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Differentiation. Author manuscript; available in PMC 2010 December 1.
Published in final edited form as:
PMCID: PMC2945291
NIHMSID: NIHMS140391

Desmoglein 4 is Regulated by Transcription Factors Implicated in Hair Shaft Differentiation

Abstract

The hair fiber is made of specialized keratinocytes, known as trichocytes, that primarily express hair keratins which are cemented by a multitude of keratin-associated proteins (KAPs). The hair keratins form the intermediate filament cytoskeleton of the trichocytes, which are linked to abundant cell-cell adhesion junctions, called desmosomes. Desmoglein 4 (DSG4) is the major desmosomal cadherin expressed in the hair shaft cortex where the hair keratins are highly expressed. In humans, mutations affecting either the hair keratins or DSG4 lead to beaded hair phenotypes with features of monilethrix. In this work, we postulated that the regulatory pathways governing the expression of hair shaft components, such as hair keratins and DSG4, are similar. Therefore, we studied the transcriptional regulation of DSG4 by transcription factors/pathways that are known regulators of hair keratin or KAP expression. We show that HOXC13, LEF1 and FOXN1 repress DSG4 transcription and provide in vitro and in vivo evidence correlating the Notch pathway with the activation and/or maintenance of DSG4 expression in the hair follicle.

Keywords: Desmoglein 4, Notch, Hair Follicle, Hox, Keratin, Differentiation

Introduction

In the mammalian hair follicle (HF), the hair shaft is produced during the anagen or growth phase of the hair cycle which continues in repeated cycles throughout life (Hardy, 1992). The hair shaft or hair fiber is the only part of the HF that protrudes above the skin surface and plays various physiological roles including protection of the skin and thermal insulation. The hair shaft consists of three concentric layers with a cuticle on the outside, a cortex, and a medulla on the inside (Fig 1A). In humans, the cortex makes up the bulk of the hair shaft, with the size and shape of the medulla varying greatly depending on hair type and ethnic background (Jave-Suarez et al., 2002). The hair shaft is surrounded by the inner root sheath (IRS) whose innermost layer is in direct contact with hair shaft cuticle cells, and is called the IRS cuticle. The IRS plays a pivotal and supportive role during hair shaft growth (Rogers, 2004). The outermost layers of the HF consist of the companion layer and the outer root sheath (ORS) which is produced during the downgrowth of the HF (Fuchs et al., 2001).

Figure 1
Dane and Herman staining of a normal human HF (A) and a Dsg4 −/− rat Vibrissae follicle (B). The dermal papilla is light blue. Note the abnormal keratinized mass at the tip of the abnormal precortex in (B) which is a hallmark of Dsg4 mutations ...

The production and assembly of the hair shaft layers are highly coordinated processes that involve the very rapid proliferation of the matrix cells in the HF bulb, followed by a gradual cellular differentiation program that takes place within the funnel-shaped precortex region (Fig 1A). It has been shown that the physical position of the matrix cells along the basement membrane separating the epithelial matrix cells from the mesenchymal dermal papilla cells along the proximo-distal axis determines, in part, their final differentiation fate in the concentric layers of the HF (Hardy, 1992; Langbein and Schweizer, 2005; Legue and Nicolas, 2005).

The major structural proteins found in the hair shaft are the hair keratins and keratin-associated proteins (KAPs) (Lee et al., 2006). Keratins constitute the intermediate filament cytoskeleton of both epidermal keratinocytes and the specialized hair shaft trichocytes, which is indispensable since these cells must withstand substantial and continuous mechanical stress (Rogers, 2004). To achieve this, the intermediate filaments or keratins form an intracellular network that links to the plaque proteins of cell-cell adhesion complexes called desmosomes. This intra- and inter-cellular network of keratins and desmosomes provides the hair fiber with its properties of a relatively high tensile strength as well as elasticity.

Desmosomes are calcium dependent cell junctions that are prevalent in tissues that are subjected to continuous mechanical stress such as the skin and heart (Bazzi and Christiano, 2007). The desmosomal cadherins, desmogleins (Dsg1–4) and desmocollins (Dsc1–3), are at the core of the adhesive interface of desmosomes. Of the desmogleins, desmoglein 4 (Dsg4) is highly expressed in the cortex region of the hair shaft, and its functional absence leads to localized autosomal recessive hypotrichosis (LAH) in humans and the lanceolate hair phenotype in rodents (lah) characterized by abnormal hair shaft differentiation and hair loss in these organisms (Fig 1B) (Kljuic et al., 2003; Bazzi and Christiano, 2007; Messenger et al., 2005; Wajid et al., 2007). Interestingly, some LAH patients present with an additional beaded hair phenotype resembling that of monilethrix patients, who have mutations in hair keratins (Schaffer et al., 2006; Shimomura et al., 2006; Zlotogorski et al., 2006).

The expression of hair shaft components is governed by a large number of transcription factors (e.g. LEF1, β-catenin, SMAD, NICD, FOXN1, MSX2 and HOXC13), many of which operate as downstream effectors of the major morphogenic pathways. For example, earlier studies have shown that the Wnt pathway with its downstream effector, Lef1, is one of the major regulators of the hair shaft differentiation process. Lef1 knockout mice completely lack vibrissae and display a significant reduction in pelage HFs (van Genderen et al., 1994)]. Another well-established regulator of hair keratin promoters in the precortex is Hoxc13. Similar to Lef1, Hoxc13 knockout mice lack hair fibers (Godwin and Capecchi, 1998). Foxn1 and Msx2 transcription factors have also been shown to regulate hair keratin expression and their knock out phenotypes resemble those of Lef1 and Hoxc13 (Mecklenburg et al., 2005; Ma et al., 2003). In addition, the knockout of Notch1 or γ-secretase, which is a presinilin-like protease that releases the active form of Notch (NICD), results in abnormal hair cortex differentiation and precortical swellings reminiscent of those observed in Dsg4 mutant mice (Pan et al., 2004).

Based on the close functional relationship between hair keratins and DSG4, as well as the similarity of the phenotypes that result when both genes are perturbed, we hypothesized that the transcription factors that regulate the expression of hair keratins and KAPs might also regulate the expression of DSG4 (Bazzi et al., 2006). Here, we show that HOXC13, LEF1 and FOXN1 repress DSG4 transcription in HaCa T keratinocytes in vitro and provide evidence that the Notch pathway indirectly regulates DSG4 expression in the HF and epidermis.

Materials and Methods

Animals

The rodent models used in this study have been described previously: Dsg4 −/− mice are known as lahJ−/− (Sundberg et al., 2000; Kljuic et al., 2003) and rats are known as lahIC−/− (or Iffa Credo) (Bazzi et al., 2004), presenilin 1 and 2 double conditional knockout (PSDCKO, genotype: Msx2-Cre +/−; Psn1 flox/flox; Psn2 −/−) (Pan et al., 2004).

Histology

The histological staining procedures used in this study are either standard haematoxylin and eosin staining or Dane and Herman staining where indicated (Dane and Herman, 1963). The latter staining procedure of paraffin embedded tissue sections is based on haematoxylin, phloxine B, alcian blue, and orange G interspersed with washes in water. The slides are then permanently mounted and photographed with regular light microscopy.

Immunofluorescence

Fresh frozen sections of human HFs discarded after surgery were fixed in 4% PFA for 10 minutes (mins) at room temperature. While frozen sections of mouse back skin were fixed in methanol at −20°C for 15 mins followed by acetone for 1 min at −20°C. The sections were washed in 1XPBS, blocked in 10% of the appropriate serum, and incubated with the primary antibody overnight at 4°C. After washing in 1XPBS, 594 or 688 Alexafluor® conjugated secondary antibodies (Molecular Probes, Invitrogen, Carlsbad, CA, USA) were applied and the signal was visualized using an HRC Axiocam fitted onto an Axioplan2 fluorescence microscope (Carl Zeiss, Thornwood, NY, USA). The antibodies used were guinea pig anti-HOXC13 and anti-Ha1 (1:2000, a kind gift of Dr. Lutz Langbein), rabbit anti-FOXN1 (1;200, Santa Cruz), goat anti-LEF1 (1:100, Santa Cruz Biotchnology Inc., Santa Cruz, CA, USA), rabbit anti-IRS3a.1 (1;1000, a kind gift of Dr. Rebecca Porter), and mouse monoclonal anti-DSG4 clone 18G8 (1:10) (Bazzi et al., 2006). For mouse antibodies on mouse tissues the Mouse On Mouse (M.O.M) kit from Vectorlabs was used per the manufacturer’s recommendations (Burlingame, CA, USA).

In Silico Promoter Analysis

For the analysis of the upstream region of desmogelin 4 we used the VISTA website for comparative genomics (http://genome.lbl.gov/vista/index.shtml) (Loots et al., 2002), in addition to the AliBaba2.1 tool on the Gene-Regulation website (http://www.gene-regulation.com/pub/programs/alibaba2/index.html). We also used a microsoft word search tool for known consensus binding sequences of transcription factors such as Lef1: (T/C)CTTTG(A/T)(A/T), HOXC13 : TT(A/T)AT(N)(A/G)(A/G), and RBPjK (GTGGGAA) considering all permutations.

Reporter Gene Assays

A ~3Kb and ~1Kb (~1.1 for the human) DNA fragments upstream of the translation initiation site (A in the first ATG is considered +1) of human and mouse DSG4 were cloned into the pGL3 basic luciferase reporter vector (Promega, Madison, WI, USA). The primers used were as follows:

  • DSG4 3Kb F: 5’-CTCTGGAAACAACTTGTGCT-3’
  • DSG4 1Kb F: 5’-AGTTTATTGATGTTGCTGCC-3’
  • DSG4 3Kb or 1Kb R: 5’-TCCTTTGGGTTTCTCTTGCA-3’
  • Dsg4 3Kb F: 5’-CGTGTAGGATATGTCCATCT-3’
  • Dsg4 1Kb F: 5’-CACACACCCTCATTTCTGTT-3’
  • Dsg4 3Kb or 1Kb R: 5’-TGCTTCGAGTCTCTCTTGG-3’

HaCa T keratinocytes were grown in DMEM with 10% FBS and 1% Penicillin-Streptomycin (Invitrogen) and seeded in six-well plates at a density of 3×105 /well. Twenty four hours after seeding, HaCaT cells were transfected with the indicated plasmids at 0.5ug/well each using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer’s recommendations. Total DNA was equalized per experiment by adding the same concentration of an appropriate empty vector. In addition, a β-galactosidase expressing construct was used as an internal control for transfection efficiency and normalization. Each treatment was run in triplicate and each experiment was reproduced two or three times. The cells were lysed 48 hrs after transfection and the signals were assayed using the appropriate substrates for luciferase (Steady Glo® luciferase assay system) and β-galactosidase (Promega) on a 20/20n luminometer (Turner Biosystems, Inc., Sunnyvale, CA, USA) for luciferase and a Model 680 microplate reader (BioRad, Hercules, CA, USA) for β-galactosidase. Student’s t-test was used to assess significance between two different treatment groups with a p-value of 0.05. Human β-catenin and LEF1 expression plasmids were generous gifts of Dr. Jan Kitajewski (Columbia University). MSX2 expression plasmid was a gift from Dr. Cheng-Ming Chuong (USC). HOXC13 expression plasmid was a gift of Dr. Lutz Langbein (DKFZ). Only HaCaT keratinocytes that displayed an intact differentiation program (i.e. expressed DSG4 mRNA and protein upon confluency) were used in this study.

Microarray Analysis

Backskins from six-day old lahJ−/− and lahJ+/− littermates were dissected and incubated in 0.25% dispase II (Invitrogen) at 4°C overnight. Following incubation, the epidermis was separated from the dermis and was minced using scissors and flash frozen in liquid nitrogen. Total RNA was isolated from the epidermal tissues using the RNeasy® Minikit according to the manufacturer’s instructions (Qiagen, Valencia, CA, USA). Triplicate RNA samples from three independent mice for each of WT and mutant mice were amplified once and labeled for hybridization on microarray chips (MOE430A) using the Affymetrix reagents and protocols (Affymetrix Inc., Santa Clara, CA, USA). The data output was normalized and analyzed using both GeneSpring GX 7.0 (Agilent Technologies Inc., Palo Alto, CA, USA) and GeneTraffic™ (Iobion Informatics, Stratagene, Agilent Technologies Inc.) commercial software packages which gave comparable results. The p-value cutoff was set to 0.05 and the significant fold difference was considered two-fold higher or lower than baseline (Bazzi et al., 2007).

Results

Desmoglein 4 is expressed in the hair cortex and IRS cuticle of rat and mouse hair follicles

We previously determined the expression of DSG4 within the hair cortex and IRS cuticle of the human anagen HF (Bazzi et al., 2006). Using a DSG4 specific antibody, mouse monoclonal antibody clone 18G8, we assessed the expression of mouse and rat Dsg4 in the HFs. We have also recently briefly reported the expression of mouse Dsg4 in pelage or back skin HF cortex (Owens et al., 2008). Similar to the human expression pattern, we found that Dsg4 is expressed in the cortex and IRS cuticle of rat vibrissae follicles, as well as rat and mouse pelage follicles during the anagen phase of the hair cycle (Fig 2A–C,E,F,I,J). Importantly, the protein is absent from Dsg4 null mutant HFs in the Iffa Credo rat and LahJ−/− mouse (Fig 2D,G,H,K,L). Moreover, Dsg4 expression commences with hair cortex differentiation on day1 post natum and is maintained around the forming club hair in the catagen phase of the hair cycle in normal rat follicles (Fig 3). This pattern of expression is consistent with the sites of main defects in LAH humans and lah rodents with desmoglein 4 mutations, which is abnormal hair shaft differentiation starting in the precortex region during anagen (Fig 1B).

Figure 2Figure 2Figure 2
Dsg4 is expressed in the cortex and IRS cuticle of rat vibrissae follicles and rat and mouse pelage follicles during anagen but absent from Dsg4−/− follicles. (A–L) Dsg4 is in green, the cortex hair keratin Ha1 is in red (B,E,G,I,K), ...
Figure 3
Dsg4 is expressed in the forming cortex on day 1 (d1) and is more prominent in the cortex in mid to late anagen (d11) in rat pelage follicles. Dsg4 is still expressed in the receding hair cortex and around the forming club hair during catagen on d19 in ...

Transcriptional profiling of Dsg4 null mouse epidermis

To gain mechanistic insight into how the perturbation of cell-cell adhesion or physical separation of HF keratinocytes in Dsg4 −/− mouse skin leads to failure in cell positioning and/or signaling, we performed microarray analyses on epidermis isolated from day 6 anagen skin of lahJ−/− mutant mice and lahJ+/− littermates. Interestingly, we found that the major class of downregulated genes in mutant epidermis was comprised of hair keratins (Hb6 or KRT86, Ha3-1 or KRT33A), as well as KAPs, particularly those of the Krtap-16 family (Table 1, Krtap-16 family highlighted in red). Most of the down-regulated Krtap-16 family members in Dsg4 −/− epidermis have been shown recently to be expressed in the HF cortex, coinciding with the expression domain of DSG4 (Pruett et al., 2004). Interestingly, many of the promoter regions of Krtap-16 genes are also potential downstream targets of Hoxc13 (Pruett et al., 2004).

Table thumbnail

HOXC13, LEF1 and FOXN1 are repressors of DSG4 expression

The functional relationship between hair keratins, KAPs and DSG4 during hair shaft differentiation led us to hypothesize that common transcription factors and regulatory pathways collectively control their expression. To place DSG4 into the context of a regulatory network, we investigated the upstream regulation of Dsg4 transcription. Using in silico promoter analysis, we identified 10 and 19 putative binding sites for LEF1 (red in Supplementary Fig 1) and HOXC13 (green in Supplementary Fig 1), respectively, within 3Kb of the upstream region of human DSG4. Many of these sites were conserved in the corresponding mouse and rat Dsg4 genomic regions (Supplementary Fig 1A,B). The high prevalence of these binding sites and their conservation between species suggests functional relevance. FOXN1 has an 11-base pair consensus binding sequence that includes an invariable 4 nucleotides (ACGC) core. While the statistical probability of finding this short 4 nucleotide core in a random 3 kb DNA sequence is relatively high compared to the HOXC13 and LEF1 consensus biniding sequences, it occurs only 3 times in the corresponding 3 kb DSG4 upstream segment.

In support of a functional relationship, using immunofluorescence analyses, we showed that DSG4 expression overlaps with the expression of HOXC13, LEF1 as well as FOXN1 in the precortex region of the human HF (Fig 4). HOXC13 is also co-expressed with DSG4 in the IRS cuticle (Fig 4A,B) and FOXN1 showed prominent expression in the differentiating cortex which is the major site of DSG4 expression (Fig 4C,D).

Figure 4
DSG4 expression (green) overlaps with that of HOXC13 (A,B), FOXN1 (C,D), and LEF1 (all is red) in the precortex and sometimes cortex of the human hair follicles. Note that DSG4 and HOXC13 are co-expressed also in the IRS cuticle (arrows in B) and that ...

We next cloned the upstream regions (1Kb and 3Kb, “H” for human and “m” for mouse) of DSG4 from both humans and mice and performed reporter gene assays using the luciferase system. When we transfected these reporter constructs into HaCaT keratinocytes, a non-tumorigenic human keratinocytes cell line, they showed an endogenous activity that was significantly higher than background levels (Fig 5A). This is consistent with our earlier findings that HaCaT keratinocytes express endogenous DSG4 when confluent (Bazzi et al., 2006).

Figure 5Figure 5Figure 5Figure 5
The endogenous activity of Dsg4 upstream region reporter constructs is repressed by some transcription factors in HaCaT keratinocytes. (A) ~3Kb and ~1kb upstream of human (H1,3) and mouse (m1,3) show endogenous reporter construct activity in HaCaT keratinocytes ...

We next examined the effect of each of the above mentioned three transcription factors on H3 transcriptional activity in HaCaT keratinocytes. We found that HOXC13 repressed H3 endogenous transcriptional activity to background levels in a similar manner (Fig 5B). This outcome was specific to HOXC13, since a mutant HOXC13 that lacked the DNA binding domain showed no effect on the H3 reporter construct activity (Fig 5B, Mut HOXC13). Moreover, FOXN1 repressed H3, while MSX2 had no significant effect on H3 reporter construct expression (Fig 5C). LEF1 expression also completely repressed the endogenous activity of all four constructs to pGL3 control levels (data not shown, e.g. the human 3Kb reporter construct H3 in Fig 5D). We obtained the same result upon transfecting both LEF1 and β-catenin together with H3 (data not shown).

Notch signaling is involved in DSG4 expression in vitro and in vivo

We next screened additional transcription factors that are expressed in the HF precortex or cortex in search of positive regulators of hair shaft differentiation. Our results showed that Notch (signaling through NICD), as well as the homeodomain gene Hoxc12, significantly activated H3 above endogenous levels (Fig 6A). Hoxc12 is expressed in the hair shaft cortex of mouse HFs coinciding with Dsg4 expression (Shang et al., 2002). The finding that NICD activates DSG4 transcription in vitro is intriguing given the striking similarity in the HF precortex defect in lahJ−/− and presenilin1 and 2 double conditional knockout (PSDCKO), that lack γ-secretase activity in MSX2 expressing tissues including some HFs (Pan et al., 2004), compared to WT follicles (Fig 6B–D). We then asked if Notch signaling regulates Dsg4 expression in vivo and therefore examined Dsg4 expression in the HFs of PSDCKO animals. We found that Dsg4 is markedly reduced in the abnormal precortex and accumulates beneath the keratinizing swelling compared to normal cortical expression in WT HFs (Fig 6E,F).

Figure 6Figure 6
Notch is a potential activator of Dsg4 expression in vitro and in vivo. (A) NICD, the downstream effector of Notch signaling, and Hoxc12 increase H3 reporter construct activity in HaCaT keratinocytes. (B–D) The precortical abnormal mass of keratinization ...

Discussion

The mechanisms governing the transcriptional regulation of hair shaft expressed genes are poorly understood. A major challenge in studying hair shaft cells is the lack of a cell culture model for their precursors. In silico studies backed by functional electromobility shift assays (EMSA) and/or reporter gene assays have been performed on a very limited number of hair keratin genes (Ha1 and Ha5) and KAPs (KAP 16-5) (Jave-Suarez et al., 2002; Pruett et al., 2004). Correlation of the overlap in expression patterns of transcription factors and genes in question between different HF compartments has proven to be a fruitful starting point for such studies. Here, we systematically screened a battery of transcription factors that are expressed in the precortex and cortex of the HF for possible regulatory roles on Dsg4 regulation. We found that transcription factors previously shown to activate hair keratin expression, such as HOXC13, LEF1 and FOXN1, repress DSG4 reporter activity in HaCaT keratinocytes (Fig 5). In contrast, our studies implicate Notch signaling through NICD, as well as Hoxc12 in DSG4 transcriptional activation (Fig 6).

Our finding that NICD activates the H3 reporter above endogenous levels suggests a role for Notch signaling in the regulation of DSG4 expression (Fig 6A). This is corroborated in vivo by the striking similarity in the HF phenotypes of Dsg4−/− or lah mutant mice and PSDCKO mice that lack γ-secretase activity and therefore a processed NICD (Fig 6C,D) (Pan et al., 2004; Bazzi et al., 2005). The decrease in Dsg4 expression in PSDCKO HFs also points to a role of NICD in Dsg4 expression regulation. It remains possible that the physical disruption of cell-cell contacts in Dsg4−/− HF precortex cells impairs Notch signaling by mechanical means. Since Notch signaling is mediated through the binding of a membrane-bound ligand and receptor on two adjacent cells, this could be an additional mechanism by which the phenotype in PSDCKO is reminiscent of Dsg4−/− HFs.

The crucial role of Notch signaling in epidermal differentiation is indisputable (Rangarajan et al., 2001; Blanpain et al., 2006). Notch signaling, through its downstream transcription factor RBPjK (which provides the DNA binding component for the NICD transcriptional activation domain), has been shown to act as a commitment switch from basal to suprabasal layers in the mouse epidermis (Blanpain et al., 2006). The upstream region of DSG4 up to 3Kb harbors only one RBPjK consensus binding site at −2089 of the translation start site that is not conserved in mouse and rat. Repeated trials with chromatin immunoprecipitation (ChIP) using an anti-NICD antibody (Cell Signaling) did not show any binding of an NICD-containing complex to this RBPjK site (data not shown). In addition, the γ-secretase inhibitor DAPT {N-(N-[3,5-difluorophenacetyl]-L-alanyl)-S-phenylglycine t-butyl ester}, was unable to reduce the endogenous activity of H3 in HaCaT keratinocytes (data not shown) (Geling et al., 2002). These findings suggest that the regulation of DSG4 by Notch signaling is indirect. This indirect regulation of DSG4 by Notch is supported by the expression of DSG4 in the upper spinous and granular layers of the epidermis and the role of Notch in the earlier transition step from basal to spinous differentiation (Bazzi et al., 2006; Blanpain et al., 2006).

Cadherins in general, including both classical and desmosomal cadherins, are calcium-dependent cell-cell junctions. Classical cadherins are linked to the actin cytoskeleton of the cell while desmosomal cadherins are linked to the intermediate filament cytoskeleton. Classical cadherins and the actin cytoskeleton are believed to be organized prior to and provide a framework for the desmosmal cadherins and the intermediate filament cytoskeleton in an epithelial sheet (Yin and Green, 2004). The post-translational regulation of cadherin assembly and the role of calcium in this process is relatively well understood (Peinado et al., 2004; Denning et al., 1998). The transcriptional regulation of expression of classical cadherins such as E-cadherin is also a widely studied area mainly because of E-cadherin involvement in epithelial to mesenchymal transition (EMT) during cancer invasiveness and metastasis (Peinado et al., 2004). In contrast, the transcriptional regulation of desmosomal cadherins has been largely understudied. It has been shown that DSC expression is regulated by CCAAT/enhancer binding proteins (C/EBP) in the epidermis and that DSG1 is regulated by protein kinase C (PKC) (Smith et al., 2004; Denning et al., 1998). Apart from our recent studies on the transcriptional regulation of Dsg4 by the BMP downstream effectors Smads, particularly Smad4, as well as the transcriptional regulation of Dsc2 by Hoxc13 and Foxq1, very little is known about desmosomal cadherin transcriptional regulation in the HF (Owens et al., 2008; Potter et al., 2006).

In summary, we report that desmoglein 4 expression, activation and/or maintenance correlate with Notch signaling in vitro and in vivo, as well as Hoxc12 expression in the hair cortex. We also show that dsg4 is repressed by Wnt signaling downstream effectors (LEF1 and β-catenin) and other transcription factors such as HOXC13 and FOXN1. Our findings highlight the importance of fine tuning transcriptional regulation during the transition from proliferation to differentiation in the hair shaft precortex. Desmogleins and desmocollins reside in a highly conserved genomic cluster on chromosome 18 in humans, mice, and rats (Hunt et al., 1999). DSGs are transcribed in one direction and DSCs in the opposite direction, andhe expression of desmosomal cadherins also correlates with the differentiation state of the tissue (Bazzi et al., 2006; Kurzen et al., 1998). The mechanisms of coordinated transcriptional regulation of this genomic cluster during development, differentiation, skin homeostasis and when these processes go awry undoubtedly warrant further investigation.

Supplementary Material

01

Supplementary Figure 1. Hoxc13 (green) and Lef1 (red) consensus binding sequences are predominant in ~3Kb of the upstream region of DSG4 (A) and many are conserved between mouse (m), rat (r) and human (h) (examples in B).

Acknowledgements

We thank Mr. Ming Zhang for excellent technical assistance. We also thank all our colleagues mentioned in the Materials and Methods section for their generous gifts of reagents. Many thanks to all the members of the Christiano lab for helpful assistance and discussion. This work was supported by a grant from NIH AR44924 to A.M.C.

Abbreviations

HF
hair follicle
DSG4/DSG4
human desmoglein 4 gene/protein
Dsg4/Dsg4
mouse or rat desmoglein 4 gene/protein
LAH
localized autosomal recessive hypotrichosis
lah
lanceolate hair rat or mouse
ORS
outer root sheath
IRS
inner root sheath
PSDCKO
Presenilin 1 and 2 double conditional knockout

Footnotes

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References

  • Bazzi H, Christiano AM. Broken hearts, woolly hair, and tattered skin: when desmosomal adhesion goes awry. Curr Opin Cell Biol. 2007;19:515–520. [PMC free article] [PubMed]
  • Bazzi H, Fantauzzo KA, Richardson GD, Jahoda CA, Christiano AM. Transcriptional profiling of developing mouse epidermis reveals novel patterns of coordinated gene expression. Dev Dyn. 2007;236:961–970. [PubMed]
  • Bazzi H, Getz A, Mahoney MG, Ishida-Yamamoto A, Langbein L, Wahl JK, 3rd, Christiano AM. Desmoglein 4 is expressed in highly differentiated keratinocytes and trichocytes in human epidermis and hair follicle. Differentiation. 2006;74:129–140. [PubMed]
  • Bazzi H, Kljuic A, Christiano AM, Christiano AM, Panteleyev AA. Intragenic deletion in the Desmoglein 4 gene underlies the skin phenotype in the Iffa Credo "hairless" rat. Differentiation. 2004;72:450–464. [PubMed]
  • Bazzi H, Martinez-Mir A, Kljuic A, Christiano AM. Desmoglein 4 mutations underlie localized autosomal recessive hypotrichosis in humans, mice, and rats. J Investig Dermatol Symp Proc. 2005;10:222–224. [PubMed]
  • Blanpain C, Lowry WE, Pasolli HA, Fuchs E. Canonical notch signaling functions as a commitment switch in the epidermal lineage. Genes Dev. 2006;20:3022–3035. [PubMed]
  • Dane ET, Herman DL. Haematoxylin-phloxine-Alcian blue-orange G differential staining of prekeratin, keratin and mucin. Stain Technol. 1963;38:97–101. [PubMed]
  • Denning MF, Guy SG, Ellerbroek SM, Norvell SM, Kowalczyk AP, Green KJ. The expression of desmoglein isoforms in cultured human keratinocytes is regulated by calcium, serum, and protein kinase C. Exp Cell Res. 1998;239:50–59. [PubMed]
  • Fuchs E, Merrill BJ, Jamora C, DasGupta R. At the roots of a never-ending cycle. Dev Cell. 2001;1:13–25. [PubMed]
  • Geling A, Steiner H, Willem M, Bally-Cuif L, Haass C. A gamma-secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish. EMBO Rep. 2002;3:688–694. [PubMed]
  • Godwin AR, Capecchi MR. Hoxc13 mutant mice lack external hair. Genes Dev. 1998;12:11–20. [PubMed]
  • Hardy MH. The secret life of the hair follicle. Trends Genet. 1992;8:55–61. [PubMed]
  • Hunt DM, Sahota VK, Taylor K, Simrak D, Hornigold N, Arnemann J, Wolfe J, Buxton RS. Clustered cadherin genes: a sequence-ready contig for the desmosomal cadherin locus on human chromosome 18. Genomics. 1999;62:445–455. [PubMed]
  • Jave-Suarez LF, Winter H, Langbein L, Rogers MA, Schweizer J. HOXC13 is involved in the regulation of human hair keratin gene expression. J Biol Chem. 2002;277:3718–3726. [PubMed]
  • Kljuic A, Bazzi H, Sundberg JP, Martinez-Mir A, O'Shaughnessy R, Mahoney MG, Levy M, Montagutelli X, Ahmad W, Aita VM, Gordon D, Uitto J, Whiting D, Ott J, Fischer S, Gilliam TC, Jahoda CA, Morris RJ, Panteleyev AA, Nguyen VT, Christiano AM. Desmoglein 4 in hair follicle differentiation and epidermal adhesion: evidence from inherited hypotrichosis and acquired pemphigus vulgaris. Cell. 2003;113:249–260. [PubMed]
  • Kurzen H, Moll I, Moll R, Schafer S, Simics E, Amagai M, Wheelock MJ, Franke WW. Compositionally different desmosomes in the various compartments of the human hair follicle. Differentiation. 1998;63:295–304. [PubMed]
  • Langbein L, Schweizer J. Keratins of the human hair follicle. Int Rev Cytol. 2005;243:1–78. [PubMed]
  • Lee YJ, Rice RH, Lee YM. Proteome analysis of human hair shaft: from protein identification to posttranslational modification. Mol Cell Proteomics. 2006;5:789–800. [PubMed]
  • Legue E, Nicolas JF. Hair follicle renewal: organization of stem cells in the matrix and the role of stereotyped lineages and behaviors. Development. 2005;132:4143–4154. [PubMed]
  • Loots GG, Ovcharenko I, Pachter L, Dubchak I, Rubin EM. rVista for comparative sequence-based discovery of functional transcription factor binding sites. Genome Res. 2002;12:832–839. [PubMed]
  • Ma L, Liu J, Wu T, Plikus M, Jiang TX, Bi Q, Liu YH, Muller-Rover S, Peters H, Sundberg JP, Maxson R, Maas RL, Chuong CM. 'Cyclic alopecia' in Msx2 mutants: defects in hair cycling and hair shaft differentiation. Development. 2003;130:379–389. [PMC free article] [PubMed]
  • Mecklenburg L, Tychsen B, Paus R. Learning from nudity: lessons from the nude phenotype. Exp Dermatol. 2005;14:797–810. [PubMed]
  • Messenger AG, Bazzi H, Parslew R, Shapiro L, Christiano AM. A missense mutation in the cadherin interaction site of the desmoglein 4 gene underlies localized autosomal recessive hypotrichosis. J Invest Dermatol. 2005;125:1077–1079. [PubMed]
  • Owens P, Bazzi H, Engelking E, Han G, Christiano AM, Wang XJ. Smad4-dependent desmoglein-4 expression contributes to hair follicle integrity. Dev Biol. 2008 [PMC free article] [PubMed]
  • Pan Y, Lin MH, Tian X, Cheng HT, Gridley T, Shen J, Kopan R. gamma-secretase functions through Notch signaling to maintain skin appendages but is not required for their patterning or initial morphogenesis. Dev Cell. 2004;7:731–743. [PubMed]
  • Peinado H, Marin F, Cubillo E, Stark HJ, Fusenig N, Nieto MA, Cano A. Snail and E47 repressors of E-cadherin induce distinct invasive and angiogenic properties in vivo. J Cell Sci. 2004;117:2827–2839. [PubMed]
  • Potter CS, Peterson RL, Barth JL, Pruett ND, Jacobs DF, Kern MJ, Argraves WS, Sundberg JP, Awgulewitsch A. Evidence that the satin hair mutant gene Foxq1 is among multiple and functionally diverse regulatory targets for Hoxc13 during hair follicle differentiation. J Biol Chem. 2006;281:29245–29255. [PubMed]
  • Pruett ND, Tkatchenko TV, Jave-Suarez L, Jacobs DF, Potter CS, Tkatchenko AV, Schweizer J, Awgulewitsch A. Krtap16, characterization of a new hair keratin-associated protein (KAP) gene complex on mouse chromosome 16 and evidence for regulation by Hoxc13. J Biol Chem. 2004;279:51524–51533. [PubMed]
  • Rangarajan A, Talora C, Okuyama R, Nicolas M, Mammucari C, Oh H, Aster JC, Krishna S, Metzger D, Chambon P, Miele L, Aguet M, Radtke F, Dotto GP. Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation. EMBO J. 2001;20:3427–3436. [PubMed]
  • Rogers GE. Hair follicle differentiation and regulation. Int J Dev Biol. 2004;48:163–170. [PubMed]
  • Schaffer JV, Bazzi H, Vitebsky A, Witkiewicz A, Kovich OI, Kamino H, Shapiro LS, Amin SP, Orlow SJ, Christiano AM. Mutations in the desmoglein 4 gene underlie localized autosomal recessive hypotrichosis with monilethrix hairs and congenital scalp erosions. J Invest Dermatol. 2006;126:1286–1291. [PubMed]
  • Shang L, Pruett ND, Awgulewitsch A. Hoxc12 expression pattern in developing and cycling murine hair follicles. Mech Dev. 2002;113:207–210. [PubMed]
  • Shimomura Y, Sakamoto F, Kariya N, Matsunaga K, Ito M. Mutations in the desmoglein 4 gene are associated with monilethrix-like congenital hypotrichosis. J Invest Dermatol. 2006;126:1281–1285. [PubMed]
  • Smith C, Zhu K, Merritt A, Picton R, Youngs D, Garrod D, Chidgey M. Regulation of desmocollin gene expression in the epidermis: CCAAT/enhancer-binding proteins modulate early and late events in keratinocyte differentiation. Biochem J. 2004;380:757–765. [PubMed]
  • Sundberg JP, Boggess D, Bascom C, Limberg BJ, Shultz LD, Sundberg BA, King LE, Jr, Montagutelli X. Lanceolate hair-J (lahJ): a mouse model for human hair disorders. Exp Dermatol. 2000;9:206–218. [PubMed]
  • van Genderen C, Okamura RM, Farinas I, Quo RG, Parslow TG, Bruhn L, Grosschedl 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]
  • Wajid M, Bazzi H, Rockey J, Lubetkin J, Zlotogorski A, Christiano AM. Localized autosomal recessive hypotrichosis due to a frameshift mutation in the desmoglein 4 gene exhibits extensive phenotypic variability within a Pakistani family. J Invest Dermatol. 2007;127:1779–1782. [PubMed]
  • Yin T, Green KJ. Regulation of desmosome assembly and adhesion. Semin Cell Dev Biol. 2004;15:665–677. [PubMed]
  • Zlotogorski A, Marek D, Horev L, Abu A, Ben-Amitai D, Gerad L, Ingber A, Frydman M, Reznik-Wolf H, Vardy DA, Pras E. An autosomal recessive form of monilethrix is caused by mutations in DSG4: clinical overlap with localized autosomal recessive hypotrichosis. J Invest Dermatol. 2006;126:1292–1296. [PubMed]