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2386 East Heritage Way, Suite B, Salt Lake City, Utah 84109 USA Phone +1-877-628-7300 • Email—[email protected] www.pachyonychia.org ORIGINAL ARTICLE

Mice Expressing a Mutant Krt75 (K6hf ) Allele Develop Hair and Nail Defects Resembling Pachyonychia Congenita Jiang Chen1, Karin Jaeger2,3, Zhining Den4, Peter J. Koch1,4,5, John P. Sundberg2 and Dennis R. Roop1,4,5

KRT75 (formerly known as K6hf) is one of the isoforms of the 6 (KRT6) family located within the type II gene cluster on of humans and chromosome 15 of mice. KRT75 is expressed in the companion layer and upper germinative matrix region of the hair follicle, the medulla of the hair shaft, and in epithelia of the nail bed. Dominant mutations in members of the KRT6 family, such as in KRT6A and KRT6B cause pachyonychia congenita (PC) -1 and -2, respectively. To determine the function of KRT75 in skin appendages, we introduced a dominant mutation into a highly conserved residue in the helix initiation peptide of Krt75. Mice expressing this mutant form of Krt75 developed hair and nail defects resembling PC. This mouse model provides in vivo evidence for the critical roles played by Krt75 in maintaining hair shaft and nail integrity. Furthermore, the phenotypes observed in our mutant Krt75 mice suggest that KRT75 may be a candidate gene for screening PC patients who do not exhibit obvious mutations in KRT6A, KRT6B, KRT16, or KRT17, especially those with extensive hair involvement. Journal of Investigative Dermatology (2008) 128, 270–279; doi:10.1038/sj.jid.5701038; published online 13 September 2007

INTRODUCTION the skin, hair follicles, and nails. However, each member of The keratin 6 (KRT6) cluster consists of several genes that the KRT6 gene family shows a cell- and tissue-type-specific encode (IF) belonging to the expression pattern. KRT6A and KRT6B are constitutively type II keratin family. In humans, functional KRT6 genes expressed in the outer root sheath of anagen-stage hair include KRT6A, KRT6B, KRT6C (formerly known as KRT6E/ follicles, epithelia of the nail bed, and stratified epithelia H), KRT75 (formerly known as K6hf) and KRT71-KRT74 lining the oral cavity and the esophagus (Moll et al., 1982; (formerly known as K6irs1-K6irs4), whereas in the mouse Ouhayoun et al., 1985; Stark et al., 1987; O’Guin et al., Krt6a, Krt6b, and Krt71-Krt75 make up this family of 1990; Langbein and Schweizer, 2005). KRT75 is expressed genes (These designations are used according to the in the companion layer, upper germinative matrix region of new nomenclature for mammalian (Schweizer the hair follicle, and medulla of the hair shaft (Winter et al., et al., 2006).). These genes share striking sequence simi- 1998; Wojcik et al., 2001; Wang et al., 2003). In addition, larities and are widely expressed in epithelial tissues, especially the mouse keratin 75 (K75) is also synthesized in the nail bed epithelia of mice (Wojcik et al., 2001). 1Department of Molecular and Cellular Biology, Baylor College of Medicine, In addition to constitutive expression, several KRT6 Houston, Texas, USA; 2The Jackson Laboratory, Bar Harbor, Maine, USA; isoforms are specifically induced in response to hyperproli- 3Department of Dermatology, Medical University of Vienna, Vienna, Austria ferative stimuli, such as wounding. For example, KRT6A is 4 and Department of Dermatology, Baylor College of Medicine, Houston, induced in all layers of the epidermis under hyperprolifera- Texas, USA tive conditions, whereas the induction of KRT6B is restricted 5Current address: Department of Dermatology and Regenerative Medicine and Stem Cell Biology Program, University of Colorado at Denver and Health to the more differentiated layers of the epidermis (Wojcik Sciences Center, Aurora, Coloardo 80045, USA et al., 2000). The in vivo functions of Krt6 genes have been Correspondence: Dr Dennis R. Roop, Department of Dermatology and evaluated in genetically engineered mouse models for Krt6a Regenerative Medicine and Stem Cell Biology Program, University of (Krt6atm1Der) and Krt6a/Krt6b (Krt6a/Krt6btm1Cou, Krt6a/ Colorado at Denver Health Sciences Center, PO Box 6511, Mail Stop 8320, Krt6btm1Der) (Wojcik et al., 2000, 2001; Wong et al., 2000). Aurora, Colorado 80045, USA. E-mail: [email protected] Interestingly, the absence of Krt6a or Krt6a/K6b did not Abbreviations: BAC, bacterial artificial chromosome; ES, embryonic stem IF, intermediate filament; K75, human and mouse keratin 75 (formerly known prevent normal development and function of epithelial as K6hf) protein; KRT6, keratin 6; KRT75, human keratin 75 (formerly known tissues and ectodermal appendages. However, loss of these as human K6hf) gene; Krt75, mouse keratin 75 (formerly known as mouse tm1Der proteins altered the response of these tissues to injury and K6hf) gene; Krt75 , mouse keratin 75-targeted mutation; PC, mechanical stress. For instance, delayed re-epithelialization pachyonychia congenita; PC-1, Type I PC; SEM, scanning electron tm1Der microscopy; SM-CSM, selection counter selection marker of the skin was observed in Krt6a mice following Received 7 May 2007; accepted 24 June 2007; published online wounding (Wojcik et al., 2000), and hyperplastic changes 13 September 2007 secondary to mechanical stress associated with food intake

270 Journal of Investigative Dermatology (2008), Volume 128 & 2007 The Society for Investigative Dermatology J Chen et al. Mouse Model for a Dominant Krt75 Mutation

were reported in the oral cavities of Krt6a/Krt6btm1Cou and To explore the role of mouse Krt75 in hair and nail Krt6a/Krt6btm1Der mice (Wong et al., 2000; Wojcik et al., formation and generate a mouse model that more closely 2001). In contrast to Krt6a and Krt6b, Krt75 expression is not mimicked human PC, we genetically engineered a dominant induced in the epidermis in response to mechanical stress or mutation into the mouse Krt75 locus. We present, the first wounding (Wojcik et al., 2001). reported ‘‘knock-in’’ model for a dominant type II keratin Mutations in KRT6A and KRT6B result in pachyonychia mutation. We report that deletion of the highly conserved congenita (PC) (OMIM #167200 and #167210). PC is a rare, asparagine residue (N159) in the initiation motif of the helical autosomal dominant form of ectodermal dysplasia, charac- rod domain of Krt75 caused collapse of the keratin IFs terized by distally progressive hypertrophic onychodystrophy in vitro, and produced hair and nail abnormalities in vivo. and focal hyperkeratosis of palms and soles (Leachman et al., 2005). Germline mutations in KRT6A and KRT16 are RESULTS associated with Type I PC (PC-1), whereas mutations in Generation of a Krt75 dominant mutation that interferes with KRT6B and KRT17 are associated with Type II PC (PC-2) normal IF assembly in cultured cells (Smith et al., 2005). KRT16 and KRT17 encode type I keratins Dominant-negative mutations have been identified in the (K16 and K17) that form keratin IF together with K6A and KRT6A gene of PC-1 patients. The most frequent mutation is a K6B, respectively. Codon 171 (AAC) and 172 (AAC) of deletion of codon 172, encoding asparagine (N). It is thought KRT6A both encode asparagines (N). Previous reports that this mutation leads to the synthesis of a defective K6a referred to deletion of one of these codons as either protein that interferes with the assembly of a functional N171del or N172del. The Human Genome Variation Society keratin IF . A comparison of the NH2-terminal (www.hgvs.org) has recently recommended that when a rod domains of human K6A and mouse K75 reveals a high codon is deleted from a series of identical codons, the degree of amino-acid sequence homology (Figure 1a). N172 deletion should be designated as the last codon in the series. of human K6A corresponds to N159 of mouse K75. There- Therefore, in accordance with this recommendation, we have fore, we hypothesized that a deletion of N159 (N159del) now designated N171del as N172del. We previously suggested that N172del was a mutational ‘‘hotspot’’ KRT6A GAGCGTGAACAGATCAAGACCCTCAACAACAAGTTTGCCTCCTTCATC (Lin et al., 1999) and a recent review as confirmed that K6A -E--R--E--Q--I--K--T--L--N--N--K--F--A--S--F--I-- N172del is the most common mutation in PC-1 (Lane and (172) McLean, 2004). This residue is located at the beginning of the KRT75 GAACGGGAGCAGATCAAGACTCTGAACAACAAGTTCGCCTCCTTCATT K75 -E--R--E--Q--I--K--T--L--N--N–--K--F--A--S--F--I-- initiation motif of the highly conserved helical rod domain (159) (also called the helix initiation peptide) of keratins. The short initiation motif is believed to be critical in mediating keratin assembly and maintaining IF stability. Analogous mutations in this evolutionarily conserved amino-acid residue have been reported in white sponge nevus (OMIM #193900), caused by deletion of the corresponding asparagine in KRT4 (Rugg et al., 1995), ichthyosis bullosa of Siemens caused by mutations in KRT2 (Whittock et al., 2001), and epidermolytic palmoplantar keratoderma caused by mutations in KRT9 (Reis et al., 1994). Many studies have noted that there is considerable variation in clinical severity of diseases caused by these mutations. Consequently, other genetic and/or environmental modifiers have been suggested to contribute significantly to disease severity. Mouse models with targeted deletions of Krt6a, Krt6b, and Krt17 have been developed (Wojcik et al., 2000, 2001; Wong et al., 2000; McGowan et al., 2002). However, these models did not fully recapitulate the clinical features of PC as seen in Figure 1. Alignment of nucleotide and peptide sequences of human KRT6A humans, in particular the nail changes, which occur in almost and mouse Krt75, and immunofluorescence labeling of PtK2 cells transfected with wild-type and mutant Krt75 expression vectors. (a) The 16 amino acids all PC patients (Leachman et al., 2005). One explanation for at the beginning of the initiation motif of the human K6A (NM005554) and this discrepancy is that PC cases are caused by dominant mouse K75 (XM128143) are identical. The amino acid corresponding to N172 mutations, which result in the accumulation of mutated of human KRT6A is underlined for mouse Krt75. Deletion of codon 159 in keratins acting in a dominant interfering manner to prevent mouse Krt75(N159del) was generated to reproduce the corresponding the normal assembly of IF within affected cells. The Krt6a-, mutation in human KRT6A that is frequently detected in PC patients. Krt6b-, and Krt17-targeted deletion mouse models represent (b, c) The endogenous keratin IF network was labeled with an anti-keratin18 (K18) antibody (green). Ectopically expressed K75 was labeled with a recessive mutations, which result in a lack of expression. The polyclonal antibody (red). Both (b) wild-type ( / ) and (c) mutant þ þ lack of K6a, K6b, or K17 proteins in these null mouse models (K75tm1Der) K75 were colocalized with the endogenous keratin filament was partially compensated for by other keratins, which we structure (yellow). (c) Mutant K75 caused the collapse of the keratin believe prevented the development of overt PC phenotypes. IF network around the nucleus. Bar 50 mm. ¼

www.jidonline.org 271 J Chen et al. Mouse Model for a Dominant Krt75 Mutation

tm1Der would lead to a mutant K75 protein (K75 ) with / 123456789 tm1Der tm1Der dominant-negative properties. tm1Der +/+ Krt75 Krt75 Wild-type and mutated versions of mouse Krt75 were +/+ +/ Krt75 expressed in PtK2 cells. This rat kangaroo kidney cell line Krt75tm1Der Krt75tm1Der +/+ expresses simple epithelia-type keratins, such as +/+ (K8) and (K18). Ectopically expressed K75 co- 123456 assembled into the endogenous keratin IF cytoskeleton of Krt75tm1Der tm1Der these cells (Figure 1b, also see Wojcik et al., 2001). Krt75 /Xmn I Expression of the N159del mutant, however, resulted in a neo collapse of the endogenous IF cytoskeleton around the nucleus (Figure 1c). These experiments demonstrate that / / tm1Der tm1Der tm1Der tm1Der deletion of codon N159 leads to a mutant protein that 5 tm1Der tm1Der 5 Krt75 Krt75 Krt7 Krt75 interferes with normal cytoskeleton architecture. Interest- +/+ +/ +/ Krt7 Krt75 ingly, this dominant-negative effect is not restricted to a loss +/+ of codon N159. Expression of Krt75 cDNA constructs with an Krt75tm1Der N159K missense mutation (corresponding to the N172K Figure 2. Characterizations of recombinant ES cells and mice. mutation in KRT6A, which is also found in PC patients) (a) Genotyping of targeted ES cells with the 30 Krt75 probe. Southern blots induced a similar collapse of the keratin IF cytoskeleton in using MfeI- and PmeI-digested recombinant ES cells DNA (heterozygous) PtK2 cells (data not shown). identified 12.1 kb (wild-type allele) and 7.3 kb (recombinant allele) bands (lane 1, 6 and 9) (Figure S2). The 30 probe was generated by PCR with primers K6hf-3 -Probe2-F and K6hf-3 -Probe2-R shown in Table S1. (b) Genotyping Generation of N159del mutant Krt75 mice 0 0 of recombinant ES cells lacking the neo-cassette after cre-mediated Our in vitro experiment suggested that a deletion of codon recombination. Cre-recombinase was transiently expressed in the above N159 in Krt75 would lead to the synthesis of a mutant protein targeted ES cell clones. Daughter ES cell clones were genotyped by Southern with dominant-negative properties, which could potentially blot for the excision of the neo-cassette (lane 5). (c) Genotyping of mutant result in hair and nail defects in vivo. To test this hypothesis, mice by PCR using genomic DNA as a template. The same primer pair we generated a mouse line in which codon N159 is deleted (K6hf-Exon1-F and K6hf-Intron1-R in Table S1) amplified a slightly larger from the endogenous Krt75 allele (see Figures S1 and S2 fragment from the mutant allele than from the wild-type allele, due to the insertion of a loxP site in intron 1 of the mutant allele (upper panel). for a detailed description of how the targeting vector was tm1Der Heterozygous mutant mice ( /Krt75 ) carry both the wild-type and the þ generated). mutant allele. However, homozygous mutant mice (Krt75tm1Der/Krt75tm1Der) Two independent recombinant embryonic stem (ES) cell carry exclusively the mutant alleles. Digestion of the PCR products with XmnI clones were used to generate the N159del (mouse keratin 75- resulted in the cleavage of the mutant band. This is due to the introduction of targeted mutation (Krt75tm1Der)) mice (see Figure 2 for an XmnI restriction site in the mutant allele. (d) Analysis of mutant Krt75 characterization of recombinant ES clones and founder expression in mice by semi-quantitative RT–PCR. A 50 biotin-labeled primer mice). The genetically independent mouse lines derived from was used to amplify the Krt75 cDNA followed by XmnI digestion, gel separation, and chemiluminescent detection of the biotin-labeled PCR these ES cell clones showed the same phenotypes (see tm1Der product. Only the mutant transcripts could be digested. Note that only one below). Heterozygous mutant ( /Krt75 ) intercrosses primer was labeled with biotin, therefore, after digestion, only one mutant þtm1Der tm1Der yielded homozygous mutant (Krt75 /Krt75 ), hetero- DNA fragment could be detected. Of the two homozygous mutant mice zygous and wild-type progeny with a frequency consistent (Krt75tm1Der/Krt75tm1Der) shown here, only mutant DNA was detectable. In the heterozygous mice ( /Krt75tm1Der), a comparable amount of both wild-type with simple Mendelian inheritance for a dominant mutation. þ All mutant mice were affected, with homozygous mice and mutant DNA was detected, indicating that both alleles are expressed at affected more severely than heterozygous mice (see below). similar levels. Previous reports have also documented that humans homo- zygous for certain dominant keratin mutations exhibit more mutant mouse at 12 weeks of age is shown in Figure 3a. severe clinical symptoms than their heterozygous parents Heterozygous mice developed a similar coat defect; (Hu et al., 1997). The birth weight, growth rate, and fertility however, the phenotype was less prominent. The of heterozygous and homozygous mice were comparable gross hair phenotype was not restricted to a particular to those of wild-type littermates. To confirm that the mutated anatomic location or hair type and these changes persisted Krt75 allele was expressed in mutant mice, we conducted throughout life. semi-quantitative reverse transcription–PCR (RT–PCR) experi- To determine whether structural abnormalities of the hair ments. As shown in Figure 2d, wild-type and mutant alleles shaft caused the rough appearance of the mutant coat, hairs were expressed at similar levels in heterozygous mice, were manually removed and studied from the dorsal thoracic whereas only mutant transcripts were detected in homo- region of wild-type and mutant mice at 1, 3, and 8 weeks of zygous mutant mice. age, with consistent findings. As shown in Figure 3, hair shafts from 8-week-old wild-type mice had regular septation and Mutant Krt75 mice showed degenerative keratinization within septulation patterns specific for each hair type (Figure 3c and the precortex and hair shaft d). Heavily pigmented hairs of mutant littermates had defects Krt75 tm1Der mice developed an apparent rough coat within 2 ranging from loose irregular aggregation of pigment, clump- weeks of age. The typical rough coat of a homozygous ing of pigment within the medulla, clumping with focal

272 Journal of Investigative Dermatology (2008), Volume 128 J Chen et al. Mouse Model for a Dominant Krt75 Mutation

12 10 8 6 4 2 0 /

Bleb-positive hair fibers (%) Bleb-positive +/+ tm1Der tm1Der tm1Der Krt75 Krt75 +/ Krt75 cde fghijkl mn op

Figure 3. Hair-associated phenotypes of the mutant mice. (a) Gross appearance of 12-week-old mice. The wild-type ( / ) mouse had a shiny uniformly þ þ smooth coat, whereas a homozygous mutant (Krt75tm1Der/Krt75tm1Der) mouse exhibited a rough coat. (b) Percentage of ‘‘bleb’’-positive hair shafts in plucked dorsal hair of 8-week-old littermates; n 10 for homozygous mutant mice (Krt75tm1Der/Krt75tm1Der), n 4 for heterozygous mutant mice (Krt75tm1Der/ ). ¼ ¼ þ (c–p) Variations in hair shaft defects in 8-week-old mutant mice. (c, d) Normal hair shafts have well demarcated septation and septulation patterns accentuated to various degrees by pigmentation. By contrast, both heterozygous and homozygous mice carrying the mutant Krt75tm1Der allele exhibited various degrees of focal change ranging from (e–g) pigment clumping, (h–n) focal bulges, (o) structural weakness, and (p) fracture. Mean7SD. Bar 100 mm. ¼

k

Figure 4. Progressive changes leading to hair shaft rupture. (a–e) Light microscopy with the condenser removed to increase light refraction. Lightly pigmented hairs illustrated (a, b) slight focal swelling, (c) changes in medulla cells from rectangular to round clumping, (d) separation of cells with breakage of cuticle, and (e) breakage of shaft with dispersion of abnormal rounded medullary cells. (f–k) Hair shaft defects observed by SEM. Characteristic findings include (f–g) focal swellings in a subpopulation of hair shafts on unmodified skin, (h–i) split cuticles, (j) broken ends of the hair shaft exposing the rounded ghost cells, (k) and the torn cuticle. Bar 200 mm in (a–e); bar 10 mm in (f–k). ¼ ¼ distention of the shaft, segregation of pigment with a light zygous mutant mice and 7.8872.27% of homozygous mice brown colored medullary abnormality, to breakage of the hair (Figure 3b), suggesting a dose-dependent effect of the shaft in the middle of these deformities (Figure 3e–p). Hair mutation. Defects could be seen in mildly to nonpigmented shafts that had observable blebs under light microscopy hair shafts when the microscope condenser was removed to comprised 3.0270.52% of counted hair shafts in hetero- highlight features of the cuticle (Figure 4a–e). The focal

www.jidonline.org 273 J Chen et al. Mouse Model for a Dominant Krt75 Mutation

distentions of the hair shafts were associated with clusters 2004). Sulfur concentration measured as a percent of total of loose aggregates of round cells associated with fracturing elements of clinically normal, wild-type hair shafts of the cuticle, leading to breakage of the shaft. All of the (1.9870.40%) were within normal ranges for other strains 4 major mouse pelage hair types were affected, including and stocks of mice (Mecklenburg et al., 2004). By contrast, the vibrissae (Figure S3). These changes were observable sulfur levels within the focal swellings of hair shafts were in three dimensions by scanning electron microscopy significantly lower (1.2470.40%) than that of controls (SEM). While most hair shafts appeared to be normal, focal (Figure 6e). Interestingly, sulfur levels along the length of swellings were observed in some (Figure 4f and g). In other the defective hair shafts varied between 1.01 and 3.39% (data regions, the cuticle split (Figure 4h and i) and eventually broke not shown), which is consistent with the above observation (Figure 4j and k). At these break sites, the cuticle fragmented that various degrees of abnormalities can occur along the in a linear manner (Figure 4k), exposing the rounded ghost defective hair shaft. The phenotype was restricted to hair cells (Figure 4j). follicles and hair shafts. We did not observe structural defects Histological evaluation of skin sections from 8-week-old in the interfollicular epidermis as determined by histology, mutant mice revealed that while some of the hair follicles which is consistent with previous studies demonstrating that appeared to be normal (Figure 5a), others had focal swellings Krt75 expression is restricted to hair follicles. in the precortical region of anagen VI-stage follicles immediately above the dermal papilla. Cells in this region Mutant Krt75 mice developed loosely attached and were brightly eosinophilic with basophilic nuclei (Figure 5b). hypertrophic nails Fading nuclear staining in these cells resulted in the Nails on the hind feet of adult mutant mice were frequently formation of ‘‘ghost’’ cells (Figure 5c and d). More normal lost (data not shown). When mutant mice were inspected on appearing septated structures developed under these struc- a cage rack, their nail plates were often ripped off from the tures, pushing them to the surface (Figure 5e). Longitudinal nail bed during the process of removing mice from the cage sections of affected hair shafts in the histological sections rack and returning them to their cage. This was never revealed multiple defects within individual shafts that varied observed in wild-type littermates handled in a similar in severity (Figure 6a). Changes in these hematoxylin and manner. Furthermore, nails of mutant mice showed progres- eosin-stained sections demonstrated the details of ghost cells sive thickening, distally (Figure 7). This phenotype was in the focal distended areas that separated as the cuticle most frequently observed in adult mice, especially homo- ruptured (Figure 6b–d). zygous mutant males (Figure 7c and f). Close examination Element analysis of hair shafts is a useful semi-quantitative of deformed toe nails showed that these nail plates method to evaluate structural integrity (Mecklenburg et al., appeared opaque, had a rough surface, and overgrew in

med

med

med

med

dp dp dp

dp

dp bulb

Figure 5. Krt75tm1Der/Krt75tm1Der mutant mice have abnormal late anagen-stage hair follicles. (a) Normal hair follicle of 8-week-old Krt75tm1Der/Krt75tm1Der mutant mice. (b–e) Degenerative changes in the precortex of the hair bulb. (b) Initial changes include hyper-eosinophilia and rounding of precortical cells, while nuclei retain basophilic staining (yellow arrow). (c, d) These changes continue forming bulbous swellings (red arrows) consisting of round cells with ghost- like nuclear remnants. (e) A normal shaft then forms below these abnormal structures, forcing them to the surface. Bar 50 mm. dp, dermal papilla; med, ¼ medulla.

274 Journal of Investigative Dermatology (2008), Volume 128 J Chen et al. Mouse Model for a Dominant Krt75 Mutation

Reduced levels of K6a and K6b did not exacerbate the mutant Krt75 hair and nail phenotypes We were curious to know whether the hair and nail phenotypes observed in our mice might be due to an interference of the mutant K75 protein with K6a and K6b. Since we previously generated Krt6a/Krt6b-null mice (Krt6a/ Krt6btm1Der) (Wojcik et al., 2001), it was possible to test this hypothesis experimentally. Krt6a, Krt6b, and Krt75 genes are tightly linked (within 140 kb) on mouse chromosome 15. Therefore, it was not possible to generate Krt75 homozygous mutants on a Krt6a/Krt6btm1Der double homozygous mutant background through breeding. However, it was possible to generate mice that were heterozygous for the mutant Krt75 allele, and also heterozygous for Krt6a/Krt6btm1Der alleles. These mice synthesized only 50% of the K6a and K6b proteins. Therefore, if the mutant Krt75 phenotypes result in part by interfering with wild-type K6a and K6b proteins, we would have predicted that the loss of one of the wild-type Krt6a and Krt6b alleles would exacerbate the mutant Krt75 hair and nail phenotypes observed in mutant Krt75 heterozygotes. Since this was not observed, our results suggest that the mutant K75 proteins do not interfere with K6a and K6b in vivo, at least not in the heterozygous state. 2.5 P=0.038 2.0 DISCUSSION Human hair shaft abnormalities are grouped into four major 1.5 classes: fractures, irregularities, coiling and twisting, and 1.0 extraneous matter on the hair shaft (Whiting, 1994; Whiting 0.5 and Howsden, 1996). Changes seen in hair shafts from 0.0 tm1Der

Sulfur content (% weight) Krt75 mice exhibit various forms of trichorrhexis +/+ tm1Der tm1Der Krt75 /Krt75 nodosa, trichoptilosis, monilethrix, pseudomolethrix, tapered Figure 6. Abnormal bulbous hair shafts observed in longitudinal sections and hair, and bayonet hair, depending on the stage of degen- reduced sulfur content within the focal lesions. (a–d) Longitudinal sections of erative change. Other mutant mice often have weak hair hair shafts cut in paraffin sections. (a) Most of the shafts appear to be relatively shafts that twist, split, and break, but very few develop normal, with focal deformities (indicated by arrows) containing eosinophilic nodular focal swellings that break off with a bulbous end ghost-like cells (boxed area) in their central region. (b) Higher magnification of boxed area reveals ghost-like cells. (c) Lightly pigmented hairs lose (Sundberg, 1994; Hogan et al., 1995). The best mouse tm1Der pigmentation in deformed areas and (d) cells locate near poles as shafts mutations studied to date that resembles the Krt75 near rupture state. (e) Sulfur content of wild-type hair shafts measured by model described here are the allelic mutations in the energy-dispersive X-ray spectroscopy shows that the wild-type hair shafts desmoglein 4 gene originally described as lanceolate hair ( / , n 15) contain a sulfur content that is within the normal range þ þ ¼ (Montagutelli et al., 1996; Sundberg et al., 2000). These (1.9870.40%), whereas the sulfur content was reduced (1.2470.40%) within mutant mice also develop bulbous changes in the precortical the focal swellings of mutant hair shafts (Krt75tm1Der/Krt75tm1Der, n 11). ¼ region of late anagen-stage hair follicles. Cells also appear to Mean7SD. The reduction of sulfur content within the focal swellings was statistically significant (P 0.038). Note that the the condenser was removed undergo coagulative necrosis forming ghost cells, although ¼ to increase the contrast of cuticules. Bar 100 mm. the cells organize in a laminated pattern as opposed to ¼ rounding up. Hair shafts emerge with a series of irregular focal nodules that break off to form a bulbous end with a point resembling a lance head. These desmoglein 4 both lateral and vertical directions, resulting in ‘‘ski slope- spontaneous mouse mutations were later found to accurately like’’ hypertrophic nails (Figure 7e and f). Histologically, model a rare human disease called localized autosomal there was a separation of the nail bed from the underlying recessive hypotrichosis (Kljuic et al., 2003), and later found in connective tissue, with large areas of hemorrhage (Figure 7h) several other species (Jahoda et al., 2004). A variety of in some nails. This presumably is the early lesion leading to mutations are under investigation, which result in subtle hair avulsion of the nail plate. Slightly overgrown nail plates were medulla defects in genes that regulate hair precortex function associated with extension of the stratum granulosum and (Potter et al., 2006). Structural genes, such as the keratins, are corneum under the nail plate beyond normal limits. This at the end of a complex gene network. Using groups of causes a loss of adhesion and can result in detachment when mutations in genes that are involved in various stages of nails catch on rough surfaces. precortex development and hair medulla formation are

www.jidonline.org 275 J Chen et al. Mouse Model for a Dominant Krt75 Mutation

p

d e f h

p

Figure 7. Nail-associated phenotypes of the mutant mice. (a–c) Gross appearance of paws of wild-type ( / ), heterozygous ( /Krt75tm1Der), and homozygous þ þ þ (Krt75tm1Der/Krt75tm1Der) mutant mice. Note the development of hypertrophic nails in (b) heterozygous and (c) homozygous littermates (indicated by arrows). (d–f) A lateral view of framed toes in (a–c). (g–h) Histology of toe nails of wild-type and Krt75tm1Der/Krt75tm1Der mice, indicated by open arrows in (a) and (c), respectively. The mutant nail shows the severely curved nail plate and hemorrhage in the nail bed compartment. Bar 500 mm in (a–f); 200 mm in (g) and (h). p, nail plate. ¼

certainly advantageous in defining the functions of these severity of nail phenotypes can vary from individual to genes in this complex network. individual even within one family with an identical mutation In humans, a polymorphism of KRT75 was linked to (Leachman et al., 2005). In addition, a subset of PC patients pseudofolliculitis barbae, the ingrowth of facial hair triggered has no nail changes until the second or third decade of life. by frequent shaving (Winter et al., 2004). This mutation was This subtype was designated as PC tarda (Paller, Moore and located at the initiation motif of the highly conserved helical Scher, 1991). Based on a small number of cases, it was rod domain, only three residues downstream from the speculated that the late onset of PC was associated with mutational ‘‘hot spot’’ reproduced in this study (see mutations occurring in less critical regions of the keratin Introduction). It was speculated that this KRT75 mutation proteins (Connors et al., 2001; Xiao et al., 2004; Smith et al., compromised the cuticle of the hair shaft, resulting in the 2005). In our current study, the mutation introduced into the ingrowth of hair shaft. However, KRT75 is expressed in both Krt75 gene is located at the beginning of 1A, the helix the companion layer and upper matrix region of the hair initiation peptide, which is believed to be critical for follicle, as well as the medulla of the hair shaft. Whether the keratin–keratin interactions (Steinert et al., 1993). Corre- expression of KRT75 in the medulla and other regions of the sponding mutations in KRT6A and KRT6B cause PC hair follicle also contributed to hair ingrowth was not clear. In phenotypes at or shortly after birth, and are usually severe. our current study, we clearly observed pathological changes The reason that the Krt75 mutation did not affect all nails to in the precortical and hair medulla cells, as well as the the same degree is not clear, but it could be dependent on the rupture of the hair cuticle. However, we could not amount of mechanical stress that individual nails are demonstrate whether the rupture of the hair cuticle was a subjected to, that result in avulsion. sequential process secondary to the focal swelling of the In summary, we have generated a ‘‘knock-in’’ mouse medulla, or perhaps that the hair cuticle was compromised in model in which one endogenous Krt75 allele was replaced the first place. Nevertheless, this mouse model provides with an allele containing a deletion of the highly conserved in vivo evidence for the critical role played by Krt75 in asparagine residue (N159). Mice expressing this mutant form maintaining hair shaft integrity. of Krt75 developed spontaneous hair and nail phenotypes Since Krt75 is also expressed in the nail epithelia of mice, that partially resembled phenotypes observed in PC patients we expected the mutant mice to develop nail phenotypes, who harbor a corresponding mutation in KRT6A. On the basis such as congenital nail thickening, the most consistent of the phenotypes observed in our mutant Krt75 mice, we phenotype observed in PC patients, with a mutation at the suggest that KRT75 may be a candidate gene for screening PC corresponding residue of KRT6A. However, the hypertrophic patients who do not exhibit obvious mutations in KRT6A, nail phenotype of the mutant Krt75 mice was not routinely KRT6B, KRT16, or KRT17, especially those with extensive observed in all mice. It was primarily restricted to the nails of hair involvement. the hind paws of adult male mice and was more frequently observed as a result of fighting when males were housed MATERIALSANDMETHODS together. Although the observed variations in nail phenotype Mouse Krt75 expression vectors may be due to the fact that these mice were on a segregating The full-length mouse Krt75 cDNA was cloned in the expres- genetic background, it is still in line with the fact that in PC sion vector pcDNA3.1 as previously reported (Wojcik et al., patients, not all digital nails are necessarily involved and the 2001). Codon N159 of mouse Krt75 (Figure 1a) was deleted using

276 Journal of Investigative Dermatology (2008), Volume 128 J Chen et al. Mouse Model for a Dominant Krt75 Mutation

a PCR-based mutagenesis strategy (site-directed mutagenesis, ES cell targeting and generation of mutant mice Stratagene, La Jolla, CA). Similar methods were used to generate a The targeting vector (Figure S2) was electroporated into W4/129S6 cDNA clone carrying a missense mutation in which the asparagine ES cells (Taconic, Hudson, NY), and addition of G418 (geneticin) residue was replaced with a lysine residue (N159K). These mutations and FIAU (1(1-2-deoxy-2-fluoro–darabinofuransyl)-5-iodouracil) to are analogous to the N172del and N172K mutations found in KRT6A the cell culture medium was used to select for recombinant ES cell in PC-1. Primers used to generate these mutations were K6hf- clones. Southern blot analysis, using mouse Krt75-specific probes N159del-F and K6hf-N159del-R, and K6hf-N159K-F and K6hf- (Figure S2) and a neo-cassette probe, confirmed homologous N159K-R (Table S1). All cDNA clones were confirmed by DNA recombination in 25% of the ES clones. To excise the neo-cassette sequencing. from the Krt75 gene locus, recombinant ES cells were electroporated with a cre-expression vector (Arin et al., 2001), and neo-sensitive Expression of wild-type and mutant K75 in cultured cells clones were tested by Southern blot to verify loss of the neo-cassette. Wild-type or mutant mouse Krt75 cDNAs was transfected into PtK2 Two targeted ES cell clones were injected into C57BL/6J blastocysts. cells, a rat kangaroo kidney epithelial cell line (ATCC, Manassas, Chimeric mice were obtained and backcrossed with C57BL/6J mice. VA), which expresses K18 but not K6, using the FuGENE 6 Progeny carrying the targeted alleles ( /Krt75tm1Der ) were identified þ transfection reagent (Roche Diagnostics, Indianapolis, IN). Cells by PCR analysis of genomic DNA obtained from tail biopsies. were analyzed 48 hours after transfection by immunofluorescence Homozygous pups (Krt75tm1Der/Krt75tm1Der) were obtained from microscopy. heterozygous ( /Krt75tm1Der) intercrosses. þ All animal studies were approved by the Baylor College of Gene targeting vector construction Medicine Institutional Animal Care and Use Committee (IACUC) A bacterial artificial chromosome (BAC) clone containing the full- and were performed in compliance with stipulations of that body. length mouse Krt75 gene locus was identified in a 129/SvJ genomic Mice were maintained in a conventional barrier facility at 22721C library (Incyte Genomics, St Louis, MO) and used to construct the and 30–70% relative humidity, exposed to a 14-hour light cycle, gene targeting vector via bacterial homologous recombination (ET allowed free access to sterilized acidified water (pH 2.8–3.2), and fed recombination) (Zhang et al., 1998, 2000) as outlined in Figure S1. irradiated LabDiets 5053 (Pico Lab, St Louis, MO) ad libitum. The All primer sequences used in the construction of targeting vector are health status of each animal room was evaluated every 13 weeks. listed in Table S1. Briefly, the 1.3 kb RpsL-neo selection counter selection marker (SM-CSM; Gene Bridges, Dresden, Germany) was RNA extraction and semi-quantitative RT–PCR generated by PCR using primers BAC-K6hf-N159del-Selection-F and Total RNA was isolated from ear biopsies of adult mice. 0.5 mg total BAC-K6hf-N159del-Selection-R. These primers contain 50 bp Krt75 RNA was reverse transcribed into cDNA with AMV reverse sequences (underlined) corresponding to a region upstream and transcriptase (Promega, Madison, WI) and poly d(T)15 primers downstream of codon 159, respectively. After transforming pSC101- (Roche Diagnostics, Indianapolis, IN). Subsequently, the cDNA BAD-gbaAtet (Gene Bridges, Dresden, Germany) into the Escherichia was amplified with mouse Krt75-specific primers. The primers used coli host containing the Krt75 BAC, homologous recombination was to amplify the mouse Krt75 were K6hf-50-UTR-Biotin-F and K6hf- induced by adding L-arabinose. After introduction of the SM-CSM cDNA-R that were derived from different exons to avoid amplifica- PCR products into the BAC host cells, homologous recombination tion of genomic DNA (Table S1). The forward primer was 50 labeled replaced codon 159 with SM-CSM (Figure S1). Correctly targeted with biotin for semi-quantitative chemiluminescent detection. BAC clones were selected by kanamycin resistance, which is Primers used to amplify mouse b- (Actb) were b-actin-F and encoded by SM-CSM. In a second round of recombination, the b-actin-R (Table S1). Semi-quantification was performed as pre- SM-CSM was replaced by a 362 bp PCR-generated Krt75 fragment viously described (Cao et al., 2001), except that the restriction that contained the N159 deletion mutation. Primers used to generate digestion was performed with XmnI, which cleaves the mutant but this DNA fragment were BAC-K6hf-N159del-Counter-F and BAC- not the wild-type Krt75 sequence. Biotin-labeled nucleic acids were K6hf-N159del-Counter-R. detected with a Chemiluminescent Nucleic Acid Detection Module Subsequently, a PGK-neo cassette (Gene Bridges, Dresden, (Pierce, Rockford, IL) as described in the instructions of the Germany) was generated by PCR (Figure S1). The neo-cassette has manufacturer. dual prokaryotic (Tn5) and eukaryotic (PGK) promoters and is flanked by loxP sites. PCR amplification introduced a 60-bp Tissue processing, histologic analysis, and immunofluorescence sequence to each side of the cassette, which allowed insertion of Mice were euthanized by CO2 asphyxiation. Skin for both routine this marker into intron 1 sequences of the Krt75 BAC (Figure S1). histology and immunohistochemistry was fixed overnight in Fekete’s Primers used to amplify the neo-cassette were BAC-K6hf-Neo-Insert- acid–alcohol–formalin solution (61% ethanol, 3.2% formaldehyde, F and BAC-K6hf-Neo-Insert-R. Finally, the modified Krt75 locus 0.75 N acetic acid), transferred to 70% ethanol, embedded in was subcloned by bacterial homologous recombination into a paraffin, sectioned at 5–6 mm and, finally, placed on microscope pUC-TK plasmid vector, which contains a herpes simplex virus slides (Superfrost/Plus, Fisherbrand, Pittsburgh, PA) and stained with thymidine kinase (TK) gene under the control of a PGK promoter. hematoxylin and eosin for routine histopathologic analysis. Double- Sequences homologous to Krt75 were added to the pUC-TK immunofluorescence labeling was performed as described pre- vector using primers (Left and Right) by PCR, through which viously (Wojcik et al., 2001). The following primary antibodies were the modified Krt75 was cloned during recombination. The final used: guinea pig anti-K75 (Wojcik et al., 2001) and mouse anti-K18 gene targeting vector was linearized with Pvu I before electropora- (Sigma-Aldrich, St Louis, MO). Secondary antibodies were Alexa- tion into ES cells. conjugated fluorochromes 594 goat anti-guinea pig and 488 goat

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anti-mouse antibodies (Molecular Probes, Eugene, OR). Photographs Figure S2. Strategy to generate a mouse model expressing the Krt75 N159del mutation (equivalent to the human KRT6A N172del mutation). were taken with a Nikon Eclipse E600 microscope in conjunction Figure S3. Changes in vibrissae. with the MetaVue v6.1r5 imaging software (Universal Imaging Corp., Downingtown, PA). REFERENCES Arin MJ, Longley MA, Wang XJ, Roop DR (2001) Focal activation of a mutant Mounting hair allele defines the role of stem cells in mosaic skin disorders. J Cell Biol Hair samples were removed manually by plucking lightly with 152:645–9 hemostats from the dorsal and ventral thorax of mice at 1, 3, and 8 Bechtold LS (2000) Ultrastructural evaluation of mouse mutations. In: weeks of age. At least three age- and sex-matched mice were used (Sundberg JP, Boggess D, eds), Systematic Characterization of Mouse for each genotype (wild type, heterozygous, and homozygous). Mutations. 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Genes Dev 16:1412–22 Statistics Mecklenburg L, Paus R, Halata Z, Bechtold LS, Fleckman P, Sundberg JP t-test was used to calculate significance. Po0.05 was considered (2004) FOXN1 is critical for onycholemmal terminal differentiation in statistically significant. nude (Foxn1) mice. J Invest Dermatol 123:1001–11 Moll R, Moll I, Wiest W (1982) Changes in the pattern of cytokeratin CONFLICT OF INTEREST polypeptides in epidermis and hair follicles during skin development in human fetuses. Differentiation 23:170–8 The authors state no conflict of interest. Montagutelli X, Hogan ME, Aubin G, Lalouette A, Guenet JL, King LE Jr et al. (1996) Lanceolate hair (lah): a recessive mouse mutation with alopecia ACKNOWLEDGMENTS and abnormal hair. J Invest Dermatol 107:20–5 We thank Dr Maranke Koster and Daisy Dai from the Roop laboratory for critical reading of this paper. 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