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Previous Article | Next Article 
Molecular and Cellular Biology, August 2005, p. 7249-7259, Vol. 25, No. 16
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.16.7249-7259.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Changhong Gu,2 and
Pierre A. Coulombe2,3*
Predoctoral Program in Human Genetics, McKusick-Nathans Institute of Genetic Medicine,1 Department of Biological Chemistry,2 Department of Dermatology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 212053
Received 10 February 2005/ Returned for modification 17 March 2005/ Accepted 19 May 2005
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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50 conserved genes, are abundant proteins in epithelial cells, where they heteropolymerize to form cytoplasmic intermediate filaments (11, 12, 25). The pairwise and differentiation-related transcriptional regulation of keratin genes, which are relatively small (<10 kb in size), relies on proximal cis-acting determinants (48, 59, 64, 73). The 5' upstream sequences from a number of keratin genes have been exploited to direct cell type-specific expression of heterologous open reading frames in transgenic mice. For instance, the K14 promoter- and K5 promoter-based cassettes (46, 59, 62, 73) have been used to create hundreds of transgenic mouse lines which, collectively, have had an extraordinary impact on our understanding of the epidermis and related complex epithelia. The unusually broad variety of epithelial settings in which type I keratin 17 (K17) occurs likely call for intricate transcriptional regulatory mechanisms. During development, specification of epithelial appendages is marked by the onset of K17 synthesis in the embryonic ectoderm (40). Concomitant with the maturation of these precursor elements into tissues such as hair follicle, nail, gland, and tooth, K17 expression becomes restricted to specific cell types (40, 70). In the hair follicle, which undergoes a developmental cycle throughout life, K17 expression continues to be dynamically modulated in the adult setting (2, 56). K17 also has the rare distinction of being expressed in both soft and hard epithelia, and K17 protein uses various type II keratins (K6, K6hf, and K5) as polymerization partners, depending on the epithelial setting in vivo (39, 76). In addition to this constitutive regulation, K17 is strongly inducible in epidermis following acute challenges such as skin injury or in disease contexts (e.g., viral infection, psoriasis, or basal cell carcinoma [15, 41]). K17 is not transcribed in normal interfollicular epidermis (40, 67).
The green fluorescent protein (GFP) of Aequarea victoria has evolved into an unparalleled tool for visualizing cells in their natural living context and has been successfully exploited to this end in model organisms ranging from unicellular eukaryotes to mice (21, 50, 81). Here, we show that that the 5' upstream sequence from the mouse K17 gene (mK17) directs the expression of GFP, used as a reporter, in the appropriate cell types within epithelial appendages in transgenic mice. [mK17 5']-GFP is also faithfully regulated during the embryonic development of skin epithelia and can be exploited to track hair follicle cycling in adult skin. Recently, the mK17 promoter was shown to be highly responsive to the transcription factor Gli2, a terminal effector of sonic hedgehog signaling, in a heterologous system ex vivo (7). Here we show that deletion of a 48-bp fragment within the mK17 promoter reduces Gli2 responsiveness in transfected cells, as well as the expression of [mK17 5']-GFP transgene in hair follicles and other epithelial appendages in vivo. These findings have conceptual and practical implications for the study of epithelial appendages from a broad variety of angles in vivo.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1,970-bp amplicon was sequenced. The mK17 promoter was inserted into the multiple cloning site of pEGFP-N1 (Clontech, Palo Alto, CA), using AseI and BamHI restriction digests. This removed the entire simian virus 40 promoter from pEGFP-N1 while preserving the poly(A) signal at the 3' end. A 48-bp deletion, from –461 to –414 (+1 refers to the ATG start codon), was created as follows. Two promoter fragments, extending from –1974 to –461 (P1/P2 primers) and from –414 to +1 (P4/P6 primers), were generated by PCR using [mK17 5']-GFP as a template. PCR conditions were 94°C for 1 min (denaturation), 65°C for 1 min (annealing), and 72°C for 1 min (elongation) for 30 cycles. The PCR primers used were as follows and contained suitable restriction enzyme sites as indicated to facilitate subcloning: mK17delA-P1 (sense), 5'-(EcoRV)GCGATATCTTTTTTTGCTTCCCTC-3'; mK17delA-P2 (antisense), 5'-CAAGCTTGATGGGAAATGAGGTAGGG(HindIII)-3'; mK17delA-P4 (sense), 5'-(HindIII)CGCAAGAAGCTTTTCTTGTCCGTATTAGG-3'; and mK17delA-P6 (antisense), 5'-TGGATCCATGGTGGCAGCGGGCAA(BamHI)-3'. PCR products were sequenced and subcloned as EcoRV-BamHI fragments into [mK17 5']-GFP to generate [mK17 delA 5']-GFP. To generate the parent [mK17 –1950/+19]-luciferase construct, the PCR product used to produce [mK17 5']-GFP was blunt ended with Klenow polymerase and subcloned into SmaI-digested pGL3-Basic vector (Promega, Madison WI). The [mK17 delA 5']-luciferase construct was obtained by digesting [mK17 delA 5']-GFP with ApaI and EcoRI and subcloning the excised fragment into the corresponding sites within [mK17 –1950/+19]-luciferase.
Transgenic mouse lines and production of staged mouse embryos. Studies involving animals were approved by the Johns Hopkins University Animal Care and Use Committee. Transgenic founders were produced by pronuclear injection of GFP reporter DNA constructs in C57BL/6-BALB/c3 embryos (26). Founders were identified using Southern blotting and PCR with strategies aimed at detecting the GFP-coding sequence in genomic DNA. Southern blotting was used to compare transgene copy numbers between lines. Probes specific for the mK16 (2) or mK17 (5) locus were used as internal loading controls. When needed, band densities on computer-scanned Southern blots were quantitated using NIH Image software.
Transgenic lines were established by crossing the [mK17 5']-GFP and [mK17 delA 5']-GFP founders with wild-type C57BL/6-BALB/c3 mice. [mK17 5']-GFP mice were also bred into the hairless background (80) by using the HRS strain available from Jackson Laboratories (Bar Harbor, ME). Once established, [mK17 5']-GFP mice were screened using live fluorescence imaging (see below). Genomic DNA from [mK17 delA 5']-GFP mice was screened using PCR.
To obtain embryos at defined developmental stages, timed pregnancies were initiated by mixing males and females and setting 12:00 noon the next day as embryonic day 0.5 (E0.5) (80). Pregnant females were euthanatized by cervical dislocation at noon on the desired day of embryonic development (ranging from E12.5 to E.18.5; term is at E19), and embryos were surgically harvested. Live imaging was conducted or frozen sections were collected for immunolocalization studies.
RNA analyses. Back skin, paw, whisker pad, tongue, stomach, liver, kidney, lung, spleen, brain, and heart tissues were surgically collected from adult mice, snap frozen in liquid nitrogen, and stored at –80°C. Total RNA was extracted by homogenizing these tissues in TRIzol reagent, following the manufacturer's protocol (Life Technologies, Inc., Gaithersburg, MD). RNA was quantitated spectrophotometrically using absorbance at 260 nm. cDNAs were synthesized via reverse transcription from 1 µg of RNA according to the manufacturer's protocol (Advantage for PCR; Clontech, Palo Alto, CA). This cDNA was then used as a template for PCR with either one of the following pairs of primers, specific for the GFP and mK17 transcripts; in both cases the PCR conditions were 94°C for 1 min (denaturation), 66°C for 1 min (annealing), and 72°C for 1 min (elongation) for 25 to 35 cycles: GFP-forward, 5'-GGCGACGTAAACGGCCACAAGTTCAGCG-3'; GFP-reverse, 5'-CGCTTCTCGTTGGGGTCTTTGCTCAGGG-3'; K17-forward, 5'-CCACCTGACTCAGTACAAGCCAAAAG-3'; and K17-reverse, 5'-CTGTGGCCTTTGTTCTGAACACTG-3'.
For Northern blot analyses (79), 10 µg of total RNA was resolved by formaldehyde-agarose gel electrophoresis, transferred to GeneScreen nylon membrane, and probed with a 714-bp-long cDNA probe obtained by digesting the GFP transgene with HphI and XbaI. The blot was then washed and exposed to radioautographic film.
Western blot analyses. Hair clippings were harvested from the back skin of adult mice, and total proteins were extracted (39). Extracted protein samples (4 µg) were electrophoresed in either 8.5% or 10% SDS-polyacrylamide gels, to look for K17 or GFP immunoreactivity, respectively. Gels were then electroblotted onto Protran nitrocellulose (Schleicher & Schuell, Keene, NH). Bound primary antibodies directed against mouse K17 (40) or GFP (Clontech, Palo Alto, CA) were detected using enhanced chemiluminescence as described by the manufacturer (Amersham Biosciences Piscataway, NJ).
Morphological analysis and live imaging of GFP expression. A Leica MZ FL III microscope equipped with an excitation filter of 470/40 nm and a barrier filter of 525/50 nm was used to view GFP fluorescence in living skin. Data were collected using a Zeiss AxioCam charge-coupled-device camera connected to a PC running the Zeiss AxioVision software. Animals were anesthetized via intraperitoneal administration of avertin prior to live imaging.
All tissues slated for frozen sectioning were submerged in Optimal Cutting Temperature compound (Tissue-Tek; Sakura Fineteck, Torrance, CA) and frozen at –80°C. Frozen tissue blocks were sectioned (5-µm width) by a Microm International HM 505 E microtome cyrostat operated at –25°C. Cryosections mounted on microscope glass slides were stored in the dark at –20°C until viewed via fluorescence microscopy. To relate the distribution of GFP to that of K17, we used indirect immunofluorescence using a rabbit polyclonal antiserum directed against K17 (40, 67). Consecutive slides were utilized, as the soluble GFP is rapidly washed off from unfixed fresh frozen tissue sections during the antibody staining procedure.
Cell culture and transfection.
308 mouse keratinocyte cells (66) were maintained in mKer medium (61), consisting of 3 parts Dulbecco's minimum essential medium, 1 part Ham's F-12 medium, 10% premium fetal bovine serum, 5 µg/ml insulin, 0.4 µg/ml hydrocortisone, 5 µg/ml transferrin, 2 x 10–9 M 3,3,5'-triiodo-L-thyroxine, 10–10 M cholera toxin,10 ng/ml epidermal growth factor, 60 µg/ml penicillin, and 25 µg/ml gentamicin. Twenty-four-well tissue culture plates (Falcon, Franklin Lakes, NJ) were seeded with 45,000 cells per well and cultured until the cells were 80% confluent. pCMVb (Clontech, Palo Alto, CA) (50 ng), which directs ß-galactosidase gene expression, was routinely included for standardization. In addition, cells were transfected with 300 ng of an individual luciferase construct along with 150 ng of the mouse Gli2 expression vector (74), diluted in 50 µl of optiMEM medium (Invitrogen, Carlsbad, CA). Two microliters of Lipofectamine 2000 (Invitrogen) was added to 50 µl of optiMEM medium and incubated at room temperature for 5 min, and this mixture was then added to the DNA solution and left for a further 20 min. The mixture was overlaid on plated cells bathing in 400 µl of fresh optiMEM medium. After 12 h, the transfection medium was replaced with fresh mKer medium and cells harvested at 48 h posttransfection. Cell lysates were prepared by washing wells with phosphate-buffered saline, followed by addition of 150 µl of 1x Passive Lysis Buffer (Promega) and a 15-min incubation with agitation.
Analysis of luciferase activity. Lysates of transfected cells were assayed for luciferase activity using the Luminoskan Ascent luminescent plate reader (Thermo Electron Corporation, Waltham, MA). Twenty microliters of cell lysate was pipetted into the wells of a 96-well microtiter plate (Microlite 1; Thermo Labsystems, Chantilly, VA). One hundred microliters of luciferase assay buffer [20 mM Tricine-NaOH (pH 7.8), 1.07 mM (MgCO3)4Mg(OH)2 · 5H2O, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM dithiothreitol, 270 µM coenzyme A, Li salt, 470 µM d-Luciferin, K salt, 530 µM ATP] was dispensed into each well and the luciferase activity allowed to integrate for 10 s. ß-Galactosidase activity was assayed from a separate 20-µl aliquot, to which 100 µl of assay buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, and 1 mM MgCl2) and 25 µl of 2-mg/ml ortho-nitrophenyl-ß-D-galactopyranoside was added. A 5-min incubation at 37°C was followed by addition of 75 µl of 1 M Na2CO3 to quench the reaction. Absorbance at 420 nm was measured on a Bio-Tek ELx800 universal microplate reader (Bio-Tek, Winooski, VT). Each construct was transfected in duplicate, and the data reported (means ± standard errors of the means) are the results of at least three independent experiments. Data were normalized and expressed as fold induction over the value obtained in cotransfection with the pCDNA3 vector.
[mK17 5']-GFP transgene regulation during wound epithelialization in vivo. For an analysis of [mK17 5']-GFP reporter expression in adult transgenic mouse skin subjected to experimental injury or topical application of a chemical inducer and an analysis of the responsiveness of the mK17 5' upstream sequence to terminal effectors of Wnt signaling, see Fig. S1 in the supplemental material.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1,970-bp-long 5' upstream region from the mK17 gene (40), which corresponds to the entire intergenic region between mK17 and the telomeric mK17n on mouse chromosome 11 (67). Two transgenic mouse lines, designated 1 and 2 (Fig. 1B), were characterized. Line 1 shows stronger expression of GFP than line 2 (even though it has a lower copy number per genome [Fig. 1B]). Otherwise, lines 1 and 2 behave identically in the analyses reported below, which emphasize skin and oral mucosa, two major sites of K17 expression (40, 56, 70). Unless mentioned otherwise, the findings reported are from [mK17 5']-GFP line 1.
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We next analyzed the distribution of GFP fluorescence by using macroscopic live imaging coupled with microscopic analysis on frozen tissue sections (Fig. 2). Intrinsic GFP fluorescence was readily detected through live imaging in tissues known to contain K17-expressing cells, including hair follicles (Fig. 2A [ear] and B [back skin]), vibrissae (Fig. 2D), nail (Fig. 2F), dorsal tongue surface (Fig. 2H), and palate (data not shown). Such fluorescence does not occur in corresponding tissues from wild-type mice (data not shown). In the case of hair follicles, GFP fluorescence occurs in the externally visible hair shaft and beneath its point of emergence at the skin surface (Fig. 2A). Upon tissue sectioning, the distribution of intrinsic GFP fluorescence coincides with that of K17 antigens. Thus, in pelage hair follicles, GFP fluorescence occurs in the outer root sheath, medulla, and matrix compartments (Fig. 2C and C') (40, 42). Similar results were obtained for vibrissa follicles (Fig. 2 E and E'); for other epithelial appendages known to express K17 (40, 42), including nail (Fig. 2F and G), filiform and fungiform papillae of dorsal tongue epithelium (Fig. 2I to K), sweat glands (Fig. 2G), and the mucous glands embedded in the skeletal muscle of the tongue (Fig. 2L and L'); and for stomach (data not shown). These observations suggest that the [mK17 5']-GFP transgene faithfully mimics the regulation of the endogenous gene in adult mouse skin, oral epithelia, and stomach.
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A 48-bp internal deletion in the mK17 promoter abrogates its activity in hair follicles in vivo. Through luciferase reporter assays performed with human skin keratinocytes, Callahan et al. (7) recently showed that the full-length mK17 promoter can be transactivated by the transcription factor Gli2. This finding provides a likely molecular basis for the consistent induction of mK17 in basal cell carcinoma of the skin, which is often associated with mutations causing deregulated sonic hedgehog signaling (see Discussion). From deletion analyses those authors inferred that a 41-nucleotide-long segment, designated –398/–448 bp (Fig. 1A), was likely to contain a Gli2-responsive element(s) (7). This region of the promoter features potential Gli binding sites that are conserved between mouse and human K17 genes (Fig. 4A).
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–461/–414 bp or delA (Fig. 1A), encompassing these potential Gli binding sites. Under our experimental conditions, the full-length mK17 promoter-driven luciferase construct showed 69-fold activation over baseline when cotransfected with a Gli2 expression vector in a mouse skin keratinocyte cell line (Fig. 4B). In contrast, the delA construct showed a much-reduced, 16-fold activation over baseline when coexpressed with Gli2 (Fig. 4B). When considering intrinsic promoter activity, [mK17 delA 5']-luciferase is stronger than the parental [mK17 5']-luciferase construct (data not shown), pointing to the presence of a repressor element within the –461/–414 bp segment. These findings confirm the prediction of Callahan et al. (7) in showing that this short region within the mK17 promoter features an element(s) that contributes to confer strong responsiveness to Gli2. The region encompassing the delA mutation was subcloned in the [mK17 5']-GFP DNA construct (Fig. 1A). Microinjection in mouse embryos yielded two transgenic founders and their independent lines (Fig. 4C). In either case, no expression of the [mK17 delA 5']-GFP transgene could be detected in skin tissue, using either live imaging of tissue sections (Fig. 4D and D'), an RT-PCR-based screen on total RNA samples (Fig. 4F), or Western immunoblotting of hair protein extracts (Fig. 4G). Likewise, no GFP fluorescence could be detected in various types of K17-expressing glands (data not shown). This deletion did not completely abrogate transgene activity, however, as evidenced by the presence of GFP fluorescence in the anterior column of filiform papillae in dorsal tongue epithelium (Fig. 4E and E') and of GFP mRNA in tongue tissue (Fig. 4F). Interestingly, shh signaling is distinctly required for formation of fungiform papillae, where GFP expression is lost in the absence of the 48-bp fragment (23, 30). Residual activity in oral mucosa establishes that lack of expression in skin is not the result of transgene integration in a transcriptionally silent area of the mouse genome. While we cannot rule out that [mK17 delA 5']-GFP exhibits an activity below the sensitivity threshold of our assays, these data establish that deletion of this internal 48-bp segment leads to a substantial loss of mK17 promoter activity in many types of epithelial appendages in vivo.
Visualizing hair cycling events in live [mK17 5']-GFP skin tissue.
A potentially useful application of the [mK17 5']-GFP mouse model is the visualization of the fate of K17-expressing keratinocytes during dynamic processes involving skin epithelia. The mature hair follicle, for instance, undergoes a cycle composed of three phases: anagen (growth stage), catagen (an apoptosis-driven regression stage), and telogen (resting stage) (24). In mice the first two hair cycles occur synchronously (albeit with an anterior-posterior gradient) over the initial 50 days following birth (9). The distribution of K17 in hair follicles (2, 40, 56) is such that variations in GFP fluorescence should reflect progression through this cycle.
Macroscopic live imaging was conducted on adult [mK17 5']-GFP mice to test this possibility. In late-anagen-stage skin (P33), when hair follicles are fully extended within the dermis, GFP fluorescence appears as an elongated object projecting downward from the base of the protruding hair shaft at the skin surface (Fig. 5 A). By comparison, the GFP signal consists of significantly smaller dots in telogen-stage skin (P24), when resting follicles have regressed and shortened. Tissue sectioning followed by microscopic analysis confirmed that GFP fluorescence directly reflects hair follicle length, and closely mirrors that of endogenous K17, for these two stages of the hair cycle (compare Fig. 5A and B).
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DISCUSSION |
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Whereas K17 is rapidly induced after a variety of insults to human and mouse skin (41), we find that neither acute injury nor the topical application of a phorbol ester leads to [mK17 5']-GFP activation in transgenic epidermis (see Fig. S1 in the supplemental material). It follows that one or several cis-acting elements required for the manifestation of this aspect of mK17 regulation are located outside its proximal 5' upstream sequence. They could reside in an intron and/or in the 3' noncoding sequence of mK17 or, alternatively, elsewhere in the type I keratin locus. Of note, mK16, the gene centromeric to mK17 (40, 67), is wound inducible and phorbol ester inducible as well (53, 75). In contrast, the telomeric mK17n is not wound inducible (67). The regulatory elements responsible for induction following injury and other environmental challenges are expected to be conserved between the human and mouse K17 genes and are under investigation. Meanwhile, the lack of inducibility in vivo simplifies further study and exploitation of the mK17 gene promoter.
Implications for the molecular mechanisms regulating K17 gene expression. Several lines of evidence point to mK17 as a likely target of shh signaling in vivo. shh-expressing cells are asymmetrically located within the hair matrix at the early anagen stage of the hair cycle (17), and these cells show higher levels of K17 antigen (39). Also, K17 is consistently expressed in basal cell carcinoma, which often arises from mutations resulting in enhanced shh signaling, in both human skin (34) and mouse models thereof (18, 19, 52). Above all, however, is the recent finding that the activity of the full-length mK17 promoter is stimulated when coexpressed alongside Gli2 in transfected human and mouse keratinocytes (7; this study). Here, we provide direct evidence that a 48-bp fragment, which is located between nucleotides –461 and –414 in the mK17 promoter and exhibits several potential Gli binding sites (7), contributes a significant fraction of the Gli responsiveness of mK17 in cultured skin keratinocytes.
Whether the loss of reporter activity in most epithelial appendages of [mK17 delA 5']-GFP mice directly reflects decreased Gli2 responsiveness is an enticing prospect that requires further testing through more refined molecular studies. Importantly, the deleted 48-bp motif in [mK17 delA 5']-GFP possesses potential binding sites for other transcription factors, such as Sp1 and AP2 (data not shown). Sp1 has been shown to modulate keratin expression in the hair follicle (4), while AP2 has defined roles in hair follicle development and cycling (55). Both Sp1 and AP2 have been shown to bind sequences located within the proximal 450 bp in the human K17 promoter (43). However, the binding sites for these factors are not conserved in mK17 (43). Again, more refined studies on this region of the mK17 and hK17 promoters will be required to define the role of these and other transcription factors in regulating their activity.
The significance of K17 as an shh target gene is an issue worth considering. Expression of K17 has been inferred to promote the survival of hair keratinocytes during the anagen phase of the hair cycle (42). The occurrence of mK17 transcription in keratinocytes exposed to shh, a signal leading to cell growth in hair and other epithelial appendages (27, 44, 51), therefore seems logical. shh responsiveness cannot, however, account for the onset of mK17 expression during development. Indeed, K17 expression can be detected in the early-stage epithelial placodes that form in shh null embryonic ectoderm (10, 65). The signal(s) activating mK17 transcription at the precursor placode stage thus remains to be defined.
We previously suggested, based on several arguments, that K17 could be a direct Wnt target gene during the early stages of placode formation in the embryonic ectoderm (31, 40). This notion was recently reinforced by lack of detectable K17 expression in transgenic embryos whose ectoderm ectopically expresses a soluble Wnt inhibitor (1). Surprisingly, however, we find that the activity of the mK17 gene promoter is not affected by lef-1 or tcf-4 expressed alone, or in combination with activated ß-catenin, in transfected Cos1 epithelial cells (data not shown; see Fig. S1 in the supplemental material). These findings thus do not support the view that a canonical Wnt signal single-handedly underlies the onset of mK17 expression in the embryonic ectoderm. Possibly, a proximal determinant(s) conferring Wnt responsiveness could be located outside the mK17 5' upstream sequence. Alternatively, another signal could be required alongside Wnt (29, 78), or a noncanonical Wnt signal could be involved (22).
Potential applications of the mK17 promoter and [mK17 5']-GFP mice. The activity profile of the mK17 promoter is distinct from those of other promoters shown to be preferentially active in transgenic mouse skin epithelia. These include, but are not limited to, K5 and K14 (46, 59, 62, 73), whose 5' upstream sequences confer expression in the basal layer of stratified epithelia; K15, which preferentially labels the presumptive epithelial stem cells located within the hair bulge (37, 45); K1 (20, 60), which unexpectedly confers expression in all layers of the epidermis; involucrin (8, 13), which, as expected, is active in the differentiating layers of epidermis and in hair follicles; and hard keratin genes (36, 58), which are active in the hair cortex and nail plate. As such, the mK17 promoter can be exploited to drive the expression of various open reading frames, including the Cre recombinase, in transgenic mice (47). Similar K14 promoter- and K5-promoter driven "Cre" mice have been exploited for conditional gene manipulation in epidermis and other stratified epithelia (3, 72). Such K17 promoter-driven "Cre donor" mice would, upon mating to mice carrying floxed alleles, enable appendage-preferred gene manipulation.
The existing [mK17 5']-GFP mice can be used, through fluorescence-activated cell sorting, to purify subpopulations of epithelial cells from dissected embryonic and adult tissues. Other reporter mouse models, exhibiting GFP expression in a restricted and interesting pattern within skin epithelia, have been devised (5, 35, 45, 71). In two instances, fluorescence-activated cell sorting was exploited to isolate and characterize populations of hair follicle cells enriched in epithelial stem cells (69, 71). [mK17 5']-GFP mice could also be used as a test bed in which to test strategies aimed at modulating gene expression in live skin. Examples include small interfering RNA-mediated decreases in GFP expression in epithelial appendages or screens for small chemicals capable of enhancing or decreasing [mK17 5']-GFP expression in skin. Either of these approaches is potentially desirable in the context of molecular therapies aimed at treating various types of skin conditions, including keratin-based genetic disorders.
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ACKNOWLEDGMENTS |
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This work was supported by grant AR44232 from the National Institutes of Health to P.A.C.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
Present address: ExonHit Therapeutics, 217 Perry Parkway, Gaithersburg, MD 20877.
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