16, and K17 genes occurs on the same time scale. At least early on, this induction occurs in suprabasal keratinocytes, i.e., in cells that have already begun to differentiate. Thereafter, expression of K6, K16, and K17 spreads distally from the cut edge of the wound, is maintained in keratinocytes that are migrating into the wound site, and ceases upon the completion of reepithelialization. The significance of this phenomenon, and the precise contribution of K6, K16, and K17, remain to be ascertained. Schermer and colleagues (1989) proposed the interesting idea that this induction may reflect a switch to an alternative, possibly more primitive mode of epithelial differentiation. From previous work we postulated that these keratins, K16 in particular, may contribute to promote the cytoarchitectural changes that are typical of keratinocyte activation (Paladini et al., 1997; McGowan et al., 1998). In this regard, the data we report here have an impact at two distinct levels. First, the notion that K17 is synthesized in soft and hard epithelial cells and that this protein is a major component of regenerating epithelia is an intriguing one. We have preliminary data further supporting this concept that involve studies with the WE6 antigen, which is wound inducible in the surface epithelium of the newt and frogs (Estrada et al., 1993) and corresponds to a type I keratin (Klatt, K., and R. Tassava, personal communication). We detect WE6 immunoreactivity in the early differentiating cells of hard epithelia in the mouse such as hair, nail, and filiform papillae of the tongue (Coulombe, P.A., unpublished observations). Additional studies are required to further explore the notion that K17, and possibly other wound repair-associated keratins, could perform a key function in both soft and hard epithelia under resting conditions. Second, our findings argue that the program of gene expression characteristic of wound-activated adult keratinocyte (for review see Coulombe, 1997) may bear a relationship to that executed by epithelial cells at specific stages of embryonic development, before expression of true differentiation-specific genes. The existence of such a molecular relationship between embryonic development and adult tissue regeneration has already been evidenced in other tissues, liver in particular (Michapoulos, 1997).

The event that most convincingly links embryonic development and wound repair in skin epithelia is the abundant expression of K17 in developing hair placodes. In this case as in adult skin wounds, a group of K17-expressing keratinocytes coordinately expands or migrates into a connective tissue, suggesting that expression of this particular keratin may be a molecular trait of keratinocytes involved in migration or tissue expansion. Additional lines of evidence support this contention. Appendages have long been known to contribute keratinocytes to the resurfacing of wound sites in adult skin, especially in cases involving partial thickness wounds (Clark, 1993), and we find that K17 expression is upregulated in the ORS of hair follicles located proximal to the wound site (data not shown). Moreover, experimental evidence has recently been provided for a similar phenomenon involving epithelial cells originating from eccrine sweat glands (Miller et al., 1998). An exciting possibility is that the cell type involved in this case would be the myoepithelial cells, which express K17 constitutively (see below).

In terms of keratin gene expression profile, however, the periderm appears to better mimic the activated keratinocyte of adult skin wounds. In developing skin, indeed, uniform and abundant co-expression of K6 and K17 occurs only in the periderm. The function of the periderm remains a mystery (Fisher, 1994). Periderm has been suggested to emanate from sites of epithelio–mesenchymal interactions (M'Boneko and Merker, 1988). The morphological changes taking place as it emerges (M'Boneko and Merker, 1988) bear a striking resemblance to activated keratinocytes at the edge of adult skin wounds (Paladini et al., 1996). An open question is whether K17 induction in the periderm is a byproduct of the inductive interactions that lead to appendage formation or whether it represents a critical event in the morphogenesis of this poorly understood cell type. The degree to which the molecular relationship between periderm and the wound-activated adult skin keratinocyte extends beyond keratin gene expression deserves further investigation.

Other than hair placodes and periderm, K17 is expressed transiently but uniformly in the basal layer of embryonic epidermis ∼e16.5 d. By that time, the developing epidermis already features a mature character as judged by various markers of adult epidermis (Fuchs and Byrne, 1994). This aspect of embryonic K17 expression is of significance, as it provides further support to the notion that K17 expression in epidermis is compatible with mitosis and not a manifestation of advanced differentiation. This finding raises several questions, however, including the fate adopted by these embryonic basal cells, and the mechanism(s) responsible for spatially restricting the expression of this gene to the postmitotic layers in adult epidermis subjected to injury and other forms of challenge (as discussed above).

At another level, our findings may have significant implications with regard to a recent study of perfect wound healing in K8-null embryos (Brock et al., 1996). These studies concluded, on the basis that reepithelialization is not altered in K8-null embryos, that keratin IFs are not an integral part of the fetal wound repair process. We would assert that one must look beyond the K8–K18 network, as these filaments may not play a substantial role once the skin-specific keratins have begun to accumulate. It may be possible that the keratins already present at e11.5 d, which at least include K5, K14, and K17, are sufficient to bring about wound closure. We are currently exploring the possibility that the wound repair keratins K6, K16, and K17 may be expressed during reepithelialization of fetal wounds.

Speculation on the Function of K17

A fundamental question in the field of keratin filament biology is why there exists so many homologous proteins. By virtue of its primary sequence, K17 belongs to a subgroup of type I keratin genes that includes K14 and K16. A comparison of their amino acid sequences reveals that individual substitutions tend to be grouped within defined segments of these proteins. Thus, the NH2-terminal head domains of K14 and K16 are clearly more related to one another than either is to K17. Conversely, homology within the COOH-terminal tail domain is greatest between K14 and K16 for the proximal half segment, while being greatest for K14 and K17 for the distal half segment. It is unclear how these variations result in functional differences between these type I keratins. What is known is that human K16 can not entirely complement the phenotype associate with a K14-null mutation in transgenic mice, a behavior that we could map to the COOH-terminal 105 amino acids (Paladini, R.D., and P.A. Coulombe, unpublished data).

Our findings point to a dual function for K17 that may help to explain the phenotype and ultrastructural pathology characteristic of pachyonychia congenita and steatocystoma multiplex, two clinical disorders that can arise from missense mutations affecting the primary sequence of K17 (McLean et al., 1995; Smith et al., 1997). On the one hand, the occurrence of cytolysis in at least some K17 mutant-expressing epithelia (McLean et al., 1995) is consistent with a structural role akin to that already defined for other keratin proteins. It is possible that the synthesis of K17 protein in the hair matrix and lower portions of the hair shaft impacts the formation of this structure and would explain the presence of twisted hairs (pili torti) in PC patients. On the other hand, our description of the developmental expression of K17, especially during odontogenesis and gland formation, may explain the linkage between K17 mutation the occurrence of developmental anomalies such as those characterizing the eyebrow, sweat glands, and teeth (Munro et al., 1994; McLean et al., 1995). This would render K17 unique among the keratin genes so far linked to inherited diseases by suggesting a function for this protein during the morphogenesis of these tissues.

The context and subcellular distribution of a given protein can also convey hints about its function. Many of the epithelial cell types expressing K17, whether normal or activated, show contractile or motile activity. An example of this are the myoepithelial cells of glandular tissues, whose cytoplasm displays arrays of actin and myosin filaments organized in a sarcomere-like fashion (Raubenheimer, 1987; Redman, 1994). Contraction of these star-shaped myoepithelial cells, whose long cytoplasmic projections envelop the lumenal tissue, is thought to supply the force needed for propelling the glandular contents into the ducts (Raubenheimer, 1987; Redman, 1994). Interestingly, as stated for the periderm epithelium above, myoepithelial cells resemble activated keratinocytes proximal to the wound edge (Paladini et al., 1996) in many respects. Thus it would appear K17-expressing epithelial cells often feature a prominent actin-based molecular machinery. Although this relationship remains to be established, recent work would suggest that such cross-talk between filament networks, as mediated by linker proteins like plectin (Wiche, 1989) and BPAG (Yang et al., 1996), is not only possible but essential (Bousquet and Coulombe, 1996). This raises the possibility that K17 containing intermediate filament networks possess unique properties that render them capable of making such connections.

Acknowledgments

We are grateful to members of the Coulombe laboratory for their advice and support, and wish to extend a very special thank you to E. Fuchs and R. Grosscheld for their generous gift of transgenic mouse tissues and antibodies; to J. Carroll (Genetics Institute, Andover, MA), for sharing in situ hybridization data; to R. Tassava (Ohio State University, Columbus, OH) and K. Klatt (Denison University, Granville, OH) for helpful discussions; and G. Rogers (University of Adelaide, Adelaide, Australia), for advice on hair protein extraction. We also thank I. Leigh (Imperial Cancer Research Foundation, London, UK) for providing the LL001 antibody.

Abbreviations used in this paper

     
  • e

    embryonic day

  •  
  • IF

    intermediate filament

  •  
  • K

    keratin

  •  
  • ORS

    outer root sheat

  •  
  • RT

    reverse transcriptase

References

References
Bessho
Y
,
Nakanishi
S
,
Nawa
H
Glutamate receptor agonists enhance the expression of BDNF mRNA in cultured cerebellar granule cells
Mol Brain Res
1993
18
201
208
[PubMed]
Bonnekoh
B
,
Huerkamp
C
,
Wevers
A
,
Geisel
J
,
Sebok
B
,
Bange
FC
,
Greenhalgh
DA
,
Bottger
EC
,
Krieg
T
,
Mahrle
G
Up-regulation of keratin 17 expression in human HaCaT keratinocytes by interferon-gamma
J Invest Dermatol
1995
104
58
61
[PubMed]
Boulikas
T
A compilation and classification of DNA binding sites for protein transcription factors from vertebrates
Crit Rev Eukaryot Gene Expr
1994
4
117
322
[PubMed]
Bousquet
O
,
Coulombe
PA
Cytoskeleton: missing links found?
Curr Biol
1996
6
1563
1566
[PubMed]
Brock
J
,
McCluskey
J
,
Baribault
H
,
Martin
P
Perfect wound healing in the keratin 8 deficient mouse embryo
Cell Motil Cytoskeleton
1996
35
358
366
[PubMed]
Byrne
C
,
Tainsky
M
,
Fuchs
E
Programming gene expression in developing epidermis
Development (Camb)
1994
120
2369
2383
[PubMed]
Carroll
JM
,
Crompton
T
,
Teery
JP
,
Watt
FM
Transgenic mice expressing IFN-gamma in the epidermis have eczema, hair hypopigmentation, and hair loss
J Invest Dermatol
1997
108
412
422
[PubMed]
Clark, R.A.F. 1993. Mechanisms of Cutaneous Wound Repair. Vol. I, T.B. Fitzpatrick, A.Z. Eisen, K. Wolff, I.M. Freedberg, and M.D. Austen, editors. McGraw-Hill, New York.
Clevers
HC
,
Grosschedl
R
Transcriptional control of lymphoid development: lessons from gene targeting
Immunol Today
1996
17
336
343
[PubMed]
Coulombe
PA
The cellular and molecular biology of keratins: beginning a new era
Curr Opin Cell Biol
1993
5
17
29
[PubMed]
Coulombe
PA
Towards a molecular definition of keratinocyte activation after acute injury to stratified epithelia
Biochem Biophys Res Comm
1997
236
231
238
[PubMed]
Dale
BA
,
Holbrook
KA
,
Kimball
JR
,
Hoff
M
,
Sun
TT
Expression of epidermal keratins and filaggrin during human fetal skin development
J Cell Biol
1985
101
1257
1269
[PubMed]
Estrada, C.M., C.D. Park, M. Castilla, and R.A. Tassava. 1993. Monoclonal antibody WE6 identifies an antigen that is up-regulated in the wound epithelium of newts and frogs. In Limb Development and Regeneration. Wiley-Liss Inc., New York. 272–282.
Fisher, C. 1994. The cellular basis of development and differentiation in mammalian keratinizing epithelia. In The Keratinocyte Handbook. I.M. Leigh, B. Lane, and F. Watt, editors. Cambridge University Press, Cambridge, UK. 131–150.
Fuchs
E
,
Weber
K
Intermediate Filaments: Structure, Dynamics, Function, and Disease
Annu Rev Biochem
1994
63
345
382
[PubMed]
Fuchs
E
,
Byrne
C
The epidermis: rising to the surface
Curr Opin Genet Dev
1994
4
725
736
[PubMed]
Greer
J
,
Roop
D
Loricrin, a major keratinocyte cell envelope protein, is expressed late in development
J Invest Dermatol
1991
96
553
560
Gurdon
JB
The generation of diversity and pattern in animal development
Cell
1992
68
185
199
[PubMed]
Hardy
MH
,
Vielkind
U
Changing patterns of cell adhesion molecules during mouse pelage hair follicle development. I. Follicle morphogenesis in wilt-type mice
Acta Anat
1996
157
169
182
[PubMed]
Hardy
MH
The secret life of the hair follicle
Trends Genet
1992
8
55
61
[PubMed]
Hatzfeld
M
,
Franke
WW
Pair formation and promiscuity of cytokeratins: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides
J Cell Biol
1985
101
1826
1841
[PubMed]
Jackson
BW
,
Grund
C
,
Winter
S
,
Franke
WW
,
Illmensee
K
Formation of cytoskeletal elements during mouse embryogenesis II. Epithelial differentiation and intermediate filaments in early post-implantation embryos
Differentiation
1981
20
203
216
[PubMed]
Jiang
CK
,
Flanagan
S
,
Ohtsuki
M
,
Shuai
K
,
Freedberg
IM
,
Blumenberg
M
Disease-activated transcription factor: allergic reactions in human skin cause nuclear translocation of STAT-91 and induce synthesis of keratin K17
Mol Cell Biol
1994
14
4759
4769
[PubMed]
Jowett
AK
,
Vajno
S
,
Ferguson
MWJ
,
Sharpe
PI
,
Thesleff
I
Epithelial-mesenchymal interactions are required for msx-1 and msx-2 gene expression in the developing murine molar tooth
Development (Camb)
1993
117
461
470
[PubMed]
Kaufman, M.H. 1992. The Atlas of Mouse Development. Academic Press, New York. 1–16.
Knapp
B
,
Rentrop
M
,
Schweizer
J
,
Winter
H
Three cDNA sequences of mouse type I keratins. Cellular localization of the mRNAs in normal and hyperproliferative tissues
J Biol Chem
1987
262
938
945
[PubMed]
Kollar, E.J. 1983. Epithelial–mesenchymal interactions in the mammalian integument: Tooth development as a model instructive induction. In Epithelial-Mesenchymal Interactions in Development. R.H. Sawyer and J.F. Fallon, editors. Praeger Press, New York. 27–50.
Komine
M
,
Freedberg
IM
,
Blumenberg
M
Regulation of epidermal expression of keratin K17 in inflammatory skin diseases
J Invest Dermatol
1996
107
569
575
[PubMed]
Kopan
R
,
Fuchs
E
A new look into an old problem: Keratins as tools to investigate determination, morphogenesis, and differentiation in skin
Genes Dev
1989
3
1
15
[PubMed]
Kozak
M
An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs
Nucleic Acids Res
1987
15
8125
8148
[PubMed]
Kratochwil
K
,
Dull
M
,
Farinas
I
,
Galceran
J
,
Grosscheld
R
Lef1 expression is activated by BMP-4 and regulates inductive tissue interactions in tooth and hair development
Cell
1996
10
1382
1394
Leigh
IM
,
Navsaria
H
,
Purkis
PE
,
McKay
IA
,
Bowden
PE
,
Riddle
PN
Keratins (K16 and K17) as markers of keratinocyte hyperproliferation in psoriasis in vivo and in vitro
Br J Dermatol
1995
133
501
511
[PubMed]
Lewis-Carl
SA
,
Gillette-Ferguson
I
,
Ferguson
DG
An indirect immuno-fluorescecnce procedure for staining the same cryosection with two mouse monoclonal antibodies
J Histochem Cytochem
1993
41
1273
1278
[PubMed]
M'Boneko
V
,
Merker
H-J
Development and morphology of the periderm of mouse embryos (Days 9-12 of gestation)
Acta Anat
1988
133
325
336
[PubMed]
Mann
SJ
Prenatal formation of hair follicle types
Anat Rec
1962
144
135
142
Marchuk
D
,
McCrohon
S
,
Fuchs
E
Remarkable conservation of structure among intermediate filament genes
Cell
1984
39
491
498
[PubMed]
McGowan, K., and P.A. Coulombe. 1998. The wound repair-associated keratins K6, K16 and K17: Insights into the role of intermediate filaments in specifying keratinocyte cytoarchitecture. In Subcellular Biochemistry: Intermediate Filaments. J.R. Harris and H. Herrmann, editors. Plenum Publishing Co., London, UK. In press.
McLean
WHI
,
Lane
EB
Intermediate filaments in diseases
Curr Opin Cell Biol
1995
7
118
125
[PubMed]
McLean
WHI
,
Rugg
EL
,
Lunny
DP
,
Morley
SM
,
Lane
EB
,
Swensson
O
,
Dopping-Hepenstal
PJC
,
Griffiths
WAD
,
Eady
RAJ
et al
Keratin 16 and keratin 17 mutations cause pachyonychia congenita
Nat Genet
1995
9
273
278
[PubMed]
Michapoulos
GK
,
DeFrances
MC
Liver regeneration
Science
1997
276
60
66
[PubMed]
Miller
S
,
Burke
EM
,
Rader
MD
,
Coulombe
PA
,
Lavker
RM
Re-epithelialization of porcine skin wounds by the sweat apparatus
J Invest Dermatol
1998
110
13
19
[PubMed]
Moll
R
,
Franke
WW
,
Schiller
DL
,
Geiger
B
,
Krepler
R
The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells
Cell
1982
3
11
24
[PubMed]
Moll
R
,
Krepler
R
,
Franke
WW
Complex cytokeratin polypeptide patterns observed in certain human carcinomas
Differentiation
1983
23
256
269
[PubMed]
Munro
CS
,
Carter
S
,
Bryce
S
,
Hall
M
,
Rees
JL
,
Kunkeler
L
,
Stephenson
A
,
Strachan
T
A gene for pachyonychia congenita is closely linked to the keratin gene cluster on 17q12-q21
J Med Genet
1994
30
675
678
[PubMed]
Nadeau
JH
,
Berger
FG
,
Cox
DR
,
Crosby
JL
,
Davisson
MT
,
Ferrara
D
,
Fuchs
E
,
Hart
C
,
Hunihan
L
,
Lalley
PA
et al
A family of type I keratin genes and the homeobox-2 gene complex are closely linked to the rex locus on mouse chromosome 11
Genomics
1989
5
454
462
[PubMed]
O'Guin, W.M., A. Schermer, M. Lynch, and T.T. Sun. 1990. Differentiation-specific expression of keratin pairs. In Cellular and Molecular Biology of Intermediate Filaments. R.D. Goldman and P.M. Steinert, editors. Plenum Publishing Co., New York. 301–334.
Osborn
M
,
Weber
K
Tumor diagnosis by intermediate filament typing: A novel tool for surgical pathology
Lab Invest
1983
48
372
393
[PubMed]
Paladini
RD
,
Takahashi
K
,
Gant
TM
,
Coulombe
PA
cDNA cloning and bacterial expression of the human type I keratin 16
Biochem Biophys Res Comm
1995
215
517
523
[PubMed]
Paladini
RD
,
Takahashi
K
,
Bravo
NS
,
Coulombe
PA
Onset of re-epithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: defining a potential role for keratin 16
J Cell Biol
1996
132
381
397
[PubMed]
Panteleyev
AA
,
Paus
R
,
Wanner
R
,
Nurnberg
W
,
Eichmuller
S
,
Thiel
R
,
Zhang
J
,
Henz
BM
,
Rosenbach
T
Keratin 17 gene expression during the murine hair cycle
J Invest Dermatol
1997
108
324
329
[PubMed]
Powell
B
,
Crocker
L
,
Rogers
G
Hair follicle differentiation: expression, structure and evolutionary conservation of the hair type II keratin intermediate filament gene family
Development (Camb)
1992
114
417
433
[PubMed]
Powell, B.C., and G.E. Rogers. 1997. The role of keratin proteins and their genes in the growth, structure and properties of hair. In Formation and Structure of Human Hairs, P. Jollès, H. Zahn, and H. Höcker, editors. Birkhaüser-Verlag, Basel, Switzerland. 59–148.
Powell
BC
,
Nesci
A
,
Rogers
GE
Regulation of keratin gene expression in hair follicle differentiation
Ann NY Acad Sci
1991
642
1
20
[PubMed]
Proby
CM
,
Churchill
L
,
Purkis
PE
,
Glover
MT
,
Sexton
CJ
,
Leigh
IM
Keratin 17 expression as a marker for epithelial transformation in viral warts
Am J Pathol
1993
143
1667
1678
[PubMed]
Purkis
PE
,
Steel
JB
,
Mackenzie
IC
,
Nathrath
WB
,
Leigh
IM
,
Lane
EB
Antibody markers of basal cells in complex epithelia
J Cell Sci
1990
97
39
50
[PubMed]
Raubenheimer
EJ
The myoepithelial cell: Embryology, function, and proliferative aspects
CRC Crit Rev Clin Lab Sci
1987
25
161
193
Redman
RS
Myoepithelium of salivary glands
Micr Res Tech
1994
27
25
45
[PubMed]
Rogers
MA
,
Langbein
L
,
Praetzel
S
,
Moll
I
,
Krieg
T
,
Winter
H
,
Schweizer
J
Sequences and differential expression of three novel human type-II hair keratins
Differentiation
1997
61
187
194
[PubMed]
Sakakura
T
,
Nishizuka
Y
,
Dawe
CJ
Mesenchyme-dependent morphogenesis and epithelial-specific cytodifferentiation in mouse mammary gland
Science
1976
194
1439
1441
[PubMed]
Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 546 pp.
Sanes
JR
,
Rubenstein
JLR
,
Nicolas
J-F
Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos
EMBO (Eur Mol Biol Organ) J
1986
5
3133
3142
[PubMed]
Schermer
A
,
Jester
JV
,
Hardy
C
,
Milano
D
,
Sun
TT
Transient synthesis of K6 and K16 keratins in regenerating rabbit corneal epithelium: keratin markers for an alternative pathway of keratinocyte differentiation
Differentiation
1989
42
103
110
[PubMed]
Schweizer, J. 1993. Murine epidermal keratins. In Molecular Biology of the Skin: The Keratinocyte. M. Darmon and M. Blumenberg, editors. Academic Press, Inc., San Diego, CA. 33–78.
Shapiro
MB
,
Senapathy
P
RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression
Nucleic Acids Res
1987
15
7155
7174
[PubMed]
Smith
FJ
,
Corden
LD
,
Rugg
EL
,
Ratnavel
R
,
Leigh
IM
,
Moss
C
,
Tidman
MJ
,
Hohl
D
,
Huber
M
,
Kunkeler
L
et al
Missense mutations in keratin 17 cause either pachyonychia congenita type 2 or a phenotype resembling steatocystoma multiplex
J Invest Dermatol
1997
108
220
223
[PubMed]
Stoler
A
,
Kopan
R
,
Duvic
M
,
Fuchs
E
Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during normal and abnormal epidermal differentiation
J Cell Biol
1988
107
427
446
[PubMed]
Takahashi
K
,
Coulombe
PA
Defining a region of the human keratin 6a gene that confers inducible expression in stratified epithelia of transgenic mice
J Biol Chem
1997
272
11979
11985
[PubMed]
Takahashi
K
,
Folmer
J
,
Coulombe
PA
Increased expression of keratin 16 causes anomalies in cytoarchitecture and keratinization in transgenic mouse skin
J Cell Biol
1994
127
505
520
[PubMed]
Takahashi
K
,
Yan
B
,
Yamanishi
K
,
Imamura
S
,
Coulombe
PA
The two functional type II keratin 6 genes of mouse show a differential regulation and evolved independently from their human orthologs
Genomics
1998
53
170
183
[PubMed]
Thorey
IS
,
Meneses
JJ
,
Neznanov
N
,
Kulesh
DA
,
Pedersen
RA
,
Oshima
RG
Embryonic expression of human keratin 18 and K18-beta-galactosidase fusion genes in transgenic mice
Dev Biol
1993
160
519
534
[PubMed]
Troyanovsky
SM
,
Guelstein
VI
,
Tchipysheva
TA
,
Krutovskikh
VA
,
Bannikov
GA
Patterns of expression of keratin 17 in human epithelia: dependency on cell position
J Cell Sci
1989
93
419
426
[PubMed]
Troyanovsky
SM
,
Leube
RE
Activation of the silent human cytokeratin 17 pseudogene-promoter region by cryptic enhancer elements of the cytokeratin 17 gene
Eur J Biochem
1994
225
61
69
[PubMed]
Troyanovsky
SM
,
Leube
RE
,
Franke
WW
Characterization of the human gene encoding cytokeratin 17 and its expression pattern
Eur J Cell Biol
1992
59
127
137
[PubMed]
van Genderen
C
,
Okamura
RM
,
Farinas
I
,
Quo
R-G
,
Parslow
TG
,
Bruhn
L
,
Grosscheld
R
Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in Lef-1-deficient mice
Genes Dev
1994
8
2691
2703
[PubMed]
Vogel
U
,
Denecke
B
,
Troyanovsky
SM
,
Leube
RE
,
Bottger
EC
Transcriptional activation of psoriasis-associated cytokeratin K17 by interferon-gamma. Analysis of gamma-interferon activation sites
Eur J Biochem
1995
227
143
149
[PubMed]
Wawersik
M
,
Paladini
RD
,
Noensie
E
,
Coulombe
PA
A proline residue in the α-helical rod domain of type I keratin 16 destabilizes keratin heterotetramers and influences incorporation into filaments
J Biol Chem
1997
272
32557
32565
[PubMed]
Weiss
RA
,
Eichner
R
,
Sun
TT
Monoclonal antibody analysis of keratin expression in epidermal diseases: a 48- and 56-kd keratin as molecular markers for hyperproliferative keratinocytes
J Cell Biol
1984
98
1397
1406
[PubMed]
Wiche
G
Plectin: general overview and appraisal of its potential role as a subunit protein of the cytomatrix
Crit Rev Biochem Mol Biol
1989
24
41
67
[PubMed]
Winter
H
,
Rogers
MA
,
Langbein
L
,
Stevens
HP
,
Leigh
IM
,
Labreze
C
,
Roul
S
,
Taieb
A
,
Krieg
T
,
Schweizer
J
Mutations in the hair cortex keratin hHb6 cause the inherited hair disease monilethrix
Nat Genet
1997
16
372
374
[PubMed]
Yang
Y
,
Dowling
J
,
Yu
QC
,
Kouklis
P
,
Cleveland
DW
,
Fuchs
E
An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments
Cell
1996
86
655
665
[PubMed]
Zhou
P
,
Byrne
C
,
Jacobs
J
,
Fuchs
E
Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate
Genes Dev
1995
9
700
713
[PubMed]

P.A. Coulombe was the recipient of Junior Faculty Research Award from the American Cancer Society (A-76019). K. McGowan was supported by NRSA fellowship (CA-67513) from the National Cancer Institute. This work was supported by a National Institutes of Health grant to P.A. Coulombe (AR-44232).

Address all correspondence to P.A. Coulombe, Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: (410) 614-0510. Fax: (410) 955-5759. E-mail: coulombe@jhmi.edu