Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Feb 1.
Published in final edited form as: J Invest Dermatol. 2013 Feb 25;133(8):1990–1997. doi: 10.1038/jid.2013.84

Frizzled6 deficiency disrupts the differentiation process of nail development

Chang-Yi Cui 1,#, Joakim Klar 2,#, Patrik Georgii-Heming 2, Anne-Sophie Fröjmark 2, Shahid M Baig 3, David Schlessinger 1, Niklas Dahl’ 2
PMCID: PMC3695035  NIHMSID: NIHMS448104  PMID: 23439395

Abstract

Nails protect the soft tissue of the tips of digits. The molecular mechanism of nail (and claw) development is largely unknown, but we have recently identified a Wnt receptor gene, Frizzled6 (Fzd6) that is mutated in a human autosomal-recessive nail dysplasia. To investigate the action of Fzd6 in claw development at the molecular level, we compared gene expression profiles of digit tips of wild-type and Fzd6−/− mice, and show that Fzd6 regulates the transcription of a striking number of epidermal differentiation-related genes. Sixty-three genes encoding keratins, keratin associated proteins, and transglutaminases and their substrates were significantly down-regulated in the knockout mice. Among them, four hard keratins, Krt86, Krt81, Krt34 and Krt31; two epithelial keratins, Krt6a and Krt6b; and transglutaminase1 were already known to be involved in nail abnormalities when dysregulated. Immunohistochemical studies revealed decreased expression of Krt86, Krt6b and involucrin in the epidermal portion of the claw field in the knockout embryos. We further showed that Dkk4, a Wnt antagonist, was significantly down-regulated in Fzd6−/− mice along with Wnt, Bmp and Hh family genes; and Dkk4 transgenic mice showed a subtly but appreciably modified claw phenotype. Thus, Fzd6-mediated Wnt signaling likely regulates the overall differentiation process of nail/claw formation.

Keywords: Nail, claw, Frizzled6, Wnt, cornified envelope

Introduction

Interactions between the epidermis and underlying mesenchyme drive the development of skin and its appendages, including teeth, hair and nails/claws. The developmental mechanisms are highly conserved among species (Chuong et al., 2001). Primordial nails/claws start to appear on the dorsal surface of the developing distal digits of mouse limbs at E14-15, and morphogenesis is similar in primates and rodents (Hamrick, 2001). The nail/claw fold extends from the epidermis and folds inward to cover the proximal nail plate. The nail fold is succeeded by the nail matrix, which contains proliferating keratinocytes. Keratinocytes dorsal to the matrix start to express epithelial keratins and eventually undergo apoptosis, depositing a cornified structure on the nail plate (Figure 1a). Krt1 and Krt10 are expressed during the initial stages of keratinization and are later replaced by a network of Krt5 and Krt14 that assembles into intermediate filaments and contribute to the cytoskeleton of epithelial cells.

Figure 1.

Figure 1

Open in a new tab

Expression profiling of mouse digit tips during claw development. (a) Schematic representation of early stage claw development. A primordial claw/nail field is formed on the digit tips in mice at around E14.5. Primitive nail matrix is discernible at E16.5, and nail matrix starts to form nail plate at around E18.5. Broken lines indicate excised region for RNA collection. (b) Scatter-plots show number of genes differentially expressed in wild-type and Fzd6−/− digit tips at E14.5, E16.5 and E18.5.

Keratins constitute a major component of the cytoskeleton of the differentiated nail plate, and consequently mutations in the corresponding genes can perturb nail formation (Chamcheu et al., 2011; McLean and Moore, 2011). As development continues, cells around the nail matrix migrate into the spinous layer, stop dividing, and begin to synthesize a new set of structural proteins and enzymes characteristic of the nail plate. Additional keratins expressed in the nail matrix include epithelial keratins Krt6A and Krt17, and hard keratins Krt31, Krt33B, Krt34, Krt39, Krt81, Krt85 and Krt86 (Barthélemy et al., 2012; Rice et al., 2010). Keratin filaments aggregate in the presence of filaggrin, and a series of other proteins such as involucrin, loricrin and proline-rich proteins are synthesized and crosslinked by transglutaminases. This reinforces the cornified envelope under the membrane of terminally differentiated keratinocytes (corneocytes). Nail plate formation then ensues.

Though the precise molecular signatures and branch points and their timing for nail development remain unclear, regulation of ectodermal appendage formation is partially understood, and is clearly dependent on multiple signaling pathways (Fuchs, 2008). Induction of the nail placode is signaled from the underlying mesenchyme to the overlying epidermis. At this stage Wnt signaling plays a major role, with a system of autocrine and paracrine pathways regulated by several feedback loops (Fuchs, 2008; Widelitz, 2008). The importance of Wnt signaling for the formation of nails has been proven from molecular studies on specific congenital human disorders (Duverger and Morasso, 2008; Hill et al., 2006). Autosomal recessive anonychia or hyponychia has been reported to be caused by mutations in the Frizzled agonist R-spondin 4 (RSPO4) gene (Bergmann et al., 2006; Blaydon et al., 2006), and we have recently reported mutations in the FZD6 gene in a severe form of isolated autosomal recessive nail dysplasia (Fröjmark et al., 2011). In addition, mutations in the WNT10A gene are associated with odontoonychodermal dysplasia (OODD) (Adaimy et al., 2007; Nawaz et al., 2009), and mutations in the WNT-associated transcription factors LMX1B and MSX1 cause Nail-Patella and Witkop syndrome, respectively (Dreyer et al., 1998; Jumlongras et al., 2001).

To define further the molecular architecture of genes important for nail/claw formation we carried out gene expression profiling in a mouse model with RNAs from wild-type and Fzd6−/− digit tips at three embryonic stages, E14.5, E16.5 and E18.5, times at which early nail development is evident histologically. The Fzd6−/− mouse was originally shown to have altered hair patterning, implying that Fzd6 is involved in a planar polarity system of biological structures (Guo et al., 2004; Wang et al., 2010). Subsequent studies showed that the Fzd6-ablated mouse is a model for congenital claw dysplasia (Fröjmark et al., 2011). Here we show that the Fzd6-ablated mouse embryos show dysregulation of many genes required for epidermal differentiation. Striking abnormalities were observed in the expression of genes encoding proteins of the cornified envelope and keratins that are critical for claw/nail development and claw/nail plate formation. The results thus clarify a range of molecular mechanisms mediated by Fzd6 in normal claw/nail development in distal digits.

Results

Expression profiling reveals genes regulated by Fzd6 in developing claw

To infer candidate genes regulated by Fzd6-mediated Wnt signaling during claw development, we carried out genome-wide gene expression profiling with RNAs from digital tips of wild-type and Fzd6−/− mouse embryos at E14.5, E16.5 and E18.5 (Figure 1a). As expected, biological triplicates of hybridization were grouped with their genotypes in a hierarchical clustering dendrogram (Figure S1a), and we set criteria for significantly affected genes as log intensity>=2.0, FDR<=0.05 and fold-difference>=2.0. ANOVA analysis revealed 650 genes as significantly affected in the knockout embryos at E14.5, 747 genes at E16.5 and 387 genes at E18.5 (Figure 1b). The full list of significantly affected genes is presented in Table S1. Differentially expressed genes were validated using qRT-PCR for the selected transcripts Fzd6, Ivl, Hrnr and Rptn. Down-regulation of Ivl (p=0.0033) and Hrnr (p=0.0026) was confirmed and the expression of Rptn was reduced, although not significantly (p=0.099) at E16 (Figure S2). Consistent with array data, Ivl, Hrnr and Rptn were not differentially expressed at E14 and E18 (data not shown). Reduced expression of Fzd6 was confirmed at all time points (p<0.01), as expected (Figure S2).

Striking numbers of epidermal differentiation marker genes were significantly affected in Fzd6−/− digit tips from early developmental stages

As an overall index of expression profiles, we carried out principal component (PC) analysis (Figure S1b). Expression patterns of wild-type and Fzd6−/− mice changed progressively during development (arrows in Figure S1b). We found a large number of epidermal differentiation marker genes clustered downward in the knockout along PC1, consistent with their positive induction/regulation by Fzd6. A smaller group of additional “Fzd6-dependent” epidermal differentiation genes were seen with a somewhat different expression pattern along PC2 (Figure S1b). The regulated epidermal differentiation genes encoded many keratins (Krts, clustered on chromosomes 11 and 15); keratin associated proteins (Krtaps, clustered on chromosome 11 and 16); and transglutaminases (Tgms) and their substrates, including late cornified envelope proteins (Lces, clustered on chromosome 3); S100a calcium binding proteins (clustered on chromosome 3); small proline-rich proteins (Sprrs, clustered on chromosome 3); and trichohyalin, loricrin, involucrin and filaggrin (clustered on chromosome 3). We next analyzed these classes further.

Keratin and keratin associated protein genes significantly affected in Fzd6−/− mice

Keratins can be classified into epithelial keratins (Krt1-24, 76–80), hair follicle specific epithelial keratins (Krt25-28, 71–75), and hard keratins expressed in hair shafts and nails (Krt31-40, 81–86) (Moll et al., 2008).

By statistical analysis, we found eight hard (nail/hair) keratins significantly down-regulated in Fzd6−/− mice (Figure 2a): Krt84 and Krt81 at E14.5; Krt86, Krt34, Krt33b, Krt32 and Krt31 at E16.5; and Krt32 at E18.5. Immunohistochemical staining revealed Krt86 expression in the suprabasal layers of epidermis in the dorsal digit tips in wild-type mice that was significantly reduced in the Fzd6−/− embryos at E16.5 (Figure 4, Krt86). Among the down-regulated keratins, Krt86, Krt81, Krt34 and Krt31 (Figure 2b) have been reported to be expressed in the nail matrix, the region producing the actual nail plate (Perrin et al., 2004). Concomitantly, five keratin associated protein (Krtap) genes -- Krtap13-1, Krtap13, Krtap8-1, Krtap3-3, and Krtap3-1 -- were sharply down-regulated in the knockout mice at E16.5 (Figure 2a). Krtaps are known to cross-link with hard keratins to form rigid structures in hair (Rogers et al., 2006).

Figure 2.

Figure 2

Open in a new tab

Keratin and keratin associated protein genes were significantly affected in Fzd6−/− digit tips. (a) Twenty-nine hard and epithelial keratins, and 5 keratin associated protein genes were affected in Fzd6−/− digit tips during claw development, most of them were down-regulated. Dn, down-regulated; Up, up-regulated. Bolded genes are known genes expressed in nails or known to be responsible for nail abnormalities. Numbers in parentheses represent fold-differences. (b) Examples of the time course of expression for 4 affected hard keratin genes known to be involved in nail formation are shown. Krt86, 34, and 31 were significantly down-regulate in the knockout mice at E16.5, while Krt81 was at E14.5.

Figure 4.

Figure 4

Open in a new tab

Immunohistochemical staining examples of the hard keratin Krt86, the epithelial keratin Krt6b and the transglutaminase substrate Ivl in planar sections of digit tips E16.5. (a–c) Krt86, Krt6b, and Ivl proteins are expressed in the suprabasal layers of the wild-type digit tips. (d–f) Only background levels was detected for the corresponding proteins in Fzd6−/− embryos. Hematoxylin (blue color) was used as background staining. Boxes indicate enlarged areas. SB; stratum basale.

Additionally, sixteen epithelial keratins were also significantly down-regulated in the knockout mice (Figure 2a). Among them, Krt6a and Krt6b are mutated in the autosomal dominant pachyonychia congenital (PC) disorder, clinically characterized by hypertrophic nail dystrophy, plantar keratoderma and epidermal cysts (McLean and Moore, 2011). Krt6a was significantly down-regulated in the knockout mice at E14.5 and E18.5, while Krt6b was down-regulated at E16.5 (Figure 2a). Immunohistochemical staining with Krt6b antibody on wild-type digits at E16.5 showed strong staining mainly in the suprabasal layers of epidermis, including squamous and granular layers and stratum corneum of the nail field, but Fzd6−/− mice showed markedly reduced staining confined mainly to the stratum corneum (Figure 4, Krt6b). Notably, Krt79 and Krt77 were significantly down-regulated in knockout mice at E16.5, but then up-regulated at E18.5 (Figure 2a).

By contrast, the third keratin class, hair follicle-specific epithelial keratins (with exception of mild up-regulation of Krt28 at E18.5), was not affected in the knockout mice, presumably because claws/nails lack epithelial structures corresponding to the inner root sheath of hair follicles.

Hard keratins were previously shown to be direct transcriptional targets of Wnt signaling (Zhou et al., 1995). The sharp down-regulation of a vast number of hard keratins, epithelial keratins and Krtaps in the Fzd6-targeted mice strongly suggests that Fzd6-mediated Wnt signaling regulates formation of the cytoskeleton in developing nails.

Genes significantly dysregulated in Fzd6−/− mice that are required to form the terminally differentiated cornified cell envelope

Another general finding from expression profiling was the downregulation of expression of genes encoding transglutaminases and their cross-linking substrates for the cornified cell envelope (CE) formation, the idiosyncratic structure in terminal differentiation (Figure 3a). The cornified envelope is an insoluble and mechanically resistant membrane structure of terminally differentiated keratinocytes (Candi et al., 2005). Three transglutaminases, Tgm1, 3, and 5, which regulate cornified envelope formation, were sharply down-regulated in the Fzd6−/− mice at E14.5 and E16.5 (Figure 3a). Among them, transglutaminase 1 had previously been shown to regulate cornified envelope formation in nail plates (Rice et al., 2003), and mutation in the gene causes lamellar ichthyosis, affecting skin and sometimes nails (Huber et al., 1995). Genes encoding Tgm substrates, including involucrin, trichohyalin, loricrin; twelve late cornified envelope (Lce) proteins; six small proline-rich protein (Sprr) genes; and four S100a calcium binding protein genes were also significantly down-regulated in the knockout mice (Figure 3a). Immunohistochemical staining of involucrin indicates that it is almost absent in suprabasal layers of the claw field in Fzd6−/− mice compared to wild-type littermates (Figure 4, Ivl). Figure 3b shows expression patterns of Tgm1 and several of its substrates (Lce1h, Sprr1a and involucrin) in wild-type and knockout mice at early developmental stages (Figure 3b). Down-regulation of transglutaminases and their substrates in the knockout mice suggests that Fzd6 affects formation of the terminally differentiated cornified envelope. Abnormal expression of keratins and cornified envelope components in Fzd6−/− mice are consistent with phenotypic observations (Fröjmark et al., 2011).

Figure 3.

Figure 3

Open in a new tab

Transglutaminases and their substrates were significantly affected in Fzd6−/− mice. (a) Tgm 1, 3 and 5 were significantly down-regulated in Fzd6−/− mice along with their substrates including 12 Lces, 4 S100s, 6 Sprrs and Tchh, Lor, Csta, Ivl, Flg. Dn, Down-regulated; Up, Up-regulated. Bolded gene (Tgm1) is known to be responsible for nail abnormalities. Numbers in parentheses indicate fold-differences. (b) Expression levels of Tgm1 and its substrates Lce1h, Sprr1a and Involucrin were shown. Note a time lag of expression pattern, Tgm1 was significantly down-regulated at E14.5, Lce1h was at E16.5, Sprr1a was at E14.5 and E16.5, and Involucrin at E16.5.

Additional genes significantly altered in digit tips of Fzd6−/− mice

In addition to affecting an unusually wide range of epidermal differentiation-associated genes, ablation of Fzd6−/− also notably downregulated some signaling proteins known to control skin appendage development and differentiation. The additionally affected genes included members of Wnt, Bmp, Fgf, Tgfα and Dhh pathways (Table S2). Among them, expression changes for Bmp2K (Bmp2-inducible kinase) in the knockout mice were notable. Similar to Fzd6, Bmp2K was significantly down-regulated in the knockout mice at all three time points analyzed (Figure 5). The Bmp2-induced kinase contains a nuclear localization domain and has been shown to be involved in Bmp2-induced osteoblast differentiation (Liu et al., 2009). In addition to Bmp2K, Bmp8a, Wnt2, Wnt2b, Fgf2, Fgf7, and Dhh were all significantly less expressed in the knockout mice at E14.5 (Table S2).

Figure 5.

Figure 5

Open in a new tab

Signaling proteins down-regulated in the Fzd6−/− mice are shown. Expression patterns of Fzd6, Dkk4 and Bmp2k. Dkk4 was significantly down-regulated in the knockout mice at E16.5 and E18.5, but not at E14.5. Like Fzd6, Bmp2k was down-regulated at all 3 time points.

At E16.5, Wnt10b, Dkk4 and Tgfα were significantly down-regulated; and at E18.5, Dkk4 and Fzd10 were less expressed. Dkk4 is an antagonist of Wnt pathway; and in an initial experiment to look at interactive modulation of action of factors within the Wnt system, we examined claw formation in wild-type mice bearing a Dkk4 transgene. In previous work, although fundamental hair formation occurred unchanged in Dkk4 transgenic mice in wild-type background, hair subtypes were altered (Cui et al., 2010). For claws as well, a modulatory effect of a Dkk4 transgene was seen at the maturation stage of development. The effect was subtle but reproducible, with an appreciable change in the thickness and angle of claws, more easily seen on the 3 central digits of the hind paws (in Figure 6, a claw from a transgenic mouse is sharper and is oriented 6–7° more vertically than wild type control claws). Overall, nine of 63 epidermal differentiation marker genes, including Krt86, Krt34, Krt6a and Krt6b, which were down-regulated in Fzd6−/− claws, were also significantly down-regulated in Dkk4 transgenic hair follicles (Table S3, and see Figure S1 in Cui et al., 2010). Thus, Dkk4 may interact with a specific Wnt protein regulated by Fzd6.

Figure 6.

Figure 6

Open in a new tab

Claw phenotype of K14-Dkk4 transgenic mice. Second digit tips of wild-type and Dkk4 transgenic mice are shown. Claw is slightly thinner and the angle between lateral claw fold and horizontal skin stripes is larger in the transgenic mice compared to WT littermates.

Finally, we also assessed up-regulated genes in Fzd6−/− mice. In contrast to a wide range of differentiation marker and signal genes down-regulated in the knockout mice, up-regulated genes did not cluster by function. However, expression of a small number of differentiation related genes and genes encoding signal proteins was up-regulated. The up-regulated genes include Krt18, Krt28, Krt42, Krt77, Krt79, Lce1g (Fig. 2a, 3a), and Lrp6, Egfbp2, Wisp2 (Table S2). These might represent secondary feedback effects of down-regulation of the cohort of differentiation-related genes and signaling proteins.

Discussion

The gene expression profile at the site and time of early claw development showed that the lack of Fzd6 expression, associated with nail dysplasia in humans, dramatically affects overall expression patterns of keratins and other proteins involved in cornified envelope formation. Our data supports a model in which the Wnt/Fzd signaling pathway, specifically involving Fzd6, plays a key role in claw/nail formation through the activation of required transcription units.

We note considerable overlap in the expression of hard keratins and keratin-associated proteins in developing claw compared to shaft composition in hair follicles. They include Krt31, Krt33b, Krt34, Krt81, Krt86, Krtap3-1 and Krtap3-3 (Barthélemy et al., 2012). We also identified a broad spectrum of epithelial keratins in the developing claw, ncluding Krt1, Krt4, Krt6a, Krt9, Krt10, Krt13, Krt19 and Krt23, which are not found in the mature claw plate (Rice et al., 2010). In addition to these major differences in the composition of the claw plate and hair shaft, several transcripts encoding structural proteins appeared more strongly or exclusively associated with developing claws. They included Krt6a, Krt6b, Krt7, Krt12, Krt13, Krt18, Krt23, Krt28, Krt42, Krt77, Krt78, Krtap8-1, and Krtap13. These findings thus point to distinctive and characteristic keratin profiles during claw morphogenesis compared to formation of the claw plate.

Hallmarks of claw formation, including several keratins and keratin-associated proteins dysregulated in the Fzd6−/− mice, are consistent with previous findings that Wnt signaling regulates hard keratin expression (Zhou et al., 1995). The pattern of dysregulated keratins (and transglutaminases and their substrates; see below) varied between time-points, with the largest number at E16.5, the peak time of expression of Fzd6 in digit tips during normal development (Fröjmark et al., 2011)(Figure 2a). This finding may suggest a requirement for spatiotemporal action of epidermal differentiation proteins and related enzymes in the early developmental stage of claw plate formation, with a few genes still affected at E18.5.

As another indication of temporal succession, Krt6a and Krt6b, mutations in which are associated with pachonychia congenita, were down-regulated, but Krt6a was significantly down-regulated at E14.5 and E18.5, whereas Krt6b was significantly down-regulated at E16.5. Similar to Krt6b, Krt86, the major keratin of the claw plate, showed a “window” of down-regulation at E16.5 in the Fzd6−/− animals. Mutations in Krt86 are associated with monilethrix (fragile hair) (van Steensel et al., 2005), but this has not been reported in patients with FZD6 mutations. Thus, the reduced Krt86 expression does not seem to affect hair shaft formation significantly.

The cornification process has already been specified to involve a large number of structural proteins and enzymes expressed in sequential order (Candi et al., 2005), and interestingly, the gene expression analysis in wild-type mice at E14.5-E18.5 showed considerable overlap with the pattern from skin as well as other epidermal appendages. But idiosyncratic to the digit tips, three key enzymes for the assembly of cornified cell envelope in stratum corneum -- transglutaminase 1, 3 and 5 -- were sharply down-regulated in Fzd6 mutant mice. Similarly, a series of transglutaminase substrate genes, including those for structural proteins trichohyalin, involucrin, loricrin and several Sprrs and Lces, showed significantly reduced expression, again consistent with the dysplastic nail formation in Fzd6−/− mice.

In addition, the Fzd6-ablated mouse clarifies critical and specific pathways for claw formation. More than 500 genes were significantly dysregulated at the three time points tested in digit tips of Fzd6−/− compared to wild-type embryos. Consistent with observations during distal limb formation (Geetha-Loganathan et al., 2008), several Wnt signaling molecules were found down-regulated. Notably, Dkk4, a Wnt antagonist, has been shown to be direct target of canonical Wnt signaling (Bazzi et al., 2007), suggesting a possible feedback regulation of Wnt signaling for nail/claw development mediated by Fzd6 and Dkk4. Figure 6 shows that overexpression of Dkk4 in wild-type mice modified claw development slightly; and we found 9 differentiation markers down-regulated in Fzd6 knockout mice that are also down-regulated in Dkk4 transgenic mice. Thus, Dkk4 may inhibit expression of a specific Wnt or Wnt subset that is positively controlled by Fzd6.

In further clues to the regulatory network, the expression of several other signaling proteins was affected. Notably, Bmp2k was down-regulated at all three time-points suggesting its regulation by Fzd6-mediated signaling. The expression of Bmp2 from mesenchymal cells has earlier been shown to be important in the differentiation of epidermal appendages (Rendl et al., 2008) and for patterning of distal limbs (Maatouk et al., 2009).

Overall, however, the developmental program of claws thus diverges sharply from that of other well-studied skin appendages. In particular, in contrast to hair, teeth, and sweat glands, ectodysplasin is not obviously involved (Cui and Schlessinger, 2006), and the plethora of epithelial keratins made is largely distinct from those seen in hair follicles. In fact, Fzd6−/− mice and humans with homozygous FZD6 mutations show normal structures of epidermis and complete formation of hair follicles. Further increasing the complexity of the regulatory network, Wnt signaling has been shown to be involved in epigenetic regulation of its target genes (Sierra et al., 2006), a mechanism that is likely operative in the coordinated regulation of epidermal differentiation genes. It is suggestive that 4 clusters of implicated cornified envelop genes (Lces, Sprrs, S100s, and trichohyalin, loricrin, filaggrin) are all nearby on chromosome 3, raising the speculative possibility that they are jointly regulated by a single Wnt-dependent epigenetic alteration. The results imply that protagonist regulators in claw/nail assembly act successively in a regulatory cascade initiated by Wnt/Fzd6; the successive waves of expression (Figures 2, 3 and 5) provide an entrée to determine how the concerted action of signaling factors and epigenetic regulation establishes the pattern of gene action.

Materials and Methods

Mouse strain, timed-mating, and gene expression profiling

Heterozygous Fzd6+/− mice (kindly provided by Dr. J. Nathans) were crossed to generate Fzd6+/−, Fzd6−/− and wild-type offspring. Timed matings were set up to harvest embryos at E14.5, E16.5 and E18.5. The morning after mating was designated as E0.5. Three middle distal phalanges from each limb were excised and immediately frozen on dry ice. Nine digital tips from three limbs of the same embryo were pooled for one replicate, and stored at −80°C freezer until use. Three pooled biological replicates were prepared for each genotype at each embryonic time-point. Tails were collected to isolate genomic DNAs, and genotyping was done as described previously (Fröjmark et al., 2011). Animal experiments were approved by the ethical committee for animal trials, Uppsala County Court, Uppsala, Sweden.

Total RNAs were isolated from each of the pooled digit tips using the Trizol Reagent (Life technologies). Total RNA (2.5μg for each replicate) was labeled with Cy3-CTP using a Quick Amp Labeling Kit (Agilent) to generate the experimental target, and the Cy5-CTP-labeled reference target was produced from the mixture of the Stratagene Universal Mouse Reference RNA (composed of total RNAs from 11 mouse cell lines) and 129ES cellular RNA. Labeled targets were purified using an RNeasy Mini Kit (Qiagen), quantified on a NanoDrop scanning spectrophotometer (NanoDrop Technologies). An experimental target was then mixed with the reference target and hybridized to the NIA Mouse 44K Microarray v3.0 (whole genome 60-mer oligo arrays manufactured by Agilent Technology, design ID 015087) according to manufacturer’s protocol (Two-Color Microarray-Based Gene Expression Analysis Protocol, Product # G4140-90050, Version 5.0.1). Hybridized microarray slides were scanned with an Agilent DNA Microarray Scanner (model G2505-64120). Triplicate data were analyzed by ANOVA with universal reference for normalization and carried out principle component analysis (PCA) (http://lgsun.grc.nia.nih.gov/ANOVA/) (Sharov et al., 2005). Universal reference was not used for normalization if the slope of regression between log(Cy5) vs. log(Cy3) is >0.3, and geometric mean of Cy3 is >3 fold higher than geometric mean of Cy5. All data are MIAME compliant and raw data has been deposited in GEO (GSE40763).

Quantitative real-time PCR was performed with the Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen) then run and analyzed on the MxPro Real-Time PCR System (Stratagene). Primers for quantification were designed to span intron-exon boundaries to ensure that the reactions were cDNA specific. All reactions were performed three times and in triplicates and normalized to GAPDH. Student’s two-tailed t-test was used for statistical analysis. All primer sequences are available upon request.

Details of the generation of Dkk4 transgenic mice were reported previously (Cui et al., 2010). Briefly, Dkk4 transgene was subcloned into a Krt14 vector, microinjected into pronuclei of mouse embryos, and followed by implantion into pseudo-pregnant female mice. Potential founders were mated to C57BL/6J mice to identify those passing the transgene. Two 6 month old transgenic mice and two littermates were analyzed for nail formation in this study.

Immunohistochemistry

For histological analyses, digital tips of Fzd6−/− and wild-type littermates at day E16.5 were fixed in 4% formaldehyde and embedded in paraffin. Three μm planar sections were cut from the middle digit for staining. Immunofluorescent staining was performed using anti-human/mouse Krt6b (Proteintech group, 17391-1-AP), anti-mouse Krt86 (Santa Cruz Biotechnology, sc-168332) and anti-human/mouse Involucrin (Abcam, ab53112) using a DAKO autostainer (Dako). The antibodies were detected using a DAKO Chemmate EnVision kit (Dako) according to the manufacturer’s protocol. Sections were counterstained with hematoxylin.

Supplementary Material

Acknowledgments

Authors thank Yulan Piao for help for expression profiling, Alexei Sharov for statistical analysis. This work was supported in part by the Intramural Research Program of the NIH, National Institute on Aging, the Swedish Research Council [grant numbers K2010-66X-10829-17-3 and 621-2009-4629], Uppsala University and Science for Life laboratory. J.K. is supported by the Swedish Society for Medical Research.

Abbreviations

Fzd6

Footnotes

Conflict of interest

The authors state no conflict of interest.

References

  1. Adaimy L, Chouery E, Megarbane H, et al. Mutation in WNT10A is associated with an autosomal recessive ectodermal dysplasia: the odonto-onycho-dermal dysplasia. Am J Hum Genet. 2007;81:821–8. doi: 10.1086/520064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barthélemy NR, Bednarczyk A, Schaeffer-Reiss C, et al. Proteomic tools for the investigation of human hair structural proteins and evidence of weakness sites on hair keratin coil segments. Anal Biochem. 2012;421:43–55. doi: 10.1016/j.ab.2011.10.011. [DOI] [PubMed] [Google Scholar]
  3. Bazzi H, Fantauzzo KA, Richardson GD, et al. The Wnt inhibitor, Dickkopf 4, is induced by canonical Wnt signaling during ectodermal appendage morphogenesis. Dev Biol. 2007;305:498–507. doi: 10.1016/j.ydbio.2007.02.035. [DOI] [PubMed] [Google Scholar]
  4. Bergmann C, Senderek J, Anhuf D, Thiel CT, Ekici AB, Poblete-Gutierrez P, et al. Mutations in the gene encoding the Wnt-signaling component R-spondin 4 (RSPO4) cause autosomal recessive anonychia. Am J Hum Genet. 2006;79:1105–9. doi: 10.1086/509789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blaydon DC, Ishii Y, O’Toole EA, et al. The gene encoding R-spondin 4 (RSPO4), a secreted protein implicated in Wnt signaling, is mutated in inherited anonychia. Nat Genet. 2006;38:1245–7. doi: 10.1038/ng1883. [DOI] [PubMed] [Google Scholar]
  6. Candi E, Schmidt R, Melino G. The cornified envelope: a model of cell death in the skin. Nat Rev Mol Cell Biol. 2005;6:328–40. doi: 10.1038/nrm1619. [DOI] [PubMed] [Google Scholar]
  7. Chamcheu JC, Siddiqui IA, Syed DN, et al. Keratin gene mutations in disorders of human skin and its appendages. Arch Biochem Biophys. 2011;508:123–37. doi: 10.1016/j.abb.2010.12.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chuong CM, Hou L, Chen PJ, et al. Dinosaur’s feather and chicken’s tooth? Tissue engineering of the integument. Eur J Dermatol. 2001;11:286–92. [PMC free article] [PubMed] [Google Scholar]
  9. Cui CY, Kunisada M, Piao Y, et al. Dkk4 and Eda regulate distinctive developmental mechanisms for subtypes of mouse hair. PLoS One. 2010;5:e10009. doi: 10.1371/journal.pone.0010009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cui CY, Schlessinger D. EDA signaling and skin appendage development. Cell Cycle. 2006;5:2477–83. doi: 10.4161/cc.5.21.3403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dreyer SD, Zhou G, Baldini A, et al. Mutations in LMX1B cause abnormal skeletal patterning and renal dysplasia in nail patella syndrome. Nat Genet. 1998;19:47–50. doi: 10.1038/ng0598-47. [DOI] [PubMed] [Google Scholar]
  12. Duverger O, Morasso MI. Role of homeobox genes in the patterning, specification, and differentiation of ectodermal appendages in mammals. J Cell Physiol. 2008;216:337–46. doi: 10.1002/jcp.21491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fröjmark AS, Schuster J, Sobol M, et al. Mutations in Frizzled 6 cause isolated autosomal-recessive nail dysplasia. Am J Hum Genet. 2011;88:852–60. doi: 10.1016/j.ajhg.2011.05.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fuchs E. Skin stem cells: rising to the surface. J Cell Biol. 2008;180:273–84. doi: 10.1083/jcb.200708185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Geetha-Loganathan P, Nimmagadda S, Scaal M. Wnt signaling in limb organogenesis. Organogenesis. 2008;4:109–15. doi: 10.4161/org.4.2.5857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Guo N, Hawkins C, Nathans J. Frizzled6 controls hair patterning in mice. Proc Natl Acad Sci U S A. 2004;101:9277–81. doi: 10.1073/pnas.0402802101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hamrick MW. Development and evolution of the mammalian limb: adaptive diversification of nails, hooves, and claws. Evol Dev. 2001;3:355–63. doi: 10.1046/j.1525-142x.2001.01032.x. [DOI] [PubMed] [Google Scholar]
  18. Hill TP, Taketo MM, Birchmeier W, et al. Multiple roles of mesenchymal betacatenin during murine limb patterning. Development. 2006;133:1219–29. doi: 10.1242/dev.02298. [DOI] [PubMed] [Google Scholar]
  19. Huber M, Rettler I, Bernasconi K, et al. Lamellar ichthyosis is genetically heterogeneous--cases with normal keratinocyte transglutaminase. J Invest Dermatol. 1995;105:653–4. doi: 10.1111/1523-1747.ep12324122. [DOI] [PubMed] [Google Scholar]
  20. Jumlongras D, Bei M, Stimson JM, et al. A nonsense mutation in MSX1 causes Witkop syndrome. Am J Hum Genet. 2001;69:67–74. doi: 10.1086/321271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Liu HP, Lin YJ, Lin WY, et al. A novel genetic variant of BMP2K contributes to high myopia. J Clin Lab Anal. 2009;23:362–7. doi: 10.1002/jcla.20344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Maatouk DM, Choi KS, Bouldin CM, et al. In the limb AER Bmp2 and Bmp4 are required for dorsal-ventral patterning and interdigital cell death but not limb outgrowth. Dev Biol. 2009;327:516–23. doi: 10.1016/j.ydbio.2009.01.004. [DOI] [PubMed] [Google Scholar]
  23. McLean WH, Moore CB. Keratin disorders: from gene to therapy. Hum Mol Genet. 2011;20:R189–97. doi: 10.1093/hmg/ddr379. [DOI] [PubMed] [Google Scholar]
  24. Moll R, Divo M, Langbein L. The human keratins: biology and pathology. Histochem Cell Biol. 2008;129:705–33. doi: 10.1007/s00418-008-0435-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nawaz S, Klar J, Wajid M, et al. WNT10A missense mutation associated with a complete odonto-onycho-dermal dysplasia syndrome. Eur J Hum Genet. 2009;17:1600–5. doi: 10.1038/ejhg.2009.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Perrin C, Langbein L, Schweizer J. Expression of hair keratins in the adult nail unit: an immunohistochemical analysis of the onychogenesis in the proximal nail fold, matrix and nail bed. Br J Dermatol. 2004;151:362–71. doi: 10.1111/j.1365-2133.2004.06108.x. [DOI] [PubMed] [Google Scholar]
  27. Rendl M, Polak L, Fuchs E. BMP signaling in dermal papilla cells is required for their hair follicle-inductive properties. Genes Dev. 2008;22:543–57. doi: 10.1101/gad.1614408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rice RH, Crumrine D, Hohl D, et al. Cross-linked envelopes in nail plate in lamellar ichthyosis. Br J Dermatol. 2003;149:1050–4. doi: 10.1111/j.1365-2133.2003.05510.x. [DOI] [PubMed] [Google Scholar]
  29. Rice RH, Xia Y, Alvarado RJ, et al. Proteomic analysis of human nail plate. J Proteome Res. 2010;9:6752–8. doi: 10.1021/pr1009349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rogers MA, Langbein L, Praetzel-Wunder S, et al. Human hair keratin-associated proteins (KAPs) Int Rev Cytol. 2006;251:209–63. doi: 10.1016/S0074-7696(06)51006-X. [DOI] [PubMed] [Google Scholar]
  31. Sharov AA, Dudekula DB, Ko MS. A web-based tool for principal component and significance analysis of microarray data. Bioinformatics. 2005;21:2548–9. doi: 10.1093/bioinformatics/bti343. [DOI] [PubMed] [Google Scholar]
  32. Sierra J, Yoshida T, Joazeiro CA, et al. The APC tumor suppressor counteracts beta-catenin activation and H3K4 methylation at Wnt target genes. Genes Dev. 2006;20:586–600. doi: 10.1101/gad.1385806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. van Steensel MA, Steijlen PM, Bladergroen RS, et al. A missense mutation in the type II hair keratin hHb3 is associated with monilethrix. J Med Genet. 2005;42:e19. doi: 10.1136/jmg.2004.021030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wang Y, Chang H, Nathans J. When whorls collide: the development of hair patterns in frizzled 6 mutant mice. Development. 2010;137:4091–9. doi: 10.1242/dev.057455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Widelitz RB. Wnt signaling in skin organogenesis. Organogenesis. 2008;4:123–33. doi: 10.4161/org.4.2.5859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zhou P, Byrne C, Jacobs J, et al. Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate. Genes Dev. 1995;9:700–13. doi: 10.1101/gad.9.6.700. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS448104-supplement-supplement_1.pdf (668.8KB, pdf)

RESOURCES