WO2001044461A1 - Hoxc13 transgenic mice exhibiting hair loss and ichthyosis-like syndrome - Google Patents

Abstract

The present invention provides an isolated nucleic acid comprising the nucleic acid of GC13 which encodes a novel murine Hoxc13 protein. The invention further provides nucleic acid fragments of GC13 set forth in the Sequence Listing as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:7. The invention also provides a purified Hoxc13 polypeptide set forth in the Sequence Listing as SEQ ID NO:6. Further provided by this invention is a transgenic non-human animal whose cells overexpress a transgene coding for Hoxc13 wherein the total native and transgenic Hoxc13 expressed in the transgenic mouse is higher than the Hoxc13 expressed in a non-transgenic mouse, which transgenic mouse has increased hair loss and hyperkeratotic skin due to the overexpression of the transgene. Also provided are methods for screening compounds for an effect on hair loss, hyperkeratotic skin disorders, adipose tissue deposition and abnormal pigmentation. This invention also provides an expression construct for conferring follicle specific expression which comprises a regulatory sequence of Hoxc13 contained within GC13. Also provided is an expression construct for conferring keratinocyte specific expression which comprises a regulatory sequence of Hoxc13 contained within GC13.

Description

HOXC13 TRANSGENIC MICE EXHIBITING HAIR LOSS AND ICHTHYOSIS-LIKE SYNDROME

This application claims priority to U.S. application Serial No. 60/171,080, filed December 15, 1999, which is hereby incoφorated in its entirety by this reference.

This invention was made with government support under National Institutes of Health grant numbers GM43334 and 1R01 AR47204-01 . The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The invention relates generally to a murine Hoxcl3 gene and the nucleic acid sequences surrounding the Hoxcl3 gene. The invention also relates to transgenic mice overexpressing Hoxcl3 protein that exhibit hair loss, ichthyosis-like syndrome and reduced adipose tissue as well as to transgenic mice overexpressing Hoxcl3 protein that exhibit hair loss, ichthyosis-like syndrome, reduced adipose tissue and abnormal pigmentation.

BACKGROUND ART

Control of precursor cell differentiation is being discussed in the literature as one of several different mechanisms through which Hox genes may exert their embryonic patterning functions (Dolle et al. 1993, Krumlauf 1994,,Capecchil997). This mechanism is potentially of great relevance for presumptive roles of this group of regulatory genes during postnatal growth and development of various tissues, including skin. Studying these roles in normal and pathological development of skin and hair requires identification of Hox downstream target genes in transgenic animal models harboring defined genetic alterations. This invention provides transgenic mice overexpressing Hoxcl3 in pre- and postnatal skin that exhibit hyperproliferation of follicular, as well as epidermal, keratinocytes. Remarkably, these mice suffer from severe hair loss and develop a progressive pathological skin condion resembling ichthyosis. Large-scale analysis of differential gene expression in postnatal skin of these mice identified a total of 29 up- and skin and hair-specific genes as presumptive downstream targets for Hoxcl3. Accordingly, the Hoxcl3 overexpressing mice provide a novel model for identifying molecules affected by, or having an effect on, Hoxcl3 in normal and pathological growth and development of skin and hair.

SUMMARY OF THE INVENTION

The present invention provides an isolated nucleic acid comprising the nucleic acid of GC13 which encodes a novel murine Hoxcl3 protein.

The invention further provides nucleic acid fragments of GC13 set forth in the Sequence Listing as SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:7.

The invention also provides a purified Hoxcl3 polypeptide set forth in the

Sequence Listing as SEQ ID NO: 6.

Further provided by this invention is a transgenic non-human animal whose cells overexpress a transgene coding for Hoxcl3 wherein the total native and transgenic Hoxcl3 expressed in the transgenic animal is higher than the Hoxcl3 expressed in a non-transgenic animal, which transgenic animal has increased hair loss, hyperkeratotic skin and decreased adipose tissue due to the overexpression of the transgene.

Also provided is a transgenic non-human animal whose cells overexpress a transgene coding for Hoxcl3 wherein the total native and transgenic Hoxcl3 expressed in the transgenic animal is higher than the Hoxcl3 expressed in a non-transgenic animal, which transgenic animal has increased hair loss, hyperkeratotic skin, decreased adipose tissue due to the overexpression of the transgene and abnormal pigmentation.

Also provided is a method for screening compounds for an effect on hair loss comprising administering the compound to a transgenic animal of this invention and monitoring the animal for a change in hair growth, wherein altered hair growth is indicative of the effect of the compound on hair growth.

Further provided is a method for screening compounds for an effect on a hyperkeratotic skin disorder by administering the compound to a transgenic animal of this invention and monitoring the animal for a change in the skin disorder, wherein a change in the hyperkeratotic skin disorder is indicative of the effect of the compound on a hyperkeratotic skin disorder.

This invention also provides an expression construct for conferring follicle specific expression which comprises a regulatory sequence of Hoxcl3 contained within GC13.

Also provided is an expression construct for conferring keratinocyte specific expression which comprises a regulatory sequence of Hoxcl3 contained within GC13.

This invention also provides a method of identifying genes associated with the pathological changes observed in the skin of GC13 mice comprisingύsolating a gene differentially expressed in skin of GC13 mice by suppressive subtractive hybridization; obtaining a probe or immunohistochemical reagent for the differentially expressed gene of step and utilizing the probe to analyze the expression pattern of the differentially expressed gene in GC13 wherein altered expression of the gene as compared to a normal mouse indicates the gene is associated with the overexpression of Hoxcl3. BRIEF DESCRIPTION OF THE DRAWING

Figure 1 A provides a schematic of a transgene and Hoxcl3 protein sequence. Top: Schematic of genomic clone cos2W3H (Peterson et al., 1994) containing the Hoxc-13 transcription unit within the proximal 19 kb Notl (N) - Xhol (X) restriction fragment; map positions oϊHoxcl3, -cl2 and -ell homeoboxes are indicated as black boxed regions. Below: Close-up of 19 kb genomic fragment used as transgene. Boxed regions indicate sequences included in overlapping Hoxcl3 -specific cDNAs (see Experimental Procedures) with presumptive coding regions shown in grey and black (homeobox), respectively, and noncoding 5' and 3' flanking sequences shown as open boxes. Positions of the 4.5 kb intron and of a bonaβde AATAAA polyadenylation signal sequence (p(A)) are indicated. The LZ-GC13 reporter gene was generated by in- frame fusion of an E.coli lacZ gene into the coding region of the first exon of Hoxcl3 by replacing the 430 bp Ascl (A) fragment as indicated (see Experimental Procedures).

Figure IB is a comparison of deduced amino acid sequence oϊHoxcl3 with those of its paralogous genes Hoxal3, Hoxbl3, and Hoxdl3 (Mortlock et al., 1996; Zeltser et al., 1996; Herault et al., 1996). Signs shown at top of the alignment indicate the following degrees of sequence conservation: ' ', average weight of column pair exchanges is less than weight matrix mean value; ' . ', - is less than mean value plus one SD; ' + ', - is less than mean value plus two SD; ' * ', - is more than mean value plus two SD. Homoedomain is underlined. The Hoxcl3 coding sequence has been deposited in the GenBank database under access code AF 193796.

Figure 2A illustrates the phenotypic changes in transgenic mice overexpressing Hoxcl3. Externally discernible changes of skin and skeleton: (a) One week old GC13 (61B1) mutant pup (top) in comparison to a non-transgenic littermate (below); note the smaller size, shorter tail, and retardation of hair growth exhibited by the mutant, (b) dorsal view of 4.5 mo. old GC13 (61U4) mutant female mouse exhibiting severe scaling and crusting of the skin, as well as alopecia. Ventral/facial view of a 10 d GC13 (61B1) mutant pup (d) in comparison to a non-transgenic littermate (c) shows retarded growth of ventral hair coat and kinkiness of mutant whiskers. Alcian blue and alizarin red -staining of posterior skeletons of 2 d (6 IB 1) mutant (f) and non-transgenic littermate (e) reveals reduction in the number of tail vertebrae in the mutant.

Figure 2B illustrates the phenotypic changes in transgenic mice overexpressing Hoxcl3. Hematoxylin-Eosin (H&E) -stained sagittal sections (5μm) of dorsal neck skin derived from normal and GC13 mutant (61B1) littermates sacrificed at 5 d, 30 d and 1 year (1 y) of age; please note the thickened cellular epidermis (E) in mutant skin of each stage and the enormously thickened squamous layer (S) in older mutants; enlargement of mutant anagen hair follicles (F) seen at 30 d appears to result from thickened outer root sheaths (ORSs); please note the distended, cyst-like follicles seen in older GC13 mice; magnification bar: 200 μm.

Figure 2C are is a scanning electron micrograph of nasal whiskers from normal, and GC13 (61B1) mutant mice of 3 mo. of age reveals changes in the cuticular septation of mutant whiskers.

Figure 3A shows the analysis of Hoxcl3 and LZ-GC13 reporter gene expression patterns. Detection of Hoxcl 3 expression pattern in tail, limbs and skin of El 5 normal and GC13 mutant embryos by whole mount in situ hybridization (WMISH) with Hoxcl 3 -specific RNA probe reveals increased hybridization signal (purple) as visualized by Purple Alkaline Phosphatase (AP) Substrate (Roche Diagnostics) - staining in tail bud and vibrissae, as well as in developing coat hair follicles of mutant versus normal embryo. Scale bar: 2 mm.

Figure 3B shows the analysis of Hoxcl3 and LZ-GC13 reporter gene expression patterns. X-gal staining (dark-blue) of normal LZ-GC13 (a and c) and mutant LZ- GC13/GC13 (b and d) transgenic embryos at E13 (a and b) and E16.5 (c and d); please note the reduction of X-gal staining in neural tube (arrowhead) and tail bud of El 3 double transgenic mutant (b) and in tail bud, vibrissae, and pelage hair follicles of El 6.5 mutant (d) in comparison to the normal embryos (a and c). Magnification bars: 2 mm.

Figure 3C shows the analysis of Hoxcl 3 and LZ-GC13 reporter gene expression patterns. In situ hybridization in 10 μm sections of 5 d normal (a) and GC13 mutant (b) skin using 35S-labelled Hoxcl ^-specific antisense probe; increased hybridization signals are detectable in upper bulb and lower shaft regions (arrowheads) of mutant (b) versus normal (a) hair follicles viewed in bright- (top panels) and darkfield (below), whereas no specific hybridization signal is detectable over epidermal cells. Magnification bar: 200 μm.

Figure 3D shows the analysis of Hoxcl 3 and LZ-GC13 reporter gene expression patterns. X-gal staining (blue) in eosin-counter-stained 5 μm sections of 5 d dorsal neck skin of a normal LZ-GC13 (a) and mutant LZ-GC13/GC13 (b) pup; please note strongest staining in normal hair follicle (arrowhead) extending from upper regions of the bulb to the hairshaft (a), regions known to contain differentiating keratinocytes (Fuchs et al., 1995), whereas mutant follicle of approximately the same stage (arrowhead in b) exhibits reduced X-gal staining (blue and brownish color) in mid- regions of the shaft and no staining in the upper bulb region.

Figure 4 A shows the keratinocyte hyperproliferation and Hoxcl 3 expression in differentiating keratinocytes. BrdU -labelling of normal (a), and GC13 mutant (b) 2 d skin as visualized by fluorescence microscopy of 7 μm sagittal sections (top panels); bottom panels show the same sections counterstained with H&E. Please note increased number of BrdU-labelled nuclei in the the outer root sheath (white arrowhead) and in the basal layer of the epidermis (blue arrowhead) in mutant skin (b). Magnification bar: 100 μm. Figure 4B shows the keratinocyte hyperproliferation and Hoxcl 3 expression in differentiating keratinocytes. Spatial relationship between LZ-GC13 expression and keratinocyte hyperproliferation was revealed by double-fluorescence microscopy of 10 μm skin sections derived from 2 d normal LZ-GC13 (a) and mutant LZ-GC13/GC-13 (b) pups by using rhodamine-conjugated anti-β-gal and fluorescin-conjugated anti- BrdU antibodies; please note the non-overlap between LZ-GC13-expressing (red- labelled follicular cells marked by white arrows) and proliferating keratinocytes in normal (a) and mutant (b) hair follicle; the blue arrowhead in (b) points to increased anti-BrdU-labelling of basal epidermal keratinocytes; the white stars in (a) and (b) indicate unspecific labelling of the squamous epidermis with anti-β-gal antibodies. (C) Hoxcl 3 in situ hybridization signals in bulb and lower shaft region of anagen hair follicle in 5 d normal skin section counterstained with Giemsa; specific signal is detectable as accumulation of silver grains under bright- (dark grains in left-hand panel) and darkfield (bright grains in right-hand panel) microscopy, which is strongest over differentiating keratinocytes in the upper bulb region (arrows) and lower hairshaft (HS); no expression is detectable in lower bulb regions (arrowheads) and in dermal papilla (DP). Magnification bar: 100 μm.

Figure 5 A shows the identification of genes differentially expressed in GC13 skin. Reverse Northern quantitative hybridization. 30 ng of each of 74 plasmid DNAs were spotted in triplicate on two filters which were hybridized in parallel with normal and GC13 skin complex cDNA probes. Washed filters were exposed to an imaging plate and scanned images (STORM 860 scanner, Molecular Dynamics) were analyzed and quantified using Image Quant 1.0 software. The resulting tables containing intensities of all spots were exported to Excell and further calculations were performed using Excell worksheet. SI, series 1; S2, series 2; S3, series 3.

Figure 5B shows the identification of genes differentially expressed in GC13 skin. List of genes differentially expressed in GC13 skin. Up- and down-regulated genes in GC13 skin are indicated by showing the corresponding ratios of expression, N/GC13, in red and green, respectively. Downregulated KAP genes mapped to mouse chromosome 16 (see Figure 6B) are shown in blue. The greatest spot-to-spot variation in the intensity of hybridization signal observed for the same cDNA was 1.21±0.08 (mean±SD, n=6); therefore, only differences greater or equal to 1.5 fold are significant and shown in the table. ArrayNo, cDNA array coordinate; N/GC13, mean values for the differences in expression (normal versus GC13); SD, standard deviation (n=9).

Figure 6 A shows the phylogenetic tree analyis and genomic mapping of KAP genes controlled by Hoxcl 3. Phylogenetic tree analysis of previously known, as well as novel KAP genes found to be downregulated in GC13 mice. Phylogenetic tree was generated by using the Genebee Multiple Alignment program (Belozersky Institute, Moscow State University; http://www.genebee.msu.su/services/malign_reduced.html) and deduced amino acid sequences. The clustering of the novel Kratplό genes together with the previously known HGTp II.1 and HGTp I alpha genes suggests closer phylogenetic relationships among members of this group than to the other genes (Pmg- 1, Pmg-2 and Hacl-1) included in the analysis. GeneBank accession numbers for the sequences used are as follows: AF003691 (Pmg-1), NM013713 (Pmg-2), U03686 (Hacl-1), D86420 (HGTp III), D86422 (HGTp I alpha); accession numbers for the novel Krtapl6-1 through Krtapl6-9 sequences are pending.

Figure 6B shows the phylogenetic tree analyis and genomic mapping of KAP genes controlled by Hoxcl 3. Schematic representation of genomic mapping results: KAP genes (shown in light type) analyzed here map between the two distal markers Grikl and Sodl (shown in bold type) on mouse chromosome 16 (MMU16), a region of conserved linkage with human chromosome HSA21q22.11 region. The novel KAP genes Krtapl6-1 through Krtapl6-8 cluster together with HGTpII.l and Hacl-1 distal to the marker Tiaml, which occupies a conserved position in the HSA21q22.11 region as indicated; the more distantly related Pmg-1 and Pmg-2 genes map proximal to Tiaml. Figure 7 is an outline of experimental strategy for defining the molecular basis for the pathological changes observed in skin of GC13 transgenic mice (triangular icon at top). The main components of this strategy are depicted by circular icons A, B, and C and reflect corresponding steps described in detail in the Examples section.

Figure 8 is a schematic outline of SSH procedure. Rsal- digested tester cDNA derived from GC13 mutant skin is split into two equal batches, which is ligated either to adapter AL (top, left) or to adapter AR (top, right). Adaptors AL and AR (boxed regions) contain adapter-specific primer sequences indicated by open (AL) and grey (AR) boxed regions in addition to a stretch of shared sequence (black boxed regions). In step I, each batch of tester RNA is mixed with an equal amount of excess driver RNA derived from normal skin that has not been ligated to adapters (top, center) and two parallel hybridization reactions are performed after denaturation. This will result in four classes of single and double-stranded molecules a, b, c, and d. Please note, sequences differentially overexpressed in GC13 skin will be enriched in class a; importantly, among these subtracted molecules, the concentrations of highly, moderately and rarely expressed sequences will be equalized by virtue of the hybridization kinetics for the formation of class b, c, and d double-stranded molecules (the hybridization kinetic is a logarithmic function of the number of copies of a given sequence per mole of total DNA). Step II: at the end of the first hybridization, the two parallel reaction mixtures are combined and new, freshly denatured, driver cDNA is added; the second hybridization is performed without denaturing class b, c, and d hybrids formed in the first reaction. This step will further enrich for differentially expressed sequences among class a molecules, which at the same time will allow formation of class e hybrids of subtracted cDNAs. Step III: filling in the 3' recessed ends of class b, c, and e hybrids will exclusively render the latter with different adapter sequences (either AL or AR) at either terminus of their single strands. Step IV: During the subsequent PCR amplification, only type e molecules, which have different terminal adapter sequences, will be amplified exponentially. Type a and c molecules have only one primer annealing sequence and can thus be amplified only linearly. Type b molecules will form panhandle structures, whose formation will be favored over primer annealing due to the much faster intramolecular first order reassociation kinetic of the two complementary terminal adapter sequences. Using nested primers, a secondary PCR reaction will further amplify differentially expressed sequences. Note, for the subtraction of cDNA sequences differentially underexpressed in GC13 skin, roles will have to be reversed, i.e. GC13 cDNA will be driver instead of being tester. This schematic has been adapted and modified from the Clontech PCR-Select user manual (www. clontech. com) . Restriction enzymes such as Rsal, Eael, Smal are utilized to remove adapters as described in the Examples. After digestion, the samples are purified using the QIAquick PCR Purification Kit (QIAGEN) to remove salts, proteins and adapters. 2 μl of each sample is 32P-labeled using Ready-To-Go DNA Labeling Beads (Pharmacia Biotech) and hybridization will be performed overnight using 8 -10 x 107 dpm of probe per filter. The filters are then washed twice for 20 min in 1 x SSPE/0.2% SDS at room temperature followed by three 20 min washes in 0.1 x SSPE/0.2% SDS at 65°C and exposed to X-ray film overnight with intensifying screens. Differential clones will be sequenced by the MUSC DNA sequencing core facility and analyzed using GCG software. Redundant clones are eliminated.

Figure 9A illustrates the effect of Hoxcl3 over expression on adipose tissue. Three month old normal FVB x C57BL6 (top) and mutant GC13/FVB x C57BL6 (below) mice.

Figures 9B shows the mutant animal shown in Figure 9A after opening inner and outer body wall.

Figure 9C shows the normal animal shown in Figure 9A after opening inner and outer body wall. Note the easily discernible fat pads (arrows) in the normal mouse (Figure 9C) which are nearly absent in the mutant (Figure 9B). Figure 10A shows that GC13 X C57BL6 FI hybrid mice exhibit pigmentation defects. Mutant adult (> 3 months) FVB/GC13 x C57BL6 (bottom) in comparison to normal FVB x C57BL6 mouse; note the irregular pigmentation in the mutant.

Figure 1 OB is a hematoxylin and eosin-stained cross section of dorsal skin derived from mutant shown in Figure 10A exhibiting highly irregular accumulation of dark melanin granules (arrows).

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Example included therein.

Before the present compounds and methods are disclosed and described, it is to be understood that this invention is not limited to specific proteins, specific methods, or specific nucleic acids, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid" includes multiple copies of the nucleic acid and can also include more than one particular species of molecule.

The present invention provides an isolated nucleic acid comprising a 19 kb genomic fragment (GC13) that contains the Hoxcl 3 transcription unit of approximately 6.7 kb, approximately 4 kb of upstream flanking sequences and approximately 8 kb of downstream flanking sequences. GC13 is contained within a murine cosmid clone, cos2W3H. PGC13 which contains the 19kb GC13 fragment was derived from cos2W3H and deposited with the American Type Culture Collection. GC13 encodes a murine Hoxcl 3 gene, which includes sequences both 5' and 3' to the coding regions of the genes. Therefore, GC13 represents the sequence of a genomic clone and therefore includes introns and regulatory sequences sufficient for the expression of the encoded gene.

The invention also provides nucleic acids that comprise fragments of GC13. These fragments include 987 nucleotides that encode the Hoxcl 3 protein coding sequence as set forth in SEQ ID NO: 1. The invention also provides a fragment of 57 nucleotides of the 5' untranslated leader sequence flanking the presumptive Hoxcl3 translation start codon as set forth in SEQ ID NO:2. Other fragments include 2200 nucleotides of noncoding sequence located upstream of the Xho 1 restriction site at the end of GC 13 as set forth as SEQ ID NO: 3. The invention also provides a nucleic acid sequence, 5'GGACCAGAAAAGGGAGGCAAATTGG3' (SEQ ID NO: 4), that was used as a primer to screen for transgenics using PCR. Also provided is a fragment of about 1400 nucleotides of 3' untranslated trailer sequence flanking the TGA stop codon and including the presumptive Hoxcl 3 polyadenylation signal sequences at the end of this sequence stretch set forth as SEQ ID NO: 5. The invention also provides a fragment set forth in the Sequence Listing as SEQ ID NO:7 which comprises SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 5. Therefore, the present invention provides a nucleic acid that comprises the nucleic acid sequence set forth in the Sequence Listing as SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5 and/or SEQ ID NO: 7.

The nucleic acids comprising SEQ ID NO: 1-7 and combinations thereof can be from 57 nucleotides to 19 kilobases in length and any size in between.

The present invention also provides an isolated nucleic acid comprising SEQ ID NO: 1 and SEQ ID NO: 2. Also provided is a nucleic acid comprising SEQ ID NO: 1 and SEQ ID NO: 3. Further provided is a nucleic acid comprising SEQ ID NO: 1 and SEQ ID NO: 4, a nucleic acid comprising SEQ ID NO: 1 and SEQ ID NO: 5 or a nucleic acid comprising SEQ ID NO: 1 and SEQ ID NO: 7. Other nucleic acids provided by this invention include: a nucleic acid comprising SEQ ID NO: 2 and SEQ ID NO: 3, a nucleic acid comprising SEQ ID NO: 2 and SEQ ID NO: 4, a nucleic acid comprising SEQ ID NO:2 and SEQ ID NO: 5, a nucleic acid comprising SEQ ID NO:2 and SEQ ID NO: 7, a nucleic acid comprising SEQ ID NO: 3 and SEQ ID NO: 4, a nucleic acid comprising SEQ ID NO: 3 and SEQ ID NO: 5, a nucleic acid comprising SEQ ID NO:3 and SEQ ID NO: 7, a nucleic acid comprising SEQ ID NO: 4 and SEQ ID NO: 5, a nucleic acid comprising SEQ ID NO: 4 and SEQ ID NO: 7, a nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, and SEQ ID NO: 5, a nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, and SEQ ID NO: 4, a nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:3, a nucleic acid comprising SEQ ID NO:l, SEQ ID NO: 2 and SEQ ID NO: 5, and SEQ ID NO: 7, a nucleic acid comprising SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID NO: 3, and SEQ ID NO: 7, a nucleic acid comprising SEQ ID NO:l, SEQ ID NO: 2 and SEQ ID NO: 4, and SEQ ID NO: 7, a nucleic acid comprising SEQ ID NO:2, SEQ ID NO: 3 and SEQ ID NO: 4, a nucleic acid comprising SEQ ID NO:3, SEQ ID NO: 4 and SEQ ID NO: 5, a nucleic acid comprising SEQ ID NO:4, SEQ ID NO: 5 and SEQ ID NO: 7.

Also provided by this invention is a nucleic acid comprising a 10 kb fragment of GC13 that includes SEQ ID NO: 1, a 9 kb fragment of GC13 that includes SEQ ID NO: 1, an 8 kb fragment of GC13 that include SEQ ID NO: 1, a 7 kb fragment of GC 13 that includes SEQ ID NO: 1 , a 6 kb fragment of GC 13 that includes SEQ ID NO: 1, a 5 kb fragment of GC13 that includes SEQ ID NO: 1, a 4 kb fragment of GC13 that includes SEQ ID NO: 1, a 3 kb fragment of GC13 that includes SEQ ID NO: 1, a 2 kb fragment of GC13 that include SEQ ID NO: 1, a 1 kb fragment that includes SEQ ID NO: 1. The invention further provides a nucleic acid encoding the polypeptide set forth in SEQ ID NO: 6 which is the protein encoded by SEQ ID NO: 1. SEQ ID NO: 6 is an example of a murine Hoxcl 3 polypeptide. This Hoxcl 3 protein and protein-coding DNA sequence has been submitted to the to the GenBank database under accession number AF 193796.

As used herein, the term "nucleic acid" refers to single-or multiple stranded molecules which may be DNA or RNA, or any combination thereof, including modifications to those nucleic acids. The nucleic acid may represent a coding strand or its complement, or any combination thereof. Nucleic acids may be identical in sequence to the sequences which are naturally occurring for any of the novel genes discussed herein or may include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence. These nucleic acids can also be modified from their typical structure. Such modifications include, but are not limited to, methylated nucleic acids, the substitution of a non-bridging oxygen on the phosphate residue with either a sulfur (yielding phosphorothioate deoxynucleotides), selenium (yielding phosphorselenoate deoxynucleotides), or methyl groups (yielding methylphosphonate deoxynucleotides).

Similarly, one skilled in the art will recognize that compounds comprising the genes, nucleic acids, and fragments of the genes and nucleic acids as disclosed and contemplated herein are also provided. For example, a compound comprising a nucleic acid can be a derivative of a typical nucleic acid such as nucleic acids which are modified to contain a terminal or internal reporter molecule and/or those nucleic acids containing non-typical bases or sugars. These reporter molecules include, but are not limited to, isotopic and non-isotopic reporters. Therefore, any molecule which may aid in detection, amplification, replication, expression, purification, uptake, etc. may be added to the nucleic acid construct. The term "gene" as used herein means a unit of heredity that occupies a specific locus on a chromosome as well as any sequences associated with the expression of that nucleic acid. For example, the gene includes any introns normally present within the coding region as well as regions preceding and following the coding region. Examples of these non-coding regions include, but are not limited to transcription termination regions, promoter regions, enhancer regions and modulation regions.

The regions upstream and downstream of Hoxcl 3 may act as promotor factors for Hoxcl 3 expression and may be involved in the pathophysiology of hyperkeratotic skin disorders or hair loss. Additionally, the regions flanking Hoxcl 3 may contain potential cis-acting elements that are required for regulation of Hoxcl 3 gene transcription. The genomic locus described herein may also encode overlapping sense and antisense RNAs. Such transcripts may be involved in the regulation of Hoxcl 3 expression and may play a role in the development of hyperkeratotic skin disorders or hair loss, through the activity of the respective gene products alone, or in combination with the other gene products, typically those surrounding the Hoxcl 3 gene. Antisense control of sense transcripts may be exerted at the level of transcription, maturation, transport, stability and translation of Hoxcl 3. For example, the 3' UTR of Hoxcl 3 may contain a region of antisense complementarity to a region of another gene whereby antisense interaction regulates the activity of Hoxcl 3. Where sufficient mutations within these genes occur, this interaction is then disrupted causing Hoxcl 3 to be incorrectly transcribed, processed, transported, translated etc. Alternatively, other genes may encode proteins that covalently or noncovalently associate with Hoxcl 3. Mutations within the Hoxcl 3 gene contemplated in this invention may therefore result in mutant proteins that are deficient in their ability to effect this protein-protein interaction.

The genes and nucleic acids provided for by the present invention may be obtained in any number of ways. For example, a DNA molecule encoding GC13 or a fragment thereof, can be isolated from the organism in which it is normally found. For example, a genomic DNA or cDNA library can be constructed and screened for the presence of the gene or nucleic acid of interest. Methods of constructing and screening such libraries are well known in the art and kits for performing the construction and screening steps are commercially available (for example, Stratagene Cloning Systems, La Jolla, CA). Once isolated, the gene or nucleic acid can be directly cloned into an appropriate vector, or if necessary, be modified to facilitate the subsequent cloning steps. Such modification steps are routine, an example of which is the addition of oligonucleotide linkers which contain restriction sites to the termini of the nucleic acid. General methods are set forth in Sambrook et al. "Molecular Cloning, a Laboratory Manual," Cold Spring Harbor Laboratory Press (1989).

Another example of a method of obtaining a DNA molecule encoding a specific gene, coding sequence, mRNA or protein of the present invention is to synthesize a recombinant DNA molecule which encodes that protein. For example, oligonucleotide synthesis procedures are routine in the art and ohgonucleotides coding for a particular protein region are readily obtainable through automated DNA synthesis. A nucleic acid for one strand of a double-stranded molecule can be synthesized and hybridized to its complementary strand. One can design these ohgonucleotides such that the resulting double-stranded molecule has either internal restriction sites or appropriate 5' or 3' overhangs at the termini for cloning into an appropriate vector. Double-stranded molecules coding for relatively large proteins can readily be synthesized by first constructing several different double-stranded molecules that code for particular regions of the protein, followed by ligating these DNA molecules together. For example, Cunningham, et al. "Receptor and Antibody Epitopes in Human Growth Hormone Identified by Homolog-Scanning Mutagenesis," Science, 243:1330-1336

(1989), have constructed a synthetic gene encoding the human growth hormone gene by first constructing overlapping and complementary synthetic ohgonucleotides and ligating these fragments together. See also, Ferretti, et al. Proc. Nat. Acad. Sci. 82:599-603 (1986), wherein synthesis of a 1057 base pair synthetic bovine rhodopsin gene from synthetic ohgonucleotides is disclosed. By constructing the desired sequence in this manner, one skilled in the art can readily obtain any particular protein such as Hoxcl 3, with desired amino acids at any particular position or positions within the protein. See also, U.S. Patent No. 5,503,995 which describes an enzyme template reaction method of making synthetic genes. Techniques such as this are routine in the art and are well documented. These nucleic acids can then be expressed in vivo or in vitro as discussed below.

Once the gene or nucleic acid sequence of the desired gene is obtained, the sequence encoding specific amino acids can be modified or changed at any particular amino acid position by techniques well known in the art. For example, PCR primers can be designed which span the amino acid position or positions and which can substitute any amino acid for another amino acid. Then a nucleic acid can be amplified and inserted into the wild-type coding sequence in order to obtain any of a number of possible combinations of amino acids at any position of the gene. Alternatively, one skilled in the art can introduce specific mutations at any point in a particular nucleic acid sequence through techniques for point mutagenesis. General methods are set forth in Smith, "In vitro mutagenesis" Ann. Rev. Gen., 19:423-462 (1985) and Zoller, "New molecular biology methods for protein engineering" Curr. Opin. Struct. Biol., 1 :605- 610 (1991). Techniques such as these can also be used to modify the genes or nucleic acids in regions other than the coding regions, such as the promoter regions or any regulatory or noncoding region.

As used herein, the term "isolated" refers to a nucleic acid separated or significantly free from at least some of the other components of the naturally occurring organism, for example, the cell structural components commonly found associated with nucleic acids and/or other nucleic acids found naturally with the nucleic acid in a cellular environment. The isolation of the native nucleic acids can be accomplished, for example, by techniques such as cell lysis followed by phenol plus chloroform extraction, followed by ethanol precipitation of the nucleic acids. The nucleic acids of this invention can be isolated from cells according to any of many methods well known in the art.

An isolated nucleic acid comprising a unique fragment of at least 10 nucleotides of GC13 or the nucleic acid set forth in the Sequence Listing as any of SEQ ID NO:l, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5 or SEQ ID NO: 7 is also provided. Unique fragments, as used herein means a nucleic acid of at least 10 nucleotides that is not identical to any other known nucleic acid sequence. Examples of the sequences of at least 10 nucleotides that are unique to GC13, SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO: 4 can be readily ascertained by comparing the sequence of the nucleic acid in question to sequences catalogued in GenBank, or any other sequence database, using the computer programs such as DNASIS (Hitachi Engineering, Inc.) or Word Search or FASTA of the Genetics Computer Group (GCG) (Madison, WI), which search the catalogued nucleotide sequences for similarities to the nucleic acid in question. If the sequence does not match any of the known sequences, it is unique. For example, the sequence of nucleotides 1-10 can be used to search the databases for an identical match. If no matches are found, then nucleotides 1-10 represent a unique fragment. Next, the sequence of nucleotides 2-11 can be used to search the databases, then the sequence of nucleotides 3-13, and so on of the nucleic acids sequences provided herein. The same type of search can be performed for sequences of 11 nucleotides, 12 nucleotides, 13 nucleotides, etc. These unique nucleic acids, as well as degenerate nucleic acids can be used, for example, as primers for amplifying nucleic acids in order to isolate allelic variants of the Hoxcl 3 proteins or as primers for reverse transcription of Hoxcl 3 mRNA, or as probes for use in detection techniques such as nucleic acid hybridization. One skilled in the art will appreciate that even though a nucleic acid of at least 10 nucleotides is unique to a specific gene, that nucleic acid fragment can hybridize to many other nucleic acids and therefore be used in techniques such as amplification and nucleic acid detection to amplify or detect nucleic acids that contain the unique nucleic acid. Also provided are allelic variants of the Hoxcl 3 protein set forth in the Sequence Listing as SEQ ID NO:6. As used herein, the term "allelic variations" or "allelic variants" is used to describe the same, or similar proteins that diverge from the Hoxcl 3 protein set forth in SEQ ID NO:6 by less than 20% in their corresponding amino acid identity. In another embodiment, these allelic variants are less than 18% divergent in their corresponding amino acid identity. In another embodiment, these allelic variants are less than 15% divergent in their corresponding amino acid identity. In another embodiment, these allelic variants are less than 12% divergent in their corresponding amino acid identity. In another embodiment, these allelic variants are less than 10% divergent in their corresponding amino acid identity. In another embodiment, these allelic variants are less than 7% divergent in their corresponding amino acid identity. In another embodiment, these allelic variants are less than 5% divergent in their corresponding amino acid identity. In another embodiment, these allelic variants are less than 3% divergent in their corresponding amino acid identity. In another embodiment, these allelic variants are less than 2% divergent in their corresponding amino acid identity. In yet another embodiment, these allelic variants are less than 1% divergent in their corresponding amino acid identity. These allelic variants can include substitutions within the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:6, they can include deletions from the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:6, and they can include additions to the amino acid sequence set forth in the Sequence Listing as SEQ ID NO:6, or any combinations thereof.

The homology between the protein coding regions of the nucleic acids encoding the allelic variants of the Hoxcl 3 protein is preferably less than 20% divergent from the region of the nucleic acid set forth in the Sequence Listing as SEQ IDNO:l encoding the protein. In another embodiment, the corresponding nucleic acids are less than 18% divergent in their sequence identity. In another embodiment, the corresponding nucleic acids are less than 15% divergent in their sequence identity. In another embodiment, the corresponding nucleic acids are less than 12% divergent in their sequence identity. In another embodiment, the corresponding nucleic acids are less than 10% divergent in their sequence identity. In another embodiment, corresponding nucleic acids are less than 7% divergent in their sequence identity. In another embodiment, the corresponding nucleic acids are less than 5% divergent in their sequence identity. In another embodiment, the corresponding nucleic acids are less than 3% divergent in their sequence identity. In another embodiment, the corresponding nucleic acids are less than 2% divergent in their corresponding sequence identity. In yet another embodiment, the corresponding nucleic acids are less than 1% divergent in their sequence identity

One skilled in the art will appreciate that nucleic acids encoding homologs or allelic variants of the protein set forth in the Sequence Listing as SEQ ID NO:6 can be isolated in a manner similar to that used to isolate the nucleic acids set forth in the Sequence Listing of the present invention as SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 7. For example, given the sequence of the primers used to amplify the nucleic acid set forth in the sequence listing as SEQ ID NO:l, one can use these or similar primers to amplify a homologous gene from other sources.

Using the sequence information provided herein, one skilled in the art can obtain the sequence of a primer that will hybridize to the desired message of genomic DNA, either in an intron, in an exon, or both, such that corresponding RNAs or DNAs from other organisms can readily be detected, and/or isolated. One can use a spliced or an unspliced message, or a corresponding DNA, or a fragment thereof, as a probe to detect homologous sequences in other organisms, or similar genes from other individuals from the same species.

The present invention also contemplates DNA probes for detecting the Hoxcl 3 gene of the locus of GC13, wherein the DNA probe hybridizes to the nucleotide sequence set forth in the Sequence Listing as SEQ ID NO: 1, and DNA probes for detecting nucleic acid sequences of GC13 wherein the DNA probe hybridizes to the nucleotide sequence set forth in the Sequence Listing as any of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:7.

As used herein, the term "DNA probe" refers to a nucleic acid fragment that selectively hybridizes under stringent conditions with a nucleic acid comprising a nucleic acid set forth in a sequence listed herein. This hybridization must be specific. The degree of complementarity between the hybridizing nucleic acid and the sequence to which it hybridizes should be at least enough to exclude hybridization with a nucleic acid encoding an unrelated protein.

Allelic variants can be identified and isolated by nucleic acid hybridization techniques. Probes selective to the nucleic acid set forth in the Sequence Listing as any of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:7 can be synthesized and used to probe the nucleic acid from various cells, tissues, libraries etc. High sequence complementarity and stringent hybridization conditions can be selected such that the probe selectively hybridizes to allelic variants of the sequence set forth in the Sequence Listing as any of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:7. For example, The selectively hybridizing nucleic acids of the invention can have at least 70%, 80%, 85%, 90%, 95%, 97%, 98% and 99% complementarity with the segment of the sequence to which it hybridizes. The nucleic acids can be at least 12, 50, 100, 150, 200, 300, 500, 750, or 1000 nucleotides in length. Thus, the nucleic acid can be a coding sequence for the Hoxcl 3 protein or fragment thereof that can be used as a probe or primer for detecting the presence of these genes. The nucleic acid can be an expression regulatory sequence of the Hoxcl 3 gene or a fragment thereof. If used as primers, the invention provides compositions including at least two nucleic acids which hybridize with different regions so as to amplify a desired region. Depending on the length of the probe or primer, target region can range between 70% complementary bases and full complementarity and still hybridize under stringent conditions. The invention provides examples of nucleic acids unique to SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:7 in the Sequence Listing so that the degree of complementarity required to distinguish selectively hybridizing from nonselectively hybridizing nucleic acids under stringent conditions can be clearly determined for each nucleic acid.

"Stringent conditions" refers to the washing conditions used in a hybridization protocol. In general, the washing conditions should be a combination of temperature and salt concentration chosen so that the denaturation temperature is approximately 5-20°C below the calculated T_m of the nucleic acid hybrid under study. The temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to the probe or protein coding nucleic acid of interest and then washed under conditions of different stringencies. The T_m of such an oligonucleotide can be estimated by allowing 2°C for each A or T nucleotide, and 4°C for each G or C. For example, an 18 nucleotide probe of 50% G+C would, therefore, have an approximate T_m of 54°C.

The present invention also contemplates any unique fragment of GC13, including but limited to the nucleic acid fragments set forth in the Sequence Listing as SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 7. To be unique, the fragment must be of sufficient size to distinguish it from other known sequences, most readily determined by comparing any nucleic acid fragment to the nucleotide sequences in computer databases, such as GenBank. Such comparative searches are standard in the art. Typically, a unique fragment useful as a primer or probe will be at least 20 to about 25 nucleotides in length depending upon the specific nucleotide content of the sequence. Additionally, fragments can be, for example, at least about 30, 40, 50, 75, 100, 200 or 500 nucleotides in length. Any of the genes, nucleic acids, and fragments of the genes and nucleic acids disclosed and contemplated herein can be single or multiple stranded, depending on the purpose for which it is intended. Once a nucleic acid encoding a particular protein of interest, such as a Hoxcl 3 protein or a nucleic acid comprising upstream or downstream control elements of the Hoxcl 3 gene, or a nucleic acid comprising upstream and downstream control elements of the Hoxcl 3 is constructed, modified, or isolated, that nucleic acid can then be cloned into an appropriate vector, which can direct the in vivo or in vitro synthesis of that wild- type and/or modified protein. The vector is contemplated to have the necessary functional elements that direct and regulate transcription of the inserted gene, or nucleic acid. These functional elements include, but are not limited to, a promoter, regions upstream or downstream of the promoter, such as enhancers that may regulate the transcriptional activity of the promoter, an origin of replication, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic resistance genes or other markers which can serve to select for cells containing the vector or the vector containing the insert, RNA splice junctions, a transcription termination region, or any other region which may serve to facilitate the expression of the inserted gene or hybrid gene. (See generally, Sambrook et al).

There are numerous E. coli (Escherichia colϊ) expression vectors known to one of ordinary skill in the art which are useful for the expression of the nucleic acid insert. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences for example, for initiating and completing transcription and translation. If necessary, an amino terminal methionine can be provided by insertion of a Met codon 5' and in- frame with the downstream nucleic acid insert. Also, the carboxy-terminal extension of the nucleic acid insert can be removed using standard oligonucleotide mutagenesis procedures.

Additionally, yeast expression can be used. There are several advantages to yeast expression systems. First, evidence exists that proteins produced in a yeast secretion systems exhibit correct disulfide pairing. Second, post-translational glycosylation is efficiently carried out by yeast secretory systems. The Saccharomyces cerevisiae pre-pro-alpha-factor leader region (encoded by the MF"-1 gene) is routinely used to direct protein secretion from yeast. (Brake, et al. "«-F actor-Directed Synthesis and Secretion of Mature Foreign Proteins in Saccharomyces cerevisiae." Proc. Nat. Acad. Sci., 81 :4642-4646 (1984)). The leader region of pre-pro-alpha-factor contains a signal peptide and a pro-segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene: this enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage signal sequence. The nucleic acid coding sequence can be fused in- frame to the pre-pro-alpha-factor leader region. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter. The nucleic acid coding sequence is followed by a translation termination codon which is followed by transcription termination signals. Alternatively, the nucleic acid coding sequences can be fused to a second protein coding sequence, such as Sj26 or β-galactosidase, used to facilitate purification of the fusion protein by affinity chromatography. The insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast. Efficient post translational glycosylation and expression of recombinant proteins can also be achieved in Baculovirus systems.

Mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active protein. Vectors useful for the expression of active proteins in mammalian cells are characterized by insertion of the protein coding sequence between a strong viral promoter and a polyadenylation signal. The vectors can contain genes conferring hygromycin resistance, gentamicin resistance, or other genes or phenotypes suitable for use as selectable markers, or methotrexate resistance for gene amplification. The chimeric protein coding sequence can be introduced into a Chinese hamster ovary (CHO) cell line using a methotrexate resistance-encoding vector, or other cell lines using suitable selection markers. Presence of the vector DNA in transformed cells can be confirmed by Southern blot analysis. Production of RNA corresponding to the insert coding sequence can be confirmed by Northern blot analysis. A number of other suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc. The vectors containing the nucleic acid segments of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transformation is commonly utilized for prokaryotic cells, whereas calcium phosphate, DEAE dextran, or lipofectin mediated transfection or electroporation may be used for other cellular hosts.

Alternative vectors for the expression of genes or nucleic acids in mammalian cells, those similar to those developed for the expression of human gamma-interferon, tissue plasminogen activator, clotting Factor VIII, hepatitis B virus surface antigen, protease Nexinl, and eosinophil major basic protein, can be employed. Further, the vector can include CMV promoter sequences and a polyadenylation signal available for expression of inserted nucleic acids in mammalian cells (such as COS-7).

Insect cells also permit the expression of mammalian proteins. Recombinant proteins produced in insect cells with baculovirus vectors undergo post-translational modifications similar to that of wild-type proteins. Briefly, baculovirus vectors useful for the expression of active proteins in insect cells are characterized by insertion of the protein coding sequence downstream of the Autographica californica nuclear polyhedrosis virus (AcNPV) promoter for the gene encoding polyhedrin, the major occlusion protein. Cultured insect cells such as Spodoptera frugiperda cell lines are transfected with a mixture of viral and plasmid DNAs and the viral progeny are plated. Deletion or insertional inactivation of the polyhedrin gene results in the production of occlusion negative viruses which form plaques that are distinctively different from those of wild-type occlusion positive viruses. These distinctive plaque morphologies allow visual screening for recombinant viruses in which the AcNPV gene has been replaced with a hybrid gene of choice.

Alternatively, the genes or nucleic acids of the present invention can be operatively linked to one or more of the functional elements that direct and regulate transcription of the inserted gene as discussed above and the gene or nucleic acid can be expressed. For example, a gene or nucleic acid can be operatively linked to a bacterial or phage promoter and used to direct the transcription of the gene or nucleic acid in vitro. A further example includes using a gene or nucleic acid provided herein in a coupled transcription-translation system where the gene directs transcription and the RNA thereby produced is used as a template for translation to produce a polypeptide. One skilled in the art will appreciate that the products of these reactions can be used in many applications such as using labeled RNAs as probes and using polypeptides to generate antibodies or in a procedure where the polypeptides are being administered to a cell or a subject.

Expression of the gene or nucleic acid, either in combination with a vector or operatively linked to an appropriate sequence, can be by either in vivo or in vitro. In vivo synthesis comprises transforming prokaryotic or eukaryotic cells that can serve as host cells for the vector. Alternatively, expression of the gene or nucleic acid can occur in an in vitro expression system. For example, in vitro transcription systems are commercially available which are routinely used to synthesize relatively large amounts of mRNA. In such in vitro transcription systems, the nucleic acid encoding the desired gene would be cloned into an expression vector adjacent to a transcription promoter. For example, the Bluescript II cloning and expression vectors contain multiple cloning sites which are flanked by strong prokaryotic transcription promoters. (Stratagene Cloning Systems, La Jolla, CA). Kits are available which contain all the necessary reagents for in vitro synthesis of an RNA from a DNA template such as the Bluescript vectors. (Stratagene Cloning Systems, La Jolla, CA). RNA produced in vitro by a system such as this can then be translated in vitro to produce the desired protein. (Stratagene Cloning Systems, La Jolla, CA).

The invention also provides an expression construct for conferring follicle specific expression which comprises a regulatory sequence of Hoxcl 3 contained within the GC13 construct. An example of a nucleic acid sequence comprising a regulatory sequence of Hoxcl 3 is the GC13 construct. One skilled in the art could insert the gene of interest, i.e. the gene to be expressed in a follicle specific manner, into the GC13 construct, insert the construct into an appropriate delivery vector and administer the construct to a cell in order to confer follicle specific expression. For example, a skilled artisan can substitute the gene of interest for the 987 nucleotides that encode Hoxcl3 in GC13, and utilize this construct to confer follicle specific expression. Examples of genes that can be delivered in a follicle specific fashion include Amphiregulin; beta- catenin; bcl2; c-fos; BMP-4; BMP-2A; BRCA I; EGF; EGF-R; FGF-1 (aFGF); FGF-2 (bFGF); FGF-7; Dsh (Dishevelled); FGF-R1; FGF-R2; FGF-7 (KGF); GSK3 (glycogen synthase kinase 3); fascin; Fz (frizzled); Hairless; Hox genes (e.g. Hoxc8, Hoxd9; Hoxdl l, Hoxdl2, Hoxcό, Hoxc4, Hoxcl2, Hoxcl 1, HoxclO, Hoxbl3, and others); ICAM; IGF-I; IL lalpha; IL lbeta; Lef-1/TCF; M-Notch; NGF-R; Neu; N-myc; MSX 1; MSX 2; NGF; NGF-R (p75); Nude (whn gene); PTHrp; ptc (patched); p53; p63; PDGF-A; PDGF-B; PDGF-Ralpha; PDGF-Rbeta; Retinoic acid receptors (RAR-alpha, -beta, -gamma); SKN-1; SKN-la; Shh (sonic hedgehog; TGF-betal; TGF-beta2; TGF- beta3; TGF-beta receptor; TIMP; TNF-alpha; Wnt genes; VEGF; Vitamin D receptor. The expression constructs of this invention can also be generated with fragments of the GC13 construct that contain a regulatory sequence of Hoxcl 3.

An expression construct for conferring keratinocyte specific expression which comprises a regulatory sequence of Hoxcl 3 contained within the GC13 construct is provided. One skilled in the art could insert the gene of interest, i.e. the gene to be expressed in a keratinocyte specific manner, into the GC13 construct, insert the construct into an appropriate delivery vector and administer the construct to a cell in order to confer keratinocyte specific expression. For example, a skilled artisan can substitute the gene of interest for the 987 nucleotides that encode Hoxcl 3 in GC13, and utilize this construct to confer keratinocyte specific expression. Examples of genes that can be delivered in a keratinocyte specific manner include Amphiregulin; beta-catenin; bcl2; c-fos; BMP-4; BMP-2A; BRCA I; EGF; EGF-R; FGF-1 (aFGF); FGF-2 (bFGF); FGF-7; Dsh (Dishevelled); FGF-R1; FGF-R2; FGF-7 (KGF); GSK3 (glycogen synthase kinase 3); fascin; Fz (frizzled); Hairless; Hox genes (e.g. Hoxc8, Hoxd9; Hoxdl l, Hoxdl2, Hoxc6, Hoxc4, Hoxcl2, Hoxcl 1, HoxclO, Hoxbl3, and others); ICAM; IGF-I; IL lalpha; IL lbeta; Lef-1/TCF; —Notch; NGF-R; Neu; N-myc; MSX 1; MSX 2; NGF; NGF-R (p75); Nude (whn gene); PTHrp; ptc (patched); p53; p63; PDGF-A; PDGF-B; PDGF-Ralpha; PDGF-Rbeta; Retinoic acid receptors (RAR-alpha, -beta, -gamma); SKN-1; SKN-la; Shh (sonic hedgehog; TGF-betal; TGF-beta2; TGF- beta3; TGF-beta receptor; TIMP; TNF-alpha; Wnt genes; VEGF; Vitamin D receptor.

The expression constructs described herein can further comprise a promoter operably linked to the regulatory sequence as well as a coding sequence for a heterologous gene operably linked to the promoter. In the case of a follicle specific or a keratinocyte specific expression construct, the promoter is preferably follicle specific or keratinocyte specific, respectively. An example of a follicle specific promoter and a keratinocyte specific promoter are provided in GC13. If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The nucleic acids of this invention can be introduced into the cells via any gene transfer mechanism, such as, for example, virus-mediated gene delivery, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into a subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.

The nucleic acids of this invention can also be utilized for in vivo gene therapy techniques. With regard to gene therapy applications, the nucleic acid can comprise a nucleotide sequence which encodes a gene product which is meant to function in the place of a defective gene product and restore normal function to a cell which functioned abnormally due to the defective gene product. Alternatively, the nucleic acid may encode a gene product which was not previously present in a cell or was not previously present in the cell at a therapeutic concentration, whereby the presence of the exogenous gene product or increased concentration of the exogenous gene product imparts a therapeutic benefit to the cell and/or to a subject. For example, the nucleic acid of this invention can include but is not limited to, a gene encoding a gene product involved in follicular development. The nucleic acid can also include regulatory elements contained within the GC13 fragment as well as a gene product under the control of these regulatory elements.

For in vivo administration, the cells can be in a subject and the nucleic acid can be administered in a pharmaceutically acceptable carrier. The subject can be any animal in which it is desirable to selectively express a nucleic acid in a cell. In a preferred embodiment, the animal of the present invention is a human. In addition, non-human animals which can be treated by the method of this invention can include, but are not limited to, mice, cats, dogs, birds, horses, cows, goats, sheep, guinea pigs, hamsters, gerbils and rabbits, as well as any other animal in which selective expression of a nucleic acid in a cell can be carried out according to the methods described herein.

In the method described above which includes the introduction of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), the nucleic acids of the present invention can be in the form of naked DNA or the nucleic acids can be in a vector for delivering the nucleic acids to the cells for expression of the nucleic acid inside the cell. The vector can be a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as Lipofectin^®, Lipofectamine^® (GIBCO-BRL, Inc., Gaithersburg, MD), Superfect^® (Qiagen, Inc. Hilden, Germany) and Transfectam^® (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art. In addition, the nucleic acid or vector of this invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a Sonoporation machine (ImaRx Pharmaceutical Corp., Tucson, AZ).

As one example, vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome. The recombinant retrovirus can then be used to infect and thereby deliver nucleic acid to the infected cells. The exact method of introducing the nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors, adeno-associated viral (AAV) vectors, lentiviral vectors, pseudotyped retroviral vectors, and pox virus vectors, such as vaccinia virus vectors. Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanism. This invention can be used in conjunction with any of these or other commonly used gene transfer methods. The nucleic acid and the nucleic acid delivery vehicles of this invention, (e.g., viruses; liposomes, plasmids, vectors) can be in a pharmaceutically acceptable carrier for in vivo administration to a subject. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vehicle, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

The nucleic acid or vehicle may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like. The exact amount of the nucleic acid or vector required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity or mechanism of any disorder being treated, the particular nucleic acid or vehicle used, its mode of administration and the like.

In another aspect, the invention provides a polypeptide encoded by the nucleic acid set forth in SEQ ID NO: 1, and the nucleic acid encoding the polypeptide of SEQ ID NO:6.

These polypeptides can be obtained in any of a number of procedures well known in the art. One method of producing a polypeptide is to link two peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA). One skilled in the art can readily appreciate that a pep tide or polypeptide corresponding to a particular protein can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a hybrid peptide can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form a larger polypeptide. (Grant, "Synthetic Peptides: A User Guide," W.H. Freeman and Co., N.Y. (1992) and Bodansky and Trost, Ed., "Principles of Peptide Synthesis," Springer- Verlag Inc., N.Y. (1993)). Alternatively, the peptide or polypeptide can be independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides may be linked to form a larger protein via similar peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segments can allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen et al. Biochemistry, 30:4151 (1991)). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. "Synthesis of Proteins by Native Chemical Ligation," Science, 266:776-779 (1994)). The first step is the chemo selective reaction of an unprotected synthetic peptide-∞-thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site. Application of this native chemical ligation method to the total synthesis of a protein molecule is illustrated by the preparation of human interleukin 8 (IL-8) (Clark-Lewis et al. FEBS Lett., 307:97 (1987), Clark-Lewis et al, J.Biol.Chem., 269:16075 (1994), Clark-Lewis et al. Biochemistry, 30:3128 (1991), and Raj arathnam et al. Biochemistry, 29:1689 (1994)). Alternatively, unprotected peptide segments can be chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer et al. Science, 256:221 (1992)). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton et al. "Techniques in Protein Chemistry IV," Academic Press, New York, pp. 257-267 (1992)).

Also provided herein are purified antibodies that selectively bind to the polypeptides provided and contemplated herein, or purified antibodies which selectively bind to a polypeptide encoded by the nucleic acid set forth in SEQ IDNO: 1 and purified antibodies which selectively bind to a polypeptide encoded by a nucleic acid encoding the polypeptide set forth in SEQ ID NO: 6. The antibody (either polyclonal or monoclonal) can be raised to any of the polypeptides provided and contemplated herein, in its naturally occurring form and in its recombinant form. The antibody can be used in techniques or procedures such as diagnostics, treatment, or vaccination.

Antibodies can be made by many well-known methods (See, e.g. Harlow and Lane, "Antibodies; A Laboratory Manual" Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1988)). Briefly, purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells can then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen nucleic acid clone libraries for cells secreting the antigen. Those positive clones can then be sequenced. (See, for example, Kelly et al. Bio/Technology, 10:163-167 (1992); Bebbington et al. Bio/Technology, 10:169-175 (1992)). The phrase "specifically binds" with the polypeptide refers to a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bound to a particular protein do not bind in a significant amount to other proteins present in the sample. Selective binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats may be used to select antibodies that selectively bind with a particular protein. For example, solid-phase ELIS A immunoassays are routinely used to select antibodies selectively immunoreactive with a protein. See Harlow and Lane "Antibodies, A Laboratory Manual" Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding.

Several conserved regions of Hoxcl3 are of relevance: (1) the DNA-binding homeodomain consisting of three alpha helices located near the C-terminus (see Fig. lb); (2) a conserved ...HLWKS...pentapeptide, which presumably is required for cofactor interaction (Fig. 1 b); (3) the conserved N-terminus of unknown function shared with the other three homeodomain proteins of the paralogous group 13 (Fig. 1 b); (4) a stretch of aliphatic amino acid residues (poly-glycine) downstream of the N- terminus which is located at an equivalent position as the poly-alanine stretch found in Hoxdl3 and whose length variation has been found to be linked to the human congenital malformation synpoydactyly (Muragaki et al. Science 272, 548, 1996).

Hoxcl 3 oligopeptides can be used as antigens to raise polyclonal antibodies in rabbits that are specific for the polypeptide set forth as SEQ ID NO: 6. This approach has been used successfully for raising Hoxc 8 (previously known as Hox-3.1)-specific antibodies (Awgulewitsch and Jacobs, Development, 108:441 (1990)). Peptides from the middle region of the exon 1 coding region can be selected for this purpose. An example of a peptide that can be used is SHNVNLQQKPCAYHPGDKY (SEQ ID NO: 34). This peptide corresponds to amino acids 133 to 151 of the Hoxc 13 protein sequence. According to standard DNA protein data base searches, this peptide was found to be unique. Appropriate peptide antigens can be selected by using PEPTIDE STRUCTURE of the GCG software package.

This invention also contemplates producing a selected cell line or a non-human transgenic animal model for the analysis of the function of a gene comprising introducing into an embryonic stem cell a vector having a selectable marker which, when the vector is inserted within a gene, the inserted vector can inhibit the expression of the gene, selecting embryonic stem cells expressing the selectable marker, excising the vector from the embryonic stem cells expressing the selectable marker such that host DNA from the gene is linked to the excised vector, sequencing the host DNA in the excised vector, comparing the sequence of the host DNA to known gene sequences to determine which host DNA is from a gene for which a model for the analysis of the function the gene is desired, selecting the embryonic stem cell containing the inhibited gene for which a model for the analysis of gene function is desired, and forming a cell line or a non-human transgenic animal from the selected embryonic stem cell.

It is also contemplated in this invention that transgenic animals can be produced which either overproduce the polypeptides of this invention or fail to produce the polypeptides of this invention in a functional form. For example, a transgenic animal which overproduces the Hoxc 13 of this invention can be produced according to the methods taught in the Examples, as well as by methods well known in the art whereby nucleic acid encoding Hoxc 13 is introduced into embryonic stem cells, at which stage it is incoφorated into the germline of the animal, resulting in the production of Hoxc 13 in the transgenic animal in increased amounts relative to a normal animal of the same species. One skilled in the art can determine if oveφroduction or undeφroduction of Hoxc 13 results in altered phenotypes or genotypes.

A transgenic animal in which the expression of Hoxcl 3, for example, is tissue specific is also contemplated for this invention. For example, transgenic animals that express or overexpress these genes at specific sites such as the skin can be produced by introducing a nucleic acid into the embryonic stem cells of the animal, wherein the nucleic acid is under the control of a specific promoter which allows expression of the nucleic acid in specific types of cells (e.g., a neuronal promoter which allows expression only in neuronal cells. One skilled in the art can determine if a tissue- specific alteration in Hoxc 13 expression results in altered phenotypes or altered expression of other genes by assaying for the expression of the gene of interest in both the non-transgenic and the transgenic animal.

Alternatively, the transgenic animal of this invention can be a "knock out" animal (see, e.g., Willnow et al., 1996), which can be an animal that, for example, normally produces Hoxc 13 but has been altered to prevent the expression of the animal's nucleic acid which encodes Hoxcl 3, thereby resulting in an animal which does not produce Hoxc 13 in a functional form. Such an animal may lack the ability to express all of the nucleic acids encoding Hoxc 13 or the transgenic animal may lack the ability to express some (one or more than one) but not all of the nucleic acids encoding the Hoxcl 3.

For example, the transgenic "knock out" animal of this invention can have the expression of a gene or genes knocked out in specific tissues. This approach obviates viability problems that can be encountered if the expression of a widely expressed gene is completely abolished in all tissues. One skilled in the art could determine whether or not the "knock out" has influenced the expression of other genes by assaying for proteins encoded by these genes or detecting mRNA expression of these genes in both the non-transgenic and transgenic animal.

The invention also provides a transgenic non-human animal whose cells overexpress a transgene coding for Hoxc 13 wherein the total native and transgenic Hoxc 13 expressed in the transgenic animal is higher than the Hoxc 13 expressed in a non-transgenic animal which transgenic animal has increased hair loss, hyperkeratotic skin due and reduced adipose tissue due to the overexpression of the transgene.

The transgenic animals of the following invention can have one or more of the phenotypes selected from the group consisting of: increased hair loss, hyperkeratotic skin, reduced adipose tissue and abnormal pigmentation.

An example of a transgenic non-human animal whose cells overexpress a transgene coding for Hoxc 13 is provided in the Examples where transgenic mice that express the GC13 construct are described. Transgenic animals whose cells overexpress Hoxc 13 can be generated as described in the Examples. The cells of the transgenic animals that overexpress Hoxc 13 can be, but are not limited to, epidermal keratinocytes and keratinocytes of the bulb region of hair follicles and the developing hair shaft. There are numerous cells that can be monitored for overexpression of the transgene as described below.

Similar to other Hox genes, the Hoxc 13 expression pattern is very complex and undergoes dynamic changes during development. The early pattern includes primarily posteriorly restricted expression in the tailbud and caudal central nerve cord, as well as the epidermis over developing limbs (Peterson et al. 1994, Godwin & Capecchi 1998). Accordingly, the early pattern involves at least three different cell lineages, including mesenchymal cells, cells of the CNS, and primitive epidermal cells. GC13 overexpression construct is expressed in the tailbud of the transgenic mice of this invention.

In addition to those sites, the late pattern (> E13.5 d) includes expression in developing whisker and coat hair follicles, the filiform papillae of the tongue, the developing nail beds of the digits (Godwin & Capecchi 1998) and the eyelid . Of greatest interest for the skin pathologies exhibited by the GC13 transgenic mice is the expression in the hair and whisker follicles. Reporter gene data and in situ hybridization data, provided herein, provide direct evidence for expression in differentiating and differentiated keratinocytes of the bulb region of hair follicles and the developing hair shaft. There is also evidence for expression in the companion layer, i.e. the innermost layer of the outer root sheath of hair follicles (Godwin & Capecchi 1998). Follicle expression persists during postnatal development and presumably into adulthood.

The invention also provides a transgenic non-human animal whose cells overexpress a transgene coding for Hoxc 13 wherein the total native and transgenic Hoxc 13 expressed in the transgenic mouse is higher than the Hoxc 13 expressed in a non-transgenic mouse, which transgenic mouse has increased hair loss, hyperkeratotic skin and reduced adipose tissue due to the overexpression of the transgene.

The invention also provides a transgenic non-human animal whose cells overexpress a transgene coding for Hoxc 13 wherein the total native and transgenic Hoxc 13 expressed in the transgenic animal is higher than the Hoxc 13 expressed in a non-transgenic animal, which transgenic animal has increased hair loss, hyperkeratotic skin, reduced adipose tissue and abnormal pigmentation due to the overexpression of the transgene. FVB-GC13/C57BL6 mice exhibiting these characteristics can be made by the methods described in the Examples.

The invention also contemplates utilizing the transgenic animals of this invention to screen for constructs that consist of the control elements contained within the 19kb genomic region of GC13 that drive Hoxc-13 specific expression. Such constructs can then be used to produce expression vectors for tissue specific expression of a heterologous protien. The methodology described in the Examples can be used to generate transgenic mice with constructs encoding fragments of GC13. Reporter gene analysis in transgenic mice, as described in the Examples, will allow those skilled in the art to determine which constructs contain the necessary control elements that drive Hoxc-13 specific expression. For example, a construct containing SEQ ID NO: 3 which contains putative control elements can be utilized to generate a transgenic mouse. If the expression pattern observed throughout development is similar to the expression pattern observed in the GC13 transgenic mouse, one skilled in the art would know that the construct contains the control elements present in GC13 and these elements are present in SEQ ID NO: 3. Alternatively, if the expression pattern is different, one skilled in the art would know that the particular construct does not contain all of the necessary control elements and all of the necessary control elements are not present within SEQ ID NO: 3. Similar experiments can be done with constructs containing other fragments of GC13 such SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5 or GC13 fragments comprising SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 7. One skilled in the art would know that within the 19 kb fragment there may be several tissue specific control elements. For example, if the expression pattern observed with the construct containing the putative control elements is not the same as that observed with the GC13 mice because no expression is observed in the hair follicles, one skilled in the art would know that the control sequences for the hair follicles are not present in the construct. By determining which region GC13 was removed to generate the construct, one skilled in the art would then be able to take that region and perform further studies to determine exactly which sequences are necessary to direct expression to these cells in hair follicles. Similar experiments can be performed to delineate the sequences that direct expression to other cells and tissues.

The present invention also provides a method for screening compounds for an effect on hair loss comprising administering the compound to the transgenic animal of this invention and monitoring the animal for a change in hair growth, wherein altered hair growth is indicative of the effect of the compound on hair growth.

By "altered, in the context of hair growth, is meant an increase or a decrease in hair growth. By "altered", in the context of hair growth is also meant changes in hair structure and composition. Hair growth can be monitored visually by observing the amount of hair on a transgenic animal not receiving the compound (the control animal) and comparing it to the amount of hair on a transgenic animal that received the compound. If the amount of hair is greater on the transgenic animal that received the compound than the amount of hair observed on the control, one skilled in the art would know that the compound has altered hair growth in the transgenic animal. Alternatively, hairs can be plucked from a control animal and compared to a hair sample from a transgenic animal that received the compound.

To screen for compounds affecting Hoxcl3-dependent hair loss in vivo, it is possible to monitor hair density, and hair structure. The structure of the hair shaft is changed in GC13 mutant mice which is a contributing factor to hair loss. Changes in shaft moφhology can be detected by light microscopy of plucked hair. In addition to altered (irregular) septation of the shaft, the roots of plucked GC13 mutant hair are typically fractured, which is not the case with hair from normal mice. One skilled in the art would also look for changes in cuticular septation which may be denser than normal, absent, or irregular. Microscopic changes in the hair and follicle structure will allow the skilled artisan to discern whether or not the compound has altered the hair of the transgenic mouse receiving the compound.

A method for screening compounds for an effect on a hyperkeratotic skin disorder by administering the compound to the transgenic animal of this invention and monitoring the animal for a change in the skin disorder, wherein a change in the hyperkeratotic skin disorder is indicative of the effect of the compound on a hypeφroliferative skin disorder.

The hypeφroliferative skin disorders contemplated by this invention include but are not limited to, ichthyosis, hyperkeratosis, psoriasis, and hypeφlasia associated with skin cancer.

As used herein, "a change", in the context of skin disorders, can be an improvement or a deterioration of the skin condition. A skin condition can be monitored visually by observing the skin on a transgenic animal not receiving the compound (the control animal) and comparing it to the skin of a transgenic animal that received the compound. If the skin on the transgenic animal that received the compound shows an improvement as compared to the control animal, one skilled in the art would know that the compound has effected a change in the hypeφroliferative skin disorder in the transgenic animal. Alternatively, skin samples can be obtained from a control animal and compared to skin samples from a transgenic animal that received the compound. Microscopic changes in the skin cells will allow the skilled artisan to discern whether or not the compound has effected a changed in the hypeφroliferative skin disorder. At the visual level one skilled in the art would look for changes in the flakiness and ulceration of the skin. At the microscopic level one skilled in the art would look for changes in the thickness of the epidermis, including cellular and cornified (squamous layers).

A method for screening compounds for an effect on reduced adipose tissue by administering the compound to a transgenic animal of this invention and monitoring the animal for a change in adipose tissue, wherein a change in the adipose tissue of the animal is indicative of the effect of the compound on adipose tissue.

As used herein, "a change", in the context of adipose tissue, can be an increase or a decrease in the amount of adipose tissue in a transgenic animal. The amount of adipose tissue can monitored visually by observing the size of the transgenic animal not receiving the compound (the control animal) and comparing it to the size of a transgenic animal that received the compound. If the amount of adipose tissue in the transgenic animal that received the compound increases as compared to the control animal, one skilled in the art would know that the compound has effected a change in the amount of adipose tissue in the transgenic animal. Alternatively, a transgenic animal that received the compound can be dissected as described in the Examples and examined for the amount of adipose tissue. Any changes in the amount of adipose tissue in the transgenic animal indicates that the compound has effected a change in the amount of adipose tissue in the animal. One skilled in the art could also measure serum levels of lipolytic enzymes to assess the effects of compounds on adipose deposition.

A method for screening compounds for an effect on pigmentation by administering the compound to a transgenic animal of this inveniton and monitoring the animal for a change in pigmentation, wherein a change in pigmentation is indicative of the effect of the compound on pigmentation. The FVB-GC13/C57BL6 mice described in the Examples can be used to screen compoudns for an effect on pigmentation.

As used herein, a "change" in pigmentation can be an improvement or a deterioration in pigmentation. Pigmentation can be monitored visually by observing the skin on a transgenic animal not receiving the compound (the control animal) and comparing it to the skin of a transgenic animal that received the compound. If the skin on the transgenic animal that received the compound shows an improvement as compared to the control animal, one skilled in the art would know that the compound has effected a change in pigmentation in the transgenic animal. Alternatively, skin samples can be obtained from a control animal and compared to skin samples from a transgenic animal that received the compound. Microscopic changes in the skin cells will allow the skilled artisan to discern whether or not the compound has effected a changed in pigmentation. At the visual level one skilled in the art would look for changes in skin coloration. At the microscopic level one skilled in the art would look for changes in melanin content and distribution.

To screen for compounds affecting Hoxc 13 expression in vitro, it is possible to use skin explants in culture. Changes in the Hoxc 13 expression pattern in both normal and mutant skin can be monitored by X-gal staining assays of skin samples derived from single transgenic LZ-GC13 and double transgenic GC13/LZ-GC13 mice, respectively.

This invention provides a LZ-GC13 line expressing the lacZ reporter gene in hair follicles. This line can be crossed with the mutant GC13 mice to generate LZ- GC13/GC13 double transgenic mice which allows one skilled in the art to directly link pathological changes in mutant skin to the expression of the transgene, i.e. overexpression of Hoxc 13 and monitor. This will also allow one skilled in the art to monitor changes of the bona fide Hoxc 13 expression pattern over time, which is of relevance for in vitro assays aimed at identifying compounds affecting Hoxc 13 expression.

One skilled in the art could also utilize the transgenic animals of this invention to test for the effects of Hoxc 13 overexpression on the expression patterns of other Hox genes (including Hoxc 13 itself, i.e. potential autoregulation) by crossing the GC13 mice with any of the existing or future Hox-lacZ reporter gene mice. For example, one skilled in the art could make GC13/LZ-GC13 double transgenics in this way.

The invention provides a method of identifying genes associated with the pathological changes observed in the skin of GC13 mice comprising: isolating a gene differentially expressed in skin of GC13 mice by suppressive subtractive hybridization; obtaining a probe or immunohistochemical reagent for the differentially expressed gene of step, and utilizing the probe to analyze the expression pattern of the differentially expressed gene in GC13 wherein altered expression of the gene as compared to a normal mouse indicates the gene is associated with the overexpression of Hoxcl 3.

The transgenic animals of this invention can be used to identify and define the genetic pathways and control mechanisms required for hair follicle development and keratinocyte proliferation/differentiation as affected by overexpression of Hoxcl 3. An example of an experimental strategy for identifying the molecular basis for the pathological changes observed in skin of GC13 transgenic mice is illustrated in Fig. 8.

Central to this strategy is the large-scale screen for genes differentially expressed in skin of GC 13 mice. This will be achieved by using methodology based on suppression subtractive hybridization (SSH) of cDNAs (Diatchenko et al. 1996), in this case derived form postnatal skin of GC13 mutant and normal mice, respectively (see Fig. 8) A protocol for generation of subtracted cDNA libraries using SSH is provided in the Examples.

In contrast to other subtractive hybridization methods, which usually favor enrichment of highly expressed mRNAs, a key feature of SSH is equalization of enrichment levels among highly, moderately, and rarely expressed messages. This drastically improves the chances of enriching for RNA species encoded by moφhogenetic controller genes, which often are expressed at low levels and include those encoding many signaling molecules and transcription factors, for example. Differential levels of expression of RNAs corresponding to thus enriched and cloned cDNAs are estimated by quantitative reverse Northern hybridization as described in the Examples. This step allows preselection of relevant cDNA clones corresponding to known and unknown genes.

Probes or immunohistological reagents for known differentially expressed genes, are currently available. Among these, markers for those genes suspected to be the most likely direct or indirect targets for Hoxc 13, including members of the keratin gene family, as well as members of certain groups of transcription and growth factors, for example, can be selected. The genes regulated by Hox proteins belong to different gene families including genes encoding other transcription factors, signaling molecules including growth factors, receptor molecules, kinases, adhesion molecules, structural proteins, and proteins involved in apoptosis.

Expression analyses of these potential targets for Hoxc 13 in GC13 mice allows elucidation of important aspects of the genetic control mechanisms underlying the histopathological changes observed in mutant skin. In order to define the functional properties of Hoxc 13 at a structural level one skilled in the art can determine to what extent the histopathological changes seen in GC13 mutant mice are specific for overexpression of Hoxc 13 or if overexpression of any Hox gene in a Hoxcl 3 -specific manner can cause the same effects. This can be done by overexpressing protein coding cDNAs corresponding to downstream neighbouring genes, or a different Hox gene of the same paralogous group (i.e. Hoxb 13, Hoxa 13 and Hoxd 13) in a Hoxcl3-specific fashion in skin. One skilled in the art would utilize the regulatory elements present in GC13 to direct the expression of the gene of interest in a Hoxc 13 specific fashion.

Similarly, to determine the functional specificity of conserved structural motifs of the Hoxc 13 protein for the development of the pathological changes observed in GC13 mice, one skilled in the art could could examine the effects of overexpressing modified Hoxcl3. For example, a transgenic mice can be generated as described in the Examples with a construct in which the homeodomain of Hoxc 13 has been deleted. Should this result in persistent phenotypic changes, one could conclude that the remaining structure of the protein is at least partially sufficient for affecting molecular mechanisms controlling follicle development and keratinocyte proliferation. In addition, the skilled artisan could examine the effects of altering the length of the conserved poly-glycine (G) stretch located near the N-terminus of the protein.

The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

EXAMPLES

Sequence Analysis and Comparison

The presumptive coding sequence of Hoxcl 3 was derived from partial and overlapping cDNAs obtained by 5'and 3' RACE (rapid amplification of cDNA ends) and confirmed by analysis of corresponding genomic sequences. Total RNA isolated from the posterior halves of E12 embryos was used as template for cDNA synthesis. The presumptive start codon is the most distal ATG of a continuous open reading frame positioned in a sequence context that resembles a common consensus for eukaryotic translational initiation 30. This is supported by the conserved N-terminal sequence similarities shared among all group 13 Hox proteins (Fig. lb). Differentially expressed cDNAs were sequenced and sequences were compared against public data bases using Advanced Blast program at NCBI.

Cloning of Transgenes

The GC13 overexpression construct was derived from murine cosmid clone cos2W3H (Peterson et al. 1994) as a 19 kb Notl - Xhol fragment and subcloned into cognate sites of pSafyre (see Bieberich et al. 1990). Reporter gene construct LZ-GC13 was generated by replacing a singular Ascl fragment of GC13 containing 430 bp of the 3'-region of Hoxcl 3 exon 1 and approximately 50 bp of adjacent intronic sequences (see Fig. 1 a) with a PCR -amplified E.coli LacZ gene thus creating an in-frame-fusion between Hoxcl 3 and lacZ coding sequences. The LacZ gene was amplified from plasmid pCHl 10 (Amersham Pharmacia, Piscataway, NJ) and Ascl restriction sites were incoφorated by PCR using the following forward and reverse primers, respectively: 5'GGCGCGCCAAGTCGTTTTACAACGTCGTGACTGG-3' (SEQ ID NO: 8), and 5'-GGCGCGCCCTTACGCGAAATACGGGCAGAC-3' (SEQ ID NO: 9).

Generation of Transgenic Mice

GC13 transgenic mice were generated by nuclear injection of single cell FVB mouse embryos according to described procedures (Gordon & Ruddle, 1983;

Awgulewitsch & Jacobs, 1992) by using DNA concentrations of 2 μg/μl for each of the respective transgenes. Mutant 61B1 and 61U4 transgenic lines were established by breeding with FVB mice. In both lines the mutant trait was transmitted to subsequent generations without recognizable changes. Transgenesis was determined by PCR analysis of genomic DNA isolated from tail biopsies or placentas (transient assays). Forward primer GC13-3' (5'GGAGAGCCTGGGCGTAGACATC) (SEQ ID NO: 10) and reverse primer GC13-5' (5'CCAATTTGCCTCCCTTTTCTGGTCC) (SEQ ID NO: 11) derived from sequences near the 3' and 5' end of GC13, respectively, amplified a diagnostic 400 bp fragment, assuming tandem incoφoration of multiple transgene copies. Injection of embryos with LZ-GC13 resulted in a total of 6 transgenic mice expressing lacZ in the same pattern (see Figure 3) as examined by X-gal staining essentially as described (Papenbrock et al, 1998). Three of these mice were sacrificed and examined at different embryonic stages, while the remaining three were used as founders for establishing three independent LZ-GC13 strains used for further analyses. Preliminary blood and bone marrow analysis of GC 13 mutant mice did not reveal hematopoietic defects as a potential cause for the frequent perinatal lethality.

In situ Hybridization

Whole mount in situ hybridization with E13.5 d - E15.5 d embryos was performed by using Digoxygenin-labeled (Roche Diagnostics, Indianapolis, IN) antisense RNA probes synthesized either from plasmid pcl3ISH (Peterson et al. 1994) containing a genomic 604 bp Xbal-EcoRI fragment that extended from 46 bp upstream and 377 bp downstream of the Hoxcl3 homeobox or from plasmid pC13rev containing a partial cDNA of 697 bp that included 532 bp of 3' coding sequence and 165 bp of 3' untranslated flanking sequence cloned into vector pCR®II-TOPO (Invitrogen,

Carlsbad, CA). Between the two probes, no difference in expression pattern and signal intensity was detectable.

In situ hybridization to 10 μm frozen skin sections using ³⁵S-labeled sense and antisense (control) RNA probes synthesized from pC13rev was performed at 50°C as described (Awgulewitsch and Utset, 1991). Exposure to Kodak NTB2 autoradiographic photoemulsion was for 4 days. Probe specificity was confirmed by hybridization under the same conditions to sagittal sections of E 13 embryos, which resulted in the known Hoxcl3 hybridization pattern (Peterson et al., 1994) only with antisense probe BrdU Assays

For ex vivo labeling, pieces of dorsal neck skin (~ 0.3 x 1.0 cm) of 2 d postnatal mice were dissected immediately after euthanesia by cervical dislocation, washed briefly in Dulbeccos Modified Eagles Medium (DMEM; Gibco BRL, Grand Island, NY) at 37 °C and placed in DMEM containing freshly prepared BrdU (40 μM) (Sigma Chemical Company, St. Louis, MO). Samples were incubated at 37 °C for two hours in a tissue culture incubator with periodic agitation. After labeling, the tissue was rinsed with two changes of ice cold EPBS before fixation in fresh 3% buffered paraformaldehyde for 30 minutes. Tissue was processed for paraffin embedding and sectioned at 7 μm. To immunolocalize BrdU uptake, the sections were deparaffmized in xylenes and rehydrated through graded ethanols to PBS. DNA was denatured in 4 N HC1 for 30 minutes and the pH subsequently adjusted with 100 mM sodium borate. The tissue was permeabilized with 0.1% Triton X-100 (20 minutes) and rinsed briefly in PBS. After blocking for 30 minutes in 3% bovine serum albumin in PBS, the sections were immunolabeled with FITC-conjugated monoclonal rat anti-bromodeoxyuridine (Harlan Sera-Lab., LTD, Loughborough, UK) for 1 hr. After labeling, the slides were rinsed in PBS (3x 10 minutes), mounted and imaged on a Leitz TM photomicrocsope equipped with a Photometries™ (Tuscon, AZ) Quantix CCD camera. Images were stored on an IBM PC workstation and processed using Adobe Photoshop software (Adobe Systems Inc., San Jose, CA).

Alternatively, the BrdU assay is performed as follows: Pieces of skin (= 0.3 x 1.0 cm) dissected from anterior dorsal trunk of 2 d postnatal mice were incubated in RPMI medium supplemented with 100 μM BrdU for 4 hrs, fixed in 3% paraformaldehyde for 30 min., and embedded in Tissue-Tek™ OCT compound (Sakura Finetechnical Co., Tokyo). Ten μm cryosections were stained with rabbit anti-beta- galactosidase polyclonal antibodies (Eppendorf - 5 Prime), followed by rhodamine- conjugated donkey anti-rabbit IgG (Chemicon International Inc.). For BrdU/β-gal double-staining, sections were incubated, in addition, with Fastlmmune Anti-BrdU FITC-conjugated antibodies with DNase (Becton Dickinson Immunocytometry Systems) for 45 min. at room temperature and fixed in 1% paraformaldehyde for 5 minutes before mounting in ProLong™ Antifade Reagent (Molecular Probes). Image analysis was performed using a Leitz™ photomicrocsope equipped with a Photometries™ Quantix CCD camera.

Scanning Electron Microscopy

Samples were attached onto specimen stubs using double-stick conductive tabs and sputter-coated with gold using a Polaron SEM Coating Unit # 5150. Samples were imaged using a JEOL JSM 5410 LV Scanning Electron Microscope.

Generation of Hoxc 13 Antibodies

A peptide corresponding to amino acids 133 to 151 (SHNVNLQQKPCAYHPGDKY (SEQ ID NO: 34)) of the Hoxcl3 protein was designed and conjugated to keyhole limpet hemocyanin. This conjugated peptide was utilized to raise antibodies in rabbits. Antibodies purified from rabbit serum detect Hoxc 13 in the hair follicles of mice.

Generation of GC13/FVB x C57BL6 transgenic mice

GC13 FVB mice were mated with C57BL6 mice obtained from Charles River, to obtain GC13 FVB x C57BL6 hybrid mice.

(A) Identification and Isolation of Differentially Expressed Sequences Generation of Subtracted cDNA Libraries Using SSH

Two subtracted libraries representing genes that are up- and down-regulated in GC13 mutant skin, respectively, can be generated using the CLONTECH PCR Select™ cDNA subtraction kit (Clontech Laboratories, Inc., CA) essentially by following the procedures and modifications as described in detail in Tkatchenko et al. 1999. RNA to be used for the cDNA synthesis is isolated from total skin of day 5 pnd GC13 transgenic mice and non-transgenic normal littermates, respectively. This stage is just beyond the 2 day pnd stage at which marked hypeφroliferation of follicular and interfollicular keratinocytes biochemically/immunihistochemically is detected based on BrdU assays. Yet it precedes the later stages at which histopathological changes in skin of GC13 mice are becoming very pronounced and thus may trigger secondary responses unrelated to the direct effects of Hoxc 13 overexpression. A schematic summary of the cDNA subtraction procedure based on SSH is provided in Fig. 8.

The SSH procedure is concluded by sequential PCR amplification of subtracted cDNAs using nested primers (see Fig. 8). Secondary PCR products are separated by 2% agarose gel electrophoresis, and short (200 bp - 1 kb) and long (1 - 5 kb) fractions are eluted from the gel using dialysis bags and the QIAquick PCR Purification Kit (QIAGEN). Fractionated PCR products are then cloned separately into the pCRII- TOPO vector using the TOPO-TA Cloning Kit (Invitrogen). White bacterial colonies are picked manually from the 135 mm agar dishes, transferred into 384- well plates and grown for 24 h at 37°C. Bacterial clones from master plates are arrayed onto Hybond nylon membrane filters (Amersham) placed on top of LB/ampicillin agar using a 384- pin tool (V&P Scientific, Inc.), and bacteria are grown overnight at 37°C. The arrayed cDNAs are then denatured and hybridized with subtracted probes.

Differential Screening of Subtracted Libraries Using Subtracted Probes

To prepare subtracted cDNA probes, 60 μl of each of the secondary PCR products are purified using the QIAquick PCR Purification Kit (QIAGEN) and eluted in 50 μl of water. 40 μl of the combined volume is digested with Rsal, Eael and Smal restriction enzymes to remove adapters. After digestion, the samples are purified using the QIAquick PCR Purification Kit (QIAGEN) to remove salts, proteins and adapters. 2 μl of each sample are 32P-labeled using Ready-To-Go DNA Labeling Beads (Pharmacia Biotech) and hybridization is performed overnight using 8 -10 x 107 dpm of probe per filter. The filters are then washed twice for 20 min in 1 x SSPE/0.2% SDS at room temperature followed by three 20 min washes in 0.1 x SSPE/0.2% SDS at 65°C and exposed to X-ray film overnight with intensifying screens. Differential clones are sequenced by the MUSC DNA sequencing core facility and analyzed using GCG software. Redundant clones are eliminated.

Quantitative Reverse Northern Hybridization

The selected clones are pooled, classified into functional categories, and a DNA array obtained thereof is used to analyze expression levels of corresponding genes. To minimize possible spot-to-spot variations, three identical array filters are hybridized with each probe. To make a DNA array, plasmid DNA isolated from each differentially expressed clone is diluted with the loading buffer (1.5 M NaCl/0.5% bromophenol blue) to the final concentration 20 ng/μl and the samples are arranged in 384-well plates. 1 μl of each sample is simultaneously transferred (printed) onto Hybond nylon membranes (Amersham) using a 384-pin tool (V&P Scientific, Inc.). DNA is denatured on top of filter paper soaked in 0.5 M NaOH/1.5 M NaCl, neutralized with 1 M Tris-HCl pH 7.5/1.5 M NaCl; membranes rinsed in 2xSSPE, dried, and DNA is UV crosslinked. The cDNAs to be used for preparing complex cDNA probes are synthesized using 2 μg of poly(A) RNA and the Marathon cDNA Amplification Kit (CLONTECH). The complex cDNA probes are prepared by 32P-labeling (25 ng of each cDNA sample) by using the Random Primers DNA Labeling System (Gibco BRL). Membranes are hybridized in 20 ml per membrane of hybridization buffer containing 10 x 107 dpm of the corresponding complex cDNA probe for 20 h at 42°C (50% formamide). Probes are carefully equilibrated in terms of specific activity and total radioactivity to certify that exactly the same amount of labelled cDNA is used for each hybridized filter. Filters are washed as described above and exposed to an imaging plate for 16 h. The plate is then scanned using a Phosphorlmager work station and images are analyzed and quantified using PCBAS 2.09f software. The resulting tables containing intensities of all spots, local backgrounds and surface of each spot are exported to Excel and further calculations are performed using Excel worksheet. (B) Selection of Subtracted cDNA Clones for Histopathological Analyses in Mutant

Skin

Selection of Clones

Downstream analyses focuses on correlating expression patterns of selected cDNAs subtracted with changes in Hoxc 13 expression patterns and the pathological changes seen in GC13 mutant skin. This is achieved by using ISH methods, as well as immunohistochemistry, provided suitable antibodies specific for a selected marker are available. The first criterion for selecting subtracted cDNA clones for this type of analyses is easily detectable levels of differential expression in skin of GC13 mice of at least three-fold (over- or under-expression) as estimated by reverse Northern hybridization.. This will include as many known as unknown sequences. Among the known sequences,priority is given to those corresponding to gene families that have been implicated in hair follicle and skin development. Among the unknown sequences, priority is given to those showing highly conspicuous levels (> five-fold) of differential expression in reverse Northerns.

Phylogenetic Tree Analysis and Genomic Mapping

Phylogenetic tree analysis of KAP -related cDNA sequences was performed by using the Genebee Multiple Alignment program (Belozersky Institute, Moscow State University; http://www.genebee.msu.su/services/malign_reduced.html) and deduced amino acid sequences. For GeneBank accession numbers of previously known and novel KAP gene sequences used for this analysis see Figure 6 A. Mapping of the known and novel KAP genes was accomplished by using a mouse/hamster radiation hybrid (RH) panel (Research Genetics, Inc.). PCR data sets obtained with this panel were analysed at the Jackson Laboratory RH database site, http:www.jax.org/resources/documents/cmdata, for determining genomic map positions. The PCR conditions were optimized for AmpliTaq Gold DNA Polymerase (Perkin Elmer) by using the following cycling conditions: 95°C for 7:00 min [94°C for 0:30 min; Ta (optimized annealing temperature) for 0:35 min; 72°C for 0:45 min]x32/ 72°C for 5:00 min. Under these conditions, the Ta and pair of forward (F) and reverse (R) primers used for each of the genes analysed were as follows: HGTpII.l : 62°C, F/5'GGCTGTGGTTATGGCTCCTA (SEQ ID NO: 12), R/5'GTGAAGAATGGCAAAGTCCTG (SEQ ID NO: 13);

Pmgl : 62°C, F/5'GATGTGGTACCCCTACTTTCT (SEQ ID NO: 14), R/5'GTTTTGCCAATTACAGGAACTC (SEQ ID NO: 15);

Hacll : 70°C, F/5'GCCCAGTTACCACAGCCTCAG (SEQ ID NO: 16), R/5'GGCGTAGAGCAAGGATTGGAG (SEQ ID NO: 17);

krtaplό.l : 56°C, F/5'TGGAGGATATGGGTTCTC (SEQ ID NO: 18), R/5'CATGTTCAGTTGGCTTAGTAC (SEQ ID NO: 19);

krtapl6.2: 56°C, F/5'GGACTTGGAAATACTTGTCG (SEQ ID NO: 20), R/5'TGGGAATGTGTTGTAGAGG (SEQ ID NO: 21);

krtapl6.3: 60°C, F/5'GTGGAGGATATGGGTTCTC (SEQ ID NO: 22), R/5'CTGCAACAATGTTATTGACTG (SEQ ID NO: 23);

krtapl6.4: 56°C, F/5'CTATGGCTCTGGAGGCTATG (SEQ ID NO: 24), R/5'GAGTTGCTTTCCCAGTTCAG (SEQ ID NO: 25);

krtapl6.5: 56°C, F/5 'AATCCTGTTCC AGTAACTC AG (SEQ ID NO: 26), R/5'ACATAATCCCACTCTTAGTGC (SEQ ID NO: 27);

krta_P16.6: 58°C, F/5'GTGGAGGATATGGGTTCTC (SEQ ID NO: 28), R/5'CTGCAACAATGTTATTGACTG (SEQ ID NO: 29);

krtapl6.7: 60°C, F/5'TGTGCTGCAACTACTACGG (SEQ ID NO: 30), R/5'GCCATATCCACAGCCATAT (SEQ ID NO: 31);

krtapl6.8: 56°C, F/5'CTATGGCTGTGGCTACCG (SEQ ID NO: 32), R/5'ACATGCAGTTCAGAATTGGAG (SEQ ID NO: 33):

Histopathological Analyses

Expression patterns of selected molecular markers in GC13 mutant and normal skin are examined by using ISH and immunohistochemical methods. Immunohistochemical methods as set forth in Awgulewitsch and Jacobs (Development, 108: 411-420 (1990) can be employed for these studies. As the subtracted cDNA library is cloned in the pCRII-TOPO vector (Invitrogen), where the multiple cloning site is flanked by T7 and SP6 RNA polymerase promoter sequences, respectively, digoxigenin -labeled sense and antisense RNA probes can directly be synthesized by using purified plasmid DNAs.

In cases where the sequence of a selected cDNA matches that of a molecular marker for skin and hair follicle development for which suitable antibodies are known to exist, antibodies are obtained either from a commercial supplier or other sources. In relevant cases both ISH and immunohistochemical reagents are available, RNA and protein expression data is compared. For example, should differential expression of an autocrine or paracrine growth factor be detected at the transcriptional level, it will be essential to know whether and how this might impact the level and spatial distribution of the corresponding protein. Should results indicate effects of Hoxc 13 on the expression of other known transcription factors, it might be very informative to examine potential colocalization, requiring differential labeling strategies of antibodies.

Analyses of expression patterns by ISH will initially be performed in sectioned postnatal and juvenile skin samples derived from dorsal neck regions at three different developmental stages including days 5, 15, and 30 pnd. Expression analyses at the exact stage chosen for the isolation of differentially expressed sequences is a first test for the veracity and specificity of the prior steps of cDNA subtraction. Analyses at the two later stages will allow correleation of potential continued differential expression of selected markers with the progressive pathological changes. Depending on the outcome of these initial experiments in juvenile skin, examining expression of individual markers also in adulthood is also contemplated. In order to synchronize the growth cycle of hair follicles, the coat in the dorsal neck region is depilated by wax stripping of shaven skin. This is performed on skin where hair follicles are in telogen phase of the growth cycle. Coat follicles of FVB mice to be used here appear to be in telogen phase at about 3.5 months (see Preliminary Studies). Skin samples for ISH experiments is dissected at different stages of the induced cycle after depilation ranging from 3 to 21 days. This will include the phase of maximum thickness of epidermis and dermis, as well as the peak of follicle depth, all occuring around 10 days after depilation (Hansen et al. 1984).

As controls for the ISH experiments, in addition to sense strand RNA probes, both a known positive and negative marker for expression in skin will be used. As a non-specific marker for skin, a probe for the alpha-fetal protein (AFP) will be utilized, which has been used successfully as a negative control for ISH in total body sections of newborn mice (Awgulewitsch et al. 1986). This will help in estimating the level of background staining. In order to estimate the strength of specific hybridization signal, a keratin 14 (K14) gene -specific probe known to detect expression in keratinocytes of the basal epidermal layer and the outer root sheath of the hair follicle will be utilized (Vassar et al. 1989; Guo et al. 1993).

ISH Protocol

1. Probe Preparation

Preparation of DIG-UTP-labelled antisense and sense (control) probes is according to

Boehringer Mannheim (BM) protocols. Probe lengths may vary from 200-1200 nucleotides (nt). Size adjustments are not necessary. 2. Tissue Preparation

- rinse tissue pieces rapidly in DEPC-treated PBS for ~ 2 min; 1000 ml lOx:

PBS: 0.9 % (w/v) NaC190 g 12.5 mM NaH2P0415 g lO.O mM NaOH 4 g pH 7.5 - adjust with 10 N NaOH to pH 7.5; filter, DEPC-treat, and autoclave

- embed immediately in Tissue Freezing Medium (Jung) or OCT and keep at (-) 80° C. - alternatively, fix tissue O/N in 4 % parformaldehyde/PBS;

- cryoprotect by: (1) incubation in 12 % sucrose/PBS at RT for 6hrs;

(2) incubation in 18 % sucrose/PBS at RT, O/N;

(3) embed in OCT and keep at (-) 80° C.

3. Sections

- cut 12 μm sections at (-) 35° C;

- mount on poly(L)-lysine (or silane) -coated slides;

- fix sections in 4 % paraformaldehyde/PBS at RT for 10 min;

- process sections for ISH immediately.

4.ISH

- incubate sections in PBS containing 0.1 % active DEPC (Fluka) for 2 x 15 min;

- equilibrate in 5 x SSC for 15 min; 20 x SSC: 3.0 M NaCl

0.3 M Na-citrate (trisodium citrate) (filter before use)

- prehybridize in 0.5 ml per section hybridization mix (Hyb-Mix) at 58° C for 2 hrs; Hyb-Mix: 50 % formamide 5 x SSC

40 μg/ml ssDNA (10 mg/ml stock)

- denature probes at 80° C for 5 min and add to Hyb-Mix at concentration of 400 ng/ml;

- hybridize with 20 μl probe mix/section at 58° C for 4 - 40 hrs; cover sections with Parafilm and perform prehyb/hyb incubations in sealed box saturated with 50 % formamide/5 x SSC.

5.Post-ISH Washes (1)2 x SSC at RT for 30 min, 1 x; (2)2 x SSC at 65° C for 60 min, 1 x; (3)0.1 x SSC at 65° C for 60 min, 1 x.

6. Antibody (Ab) Incubation

- equilibrate in Buffer 1 for 5 min at RT;

Buffer 1:100 mM Tris-HC160.55 g Tris base, 32.5 ml cone. HC1

150 mM NaCl pH 7.5

-incubate sections with AP-conjugated anti-DIG Ab (BM) diluted 1 : 5000 in Buffer 1 with 0.5% BM blocking reagent; then incubate at RT for 2 hrs.

7. Post Ab-Incubation Washes and Color Development (l)Buffer 1 at RT for 2 x 15 min; (2)equilibrate sections in Buffer 2 at RT for 5 min; Buffer 2: lOO mM Tris-HCl 100 mM NaCl

50 mM MgC12 (1 M stock) pH 9.5

(3) develop color in Buffer 2 containing 45 μl NBT and 35 μl BCIP (BM) per 10 ml for 0.5 - 72 hrs; alternatively, use BM Puφle Blue AP substrate for color development; (4) stop staining in 10 mM Tris / 1 mM EDTA, pH 8.0 at RT for 10 min;

(5) remove unspecific staining by incubation in 95 % Ethanol under gentle agitation at RT O/N;

(6) remove precipitated Tris by rehydration in deionized water for 15 min;

(7) dehydrate through 70 %, 95 %, 100 % EtOH and xylol for 2 x 15 min each; (8) mount in Eukitt resin.

GC13 Transgenic Mice Exhibit Severe Hair and Skin Defects

To further assess the role of Hoxc 13 in hair growth, transgenic mice overexpressing this gene were generated, employing as a transgene a 19 kb genomic fragment (GC13) containing Hoxcl 3 (Figure 1 A). Based on genomic and cDNA sequencing, it was determined that the deduced protein sequence of 328 amino acid residues contains the conserved Hoxcl3 homeodomain (Peterson et al., 1994) near its C-terminus (Figure IB). Sequence comparison revealed, outside of the homeodomain, partial similarity at the N-terminus to the other three paralogous group 13 Hox proteins (Mortlock et al., 1996; Zeltser et al., 1996; Herault et al., 1996).

Two GC13 mutant transgenic lines, 61B1, and 61U4, of essentially identical phenotype (Figure 2) were established. Mutant mice could be identified at birth by a short tail and taut skin (Figure 2Aa), as well as kinky whiskers (Figure 2Ac), and frequently an overall smaller stature in comparison to non-transgenic littermates (Figures 2Aa). Juvenile mutants exhibited retarded growth of coat hair (Figure 2Aa), while adult animals developed progressive alopecia and flakiness of the skin at varying degrees (Figure 2Ab). Regions most severely affected were ventral and anterior-dorsal trunk and head. Skeletal analysis revealed that the shorter tail was due to a reduction in the number of tail vertebrae by about ten (Figures 2Ae and 2Af). This can be inteφreted as an anterior-towards-posterior homeotic transformation, i.e. more proximal tail vertebrae assume identities of the most caudal ones. Different levels of Hoxcl 3 expression in developing tail (Figure 3 A) may be a molecular determinant for its phylogenetic variations in length.

The overtly recognizable and, with age, rapidly advancing pathological changes in mutant skin were mirrored by progressive histological abnormalities (Figure 2B). Most obvious changes were epidermal thickening and enlarged hair follicles; the former was apparent already in 5 d postnatal skin (Figure 2B) when hair follicles are in the growth phase (anagen) of the first hair cycle (Paus et al., 1999). Follicle enlargement appeared to result primarily from thickened outer root sheaths (ORSs), as observed in anagen follicles of the second hair cycle in 30 d skin. In skin of adult mutants older than 2-3 mo., many follicles had degenerated into cyst-like structures filled with large amounts of presumably keratinaceous material (Figure 2B). Both the squamous and suprabasal cellular layers, which appeared overall irregular and disorganized, contributed to the dramatic epidermal thickening underlying the scaly appearance of the skin characteristic of ichthyosis. Severely affected animals typically exhibited ulceration of the skin (data not shown). Scanning electron microscopy of mutant whisker shafts revealed changes in cuticular septation, which often exhibited lesions and appeared generally denser and less well defined (Figure 2C). These architectural defects were mirrored by lack of, or irregular septation in plucked mutant coat hair, which often showed fracturing near the root.

Mutant embryos at stages E13 through E15 exhibited higher levels of Hoxcl3 expression exclusively in those structures which were observed to be phenotypically affected, namely vibrissae and tail bud at El 3, as well as pelage hair follicles beginning at El 5 (Figure 3 A). These data show the presence of control elements required for Hoxcl 3 expression in developing hair follicles and tail bud within the GC13 transgene. This was confirmed by analyzing the expression pattern of a / cZ-tagged version of the GC13 transgene termed LZ-GC13 (Fig. 1 A), which was expressed in tailbud and vibrissae at E13 and in coat hair follicles at later (≥ E15) stages (Figures 3Ba and 3Bc). To monitor LZ-GC13 expression in GC13 mutant background, we generated LZ- GC13/GC13 double transgenic mice. Suφrisingly, these mutant mice showed reduced levels of X-gal staining in neural tube and tail at El 3 (compare Figures 3Ba and 3Bb), as well as in vibrissae and developing pelage hair follicles at El 6.5 (Figures 3Bc and 3Bd), i.e. the same sites where increased levels of Hoxcl 3 expression by WMISH in GC13 mice was detected (Figure 3 A). These results provide evidence to support a negative autoregulatory feedback loop, which is required for maintaining balanced physiological levels of Hoxcl 3 expression. To obtain evidence for this control mechanism during postnatal hair growth and development, examined Hoxcl 3 RNA and LZ-GC13 β-gal expression were examined in sectioned skin of 5 d old normal and GC13 mutant mice. While the in situ hybridization data clearly indicated substantially increased levels of Hoxcl 3 expression in the upper bulb and lower shaft regions of GC13 versus normal anagen hair follicles (Figure 3C), the levels of LZ-GC13 reporter gene activity in the same regions presented the reverse situation, i.e. LZ-GC13 expression was drastically reduced in LZ-GC13/GC13 mutant compared to normal hair follicles in LZ-GC13 mice (Figure 3D). Combined, these data suggest employment of a negative autoregulatory feedback mechanism that, either directly or indirectly, controls Hoxcl 3 expression levels during development of both axial skeleton and hair follicles.

Hyperproliferation of Keratinocytes in Skin ofGC13 Mice

To examine whether the enlargement of hair follicles and the thickening of the epidermis in mutant skin reflect changes in the proliferation rates of precursor cells, bromodeoxyuridine (BrdU) assays were performed with 2 d normal and mutant skin. The results showed an increase in the number of BrdU-labelled nuclei in the ORS of mutant hair follicles and in the basal layer of mutant epidermis (Figure 4A), regions that appear to exclude Hoxcl 3 expression as determined by X-gal staining of skin from 2 d and 5 d LZ-GC13 mice (Figure 3D), as well as by in situ hybridization in 5 d normal and GC13 mutant skin (Figures 3C and 4C). To examine more directly whether Hoxcl 3 is expressed in proliferating or differentiating keratinocytes, double- immunofluorescence analysis on BrdU-labelled sections of 2 d normal LZ-GC13 and mutant LZ-GC13/GC13 skin was performend by using anti-BrdU and anti-β-gal antibodies (Figures 4Ba and 4Bb). Since the results show no overlap between β-gal positive and BrdU-labelled cells, it was confirmed that Hoxcl 3 is expressed in differentiating keratinocytes and that the hypeφroliferation of both follicular and epidermal keratinocytes in GC13 mutant skin is an intercellular response to Hoxcl 3 overexpression in differentiating cells. The GC13 mutant hair phenotype is thus directly linked to Hoxcl 3 overexpression in differentiating keratinocytes of the upper bulb and lower shaft regions of the hair follicle, regions that are known to express hair- specific keratin genes including intermediate filament (IF) and keratin-associated protein (KAP) -encoding genes (reviewed in Fuchs, 1995; Powell and Rogers, 1997).

Identification of Hair-Specific Genes as Hoxcl 3 Downstream Targets As a first step toward a definition of the molecular mechanisms underlying the pathological changes observed in GC13 mutant skin, the skin of 5 d GC13 mice was screened for differentially expressed genes. This developmental stage was chosen because the more advanced pathological manifestations seen at later stages may result in secondary and increasingly nonspecific changes in gene expression. Using a strategy based on Suppression Subtractive Hybridization (SSH) (Diatchenko et al., 1996; Tkatchenko et al., 2000), 74 potentially differential cDNA clones were isolated. Reverse Northern quantitative hybridization of these cDNAs using complex cDNA probes (Figure 5 A), identified a total of 4 up- and 25 down-regulated sequences (Figure 5B). The overexpressed cDNAs corresponded to 4 known genes which included, most conspicuously, keratin complex 2 gene 1 (Kl) whose overexpression in mutated form in transgenic mice reproduced features of epidermolytic hyperkeratosis (Bickenbach et al., 1996), a skin disorder with similarities to the one observed here. Of apparent relevance is also the overexpression of a member of the 14-3-3 protein gene family shown in Figure 5B as the 14-3-3 protein, β subtype. The 14-3-3 proteins are known to play a role in keratinocyte differentiation and proliferation and interact with K8 and K18 keratins in a cell-cycle and phosphorylation-dependent manner (Liao and Omary, 1996).

Remarkably consistent with a Hoxcl 3 role in hair follicle development and hair growth is the finding that the 25 downregulated cDNAs included 5 sequences corresponding to previously known hair-specific keratin-associated-protein (KAP) - encoding genes, as well as 9 novel sequences that were found to have closest similarities to KAP genes of the high glycine tyrosine (HGTp) class including the murine HGTpII.l and HGTpI-alpha (Aoki et al, 1997) identified here (Figures 5B and 6A). The 9 novel HGTp-type KAP genes have been designated Krtapl6-1 through Krtapl6-9 because phylogenetic tree and genomic mapping analysis indicate that they belong to a novel cluster of structurally similar genes. The previously reported expression patterns of the identified known KAP genes, HGTpII.l and HGTpI-alpha (Aoki et al., 1997), Pmg-1, Pmg-2 (Kuhn et al., 1999), and Hacl-1 (Huh et al., 1994), as well as the IF keratin gene Krtl-1 (Bertohno et al., 1988), extensively overlap with the GC13 transgene expression pattern in differentiating keratinocytes of the hairshaft (Figures 3 and 4). This, combined with the high levels of downregulation ranging from 2.5- to 21.5-fold for this subgroup, shows that these genes are downstream targets for Hoxcl 3.

Apart from the keratin and KAP genes, a conspicuous member of the group of repressed genes is agouti-related protein (Agrp), whose 13 -fold downregulation is of relevance to the overall smaller stature and reduced body weight, as well as the skin phenotype of GC13 mice. Like its close structural relative, Agouti protein, Agφ is a paracrine signaling molecule that has been implicated in the control of body weight as a hypothalamic Mc4r melanocortin receptor antagonist as evidenced by obesity in transgenic mice ubiquitously overexpressing Agrp (Ollmann et al, 1997). Recent findings of expression in numerous tissues in chicken, including skin, may suggest a role for Agφ in regulating peripheral melanocortin systems as well (Takeuchi et al., 2000).

Genomic Mapping of a Novel KAP Gene Cluster Controlled by Hoxc 13

Insight into the structural and phylogenetic relationships among the identified KAP -related cDNAs was gained by generating a phylogenetic tree using a multiple sequence alignment program. The data revealed that the 9 novel KAP cDNAs were structurally more related to each other and to HGTpII.1 and HGTpI-alpha than to the remaining known KAP genes identified (Figure 6A), thus suggesting that they correspond to members of a new distinct subgroup which is designated Krtap 16-n according to human genome organization (HUGO) keratin gene nomenclature (http;//www. gene.ucl.ac.uk/users/hester/krt.html). Furthermore, sequence comparison of the 9 novel KAP -related cDNA sequences, Krtap 16-1 through Krtap 16-9, against public data bases revealed highest similarity to a previously unidentified cluster of KAP -related sequences located in the human chromosome 21q22.11 region (Hattori et al., 2000). Since this region is of conserved linkage with the distal region of mouse chromosome 16, clustering of the Krtap 16 genes in that part of the mouse genome was predicted. This was confirmed by mapping Krtap 16-1 through Krtap 16-8 between the two distal markers Tiaml and Sodl on mouse chromosome 16 using a mouse/hamster radiation hybrid panel (Figure 6B). Of the known KAP genes identified as Hoxcl3 targets, HGTpII.l and HGTpI-alpha, which are structurally similar to the Krtap 16 genes were mapped, to the same region, as well as the more distantly related Hacl-1. On the other hand, the structurally distinct Pmg-1 and Pmg-2 genes, which are known to be closely linked to each other (Kuhn et al., 1999), were mapped proximal to the Tiaml marker (Figure 6B). Negative Feedback Regulation of Hoxcl 3 Expression

The present invention shows that increased levels of Hoxcl 3 expression in hair follicles during pre- and postnatal development cause severe hair and skin defects including alopecia and a progressive hypeφroliferative disorder resembling ichthyosis. Accordingly, one may conclude that the level of Hoxc 13 expression is a critical parameter for proper hair growth and development.

The present invention provides evidence for the involvement of a negative feedback mechanism in the control of Hoxcl 3 expression levels which provides significant new insight into the regulation of a. Hox gene, in this case a gene that plays a pivotal role in hair growth and development.

Identification of Hoxc 13 Downstream Target Genes

As described above, suppression subtractive hybridization (SSH) of cDNAs (Diatchenko et al., 1996) in conjunction with reverse Northern quantitative hybridization was performed to identify genes differentially expressed in 5d postnatal skin of GC13 mutant mice. This is the first instance where this or a comparable approach has been applied for a large-scale screen of Hox downstream targets in either Hox gain- or loss-of-function mutant mice.

In contrast to other subtractive hybridization methods, which usually favor enrichment of highly expressed mRNAs, a key feature of SSH is equalization of enrichment levels among highly, moderately, and rarely expressed messages; this greatly increases the overall efficiency of the screening process. Second, instead of ubiquitously expressing Hoxcl 3, overexpression was restricted to the normal functional domains of this gene as judged by comparison of mutant versus normal Hoxcl 3 expression patterns and the close correlation between the sites of overexpression and the sites where phenotypic changes occurred; this strategy should minimizes the risk of isolating presumptive targets that might be irrelevant for the normal function of this gene. Third, this screening methodology is aimed at identifying targets that are up-, as well as downregulated (repressed) upon overexpression of Hoxcl 3. The latter group is highly relevant as it comprises by far most of the downstream genes identified.

Clustering of Realisator Genes The KAP genes form by far the overall largest group of presumptive Hoxc 13 targets identified in this screen, i.e. 14 out of a total of 29 genes, including 9 that have been classified as novel KAPs. All of these KAPs are downregulated ranging from approximately 2.5-fold (Pmg-1) to more than 20-fold for the novel Krtap 16-9 compared to normal levels (Figures 5A and 5B). KAP gene products presumably are involved in alignment and lateral crosslinking of keratin intermediate filaments (IFs), thus playing an important role in epidermal and follicular keratinocyte differentiation (review: Powell and Rogers, 1997; Fuchs, 1995). Structural similarities allow one to distinguish several different groups of KAPs whose distinct expression patterns reflect the functional diversity among keratinocytes. While certain structurally unique epidermal KAPs, such as fillagrin and loricrin, are encoded by spliced transcripts (e.g. Markova et al., 1993; Rothnagel et al., 1994), all follicular KAPs identified to date appear to be encoded by small intron-less genes (Powell and Rogers, 1997). Based on their amino acid composition, the latter group has been classified into high sulfur proteins containing up to 40% cysteine and high-glycine/tyrosine (HGT) proteins (Powell and Rogers, 1991; Powell and Rogers, 1997). Both classes include several subfamilies that have uniformly been designated keratin-associated proteins (Krtaps) followed by a number specifying the family and its individual member according to HUGO keratin gene nomenclature (http;//www.gene. ucl.ac.uk/users/hester/krt.html). Following this nomenclature system, the 9 novel KAP genes of the HGTp-class identified by the present invention are classified individually as Krtap 16-1 through Krtap 16-9, because they form a 16th group of structurally distinct genes located in a novel cluster on mouse chromosome 16 (Figures 6A and 6B). The map position of this new cluster is between the two distal markers Tiaml and Sodl, a region that is syntenic to the human chromosome 21q22.11 region. The conserved genomic organization and clustering of KAP genes may reflect a constraining force of relevance for coordinate expression. This view is consistent with the sequential activation of keratin and KAP genes in distinct subpopulations of keratinocytes during follicle differentiation (Powell and Rogers, 1997). Accordingly, the first class of genes being expressed is the group of IF keratin genes, followed by the HGTp class and later by high sulfur protein genes.

It has been proposed that Hox genes coordinate the activities of thousands of realisator genes in a concerted fashion because independent control of the realisators would result in chaotic development (Gehring, 1987). One would predict that during evolution this logistical demand has been coped with by clustering of functionally similar realisator genes. The present invention provides a realisator gene cluster controlled by Hoxc 13 that supports this concept and provides an experimental system for studying the mechanisms of its regulation.

Relevance of Hoxc 13 Target Genes Identified in GC13 Mutant Mouse Skin for Human Pathological Conditions

Among the genes found to be overexpressed in GC13 mice is the keratin intermediate filament protein-encoding gene Krt2-1, previously known as Kl, which has been linked to epidermolytic hyperkeratosis in humans (Chipev et al. 1992, Cell 70, 821; Rothnagel et al. 1992, Science 257, 1128). This condition has been modeled in transgenic mice overexpressing mutated Krt2-1 (Bickenbach et al. 1996, Differentiation 61, 129) whose skin phenotype shows striking similarities to the one exhibited by our GC13 mice.

Noteworthy among the genes found to be upregulated in GC13 mutant mice is β-isoform 14-3-3 protein-encoding gene, because elevated levels of this gene's product have been found in the cerebrospinal fluid of patients with Creutzfeldt- Jacob disease (Wiltfang et al. 1999, J. Neurochem. 73, 2485). Accordingly, 14-3-3 is being used as a diagnostic marker for this neurodegenerative disease. In skin, 14-3-3 proteins are known to play a role in keratinocyte proliferation and differentiation and interact with K8 and K18 keratins in a cell-cycle and phosphorylation-dependent manner (Liao and Omary, 1996, J. Cell. Biol. 133, 345).

Among the genes downregulated in GC13 mutant skin certain previously known, as well as novel keratin-associated protein (KAP) genes, constitute the largest group. Most prevalent within this group are members of the subfamily of high glycine tyrosine (HGT) protein genes. Expression of HGT proteins has been found to be markedly reduced in the mouse mutation naked (Raphael et al. 1982, Genet. Res. Camb. 39, 139; Raphael et al. 1984, Genet. Res. Camb. 44, 29) that exhibits brittle hair with cuticular defects resulting in alopecia (Sundberg 1994, in Handbook of mouse mutations with skin and hair abnormalities, Sundberg, J.P. ed., CRC Press, Boca Raton, Ann Arbor, London, Tokyo, pp. 371-377) as seen in our GC13 mice. Interestingly, naked is genomically closely linked to the embryonic lethal hair mutation velevet coat on mouse chromosome 15 (ibid), which itself has been found to be in close linkage to the the Hoxc gene cluster (Hart et al. 1992, J. Exp. Zool. 263, 83). The close genomic linkage between naked and Hoxc 13 and the pleitropic effects of naked, which are indicative of a regulatory mutation, may suggest involvement ot Hoxc 13 in the naked phenotype equivalent to the human genetic disease hydrotic ectodermal dysplasia (Sundberg 1994, as referenced above). Hydrotic ectodermal dysplasia patients suffer from fragile hair, total alopecia, mental deficiency, hyperkeratosis and hypeφigmentation; the latter is consistent with the pigmentation disorder seen in our Hoxc 13 overexpressing mice, which we believe is mediated by the downregulation of agouti-related protein (Agφ).

The close linkage of the keratin type II gene cluster to Hoxc 13 and the fact that we find one of its members (Krt2-1) to be differentially expressed in GC13 mutant skin supports the speculation that individual members of this gene cluster or the entire cluster might be regulated by Hoxc 13 and the neighboring Hoxc genes (Hart et al. 1992, J. Exp. Zool. 263, 83). These keratin genes have been implicated in a wide spectrum of cutaneous diseases including ichthyosis, epidermolytic bullosa simplex, epidermolytic hyperkeratosis, plamoplantar keratoderma, epidermal nevi, pachyonychia congenita, and skin cancer (Fuchs, 1995, Ann. Rev. Cell Dev. Biol. 11, 123).

GC13 Mice Exhibit Reduction of Adipose Tissue

GC13 mice frequently (>50%) exhibit a smaller size and stature at birth suggesting an effect of Hoxc 13 on metabolic pathways controlling body growth and weight. The present invention provides data showing dramatically reduced adipose tissue in adult GC13/FVB x C57BL6 FI hybrid mice (see Figure 9). A link between Hoxc 13 overexpression and this reduction in adipose tissue is the finding shown above of a 13-fold downregulation of agouti-related protein (Agφ) gene in skin of 5 d GC13 mice. Like its close structural relative, Agouti protein, Agφ is a paracrine signaling molecule that has been implicated in the control of body weight as a hypothalamic Mc3r/Mc4r melanocortin receptor antagonist as evidenced by obesity in transgenic mice ubiquitously overexpressing Agrp (Ollmann et al., 1997, Science 278, 135).

Recent findings of expression in numerous tissues in chicken, including skin, suggests a role for Agφ in regulating peripheral melanocortin systems as well (Takeuchi et al., 2000, Biochim. Biophys. Acta 1496, 261), a finding consistent with data revealing Agφ as a Mclr agonist (Haskell-Luevano et al. 2000, Peptides 5, 683).

While the reduced adipose tissue in GC13 mice might be linked to the downregulation of Agφ protein in the periphery, i.e. the skin, Hoxcl3 may be expressed in the hypothalamus and thus have a more direct effect on feeding behaviour through Hoxc 13 regulated Agφ via the central Mc3r/Mc4r system. Likewise, it is possible that the dramatic downregulation of peripheral Agφ (13-fold) in GC13 skin has an impact on the central system. Finally, a currently unknown effect of peripheral Agφ on fat metabolism through additional postulated melanocortin receptors (Bicknell et al. 2000, J. Neuroendocrinol. 10, 977) cannot be excluded. These results strongly suggest that Hoxc 13 expression levels negatively correlate with the amount of adipose tissue, involving Agφ, a signaling molecule implicated in the normal control of body weight. Accordingly, GC13 mice and GC13/FVB x C57BL6 FI hybrids are a useful model for dissecting regulatory pathways controlling body weight that involve Hoxcl 3. This model allows the examination of modulation of the central melanocortin receptor system and thus food intake and/or energy expenditure by Hoxc 13 and Agφ. This approach should facilitate the identification of compounds potentially useful for controlling obesity.

Potential Role for Hoxcl 3 in Melanocyte Differentiation and Melanoma

The GC13 mice -provide a unique, genetically defined animal model to study potential roles of Hoxc 13 in melanocyte differentiation and melanoma. The present invention shows that the overexpression of Hoxcl 3 in these mice in differentiating keratinocytes of hair follicles during the progressive growth results in severe hair and skin abnormalities including alopecia and epidermal hypeφlasia. We provide evidence that the hair phenotype is linked to the downregulation of novel, as well as previously known keratin-related genes, whereas the hypeφlasia is based on hypeφroliferation of follicular and epidermal keratinocytes. While these studies have been performed in an inbred FVB albino background, results obtained by crossing these mice with pigmented C57BL6 mice revealed a severe pigmentation disorder in the FI offspring (Figure 10). Therefore, the Hoxcl 3 is expressed in melanocytes in addition to follicular keratinocytes. A potential role for Hoxcl 3 in regulating pigmentation and melanocyte differentiation is indicated by the 13-fold downregulation of the agouti-related protein (Agrp) gene as one of the Hoxc 13 target genes identified in 5 d postnatal skin of GC13. This is independently being supported by data showing differential expression of

HOXCl 3, as well as some of its downstream neighbors in tumor clones derived from a single human melanoma metastasis (Cillo et al., 1996, Int. J. Cancer 66, 692). Interestingly, and in keeping with our data suggesting primarily a repression of target genes upon Hoxcl 3 overexpression in hair follicles of mice, expression of HOXC 13 together with HOXCl 1 and HOXC 10 in the melanoma cells was shown to be associated with downregulation of VLA-2, VLA-5 and VLA-6 integrins and ICAM-1. Combined, these data suggest a role for Hoxcl 3 in melanocyte differentiation and melanoma development.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties, as well as the references cited in these publications, are hereby incoφorated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

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Claims

What is claimed is:

1. An isolated nucleic acid comprising the nucleic acid of GC 13.

2. An isolated nucleic acid comprising the nucleic acid set forth in the Sequence

Listing as SEQ ID NO: 1.

3. An isolated nucleic acid comprising the nucleic acid set forth in the Sequence

Listing as SEQ ID NO: 2.

4. An isolated nucleic acid comprising the nucleic acid set forth in the Sequence

Listing as SEQ ID NO: 3.

5. An isolated nucleic acid comprising the nucleic acid set forth in the Sequence

Listing as SEQ ID NO: 4.

6. An isolated nucleic acid comprising the nucleic acid set forth in the Sequence

Listing as SEQ ID NO: 5.

7. An isolated nucleic acid comprising the nucleic acid set forth in the Sequence

Listing as SEQ ID NO: 7.

8. The isolated nucleic acid of claim 1, 2, 3, 4, 5, 6 or 7 in a vector suitable for

expressing the nucleic acid.

9. The vector of claim 8 in a host suitable for expressing the nucleic acid.

10. A purified polypeptide encoded by the nucleic acid of claim 1, 2, 3, 4, 5, 6, or 7.

11. A purified polypeptide set forth in the Sequence Listing as SEQ ID NO: 6

12. An isolated nucleic acid encoding the polypeptide of claim 10 or 11.

13. A purified antibody which specifically binds to the polypeptide of claim 10 or 11.

14. A transgenic non-human animal whose cells overexpress a transgene coding for

Hoxc 13 wherein the total native and transgenic Hoxc 13 expressed in the

transgenic animal is higher than the Hoxc 13 expressed in a non-transgenic

animal, which transgenic animal has increased hair loss, hyperkeratotic skin and

decreased adipose tissue due to the overexpression of the transgene.

15. The transgenic animal of claim 14, wherein the animal has decreased adipose

tissue.

16. A transgenic non-human animal whose cells overexpress a transgene coding for

Hoxc 13 wherein the total native and transgenic Hoxc 13 expressed in the

transgenic animal is higher than the Hoxc 13 expressed in a non-transgenic

animal, which transgenic animal has increased hair loss, hyperkeratotic skin,

decreased adipose tissue and abnormal pigmentation due to the overexpression of

the transgene.

17. The transgenic animal of claim 14, wherein the animal is a mouse and the

transgene comprises GC13.

18. A method for screening compounds for an effect on hair loss comprising

administering the compound to the transgenic animal of claim 14 and monitoring

the animal for a change in hair growth, wherein altered hair growth is indicative

of the effect of the compound on hair growth.

19. A method for screening compounds for an effect on a hyperkeratotic skin

disorder by administering the compound to the transgenic animal of claim 14 and

monitoring the animal for a change in the skin disorder, wherein a change in the

hyperkeratotic skin disorder is indicative of the effect of the compound on a

hyperkeratotic skin disorder.

20. A method for screening compounds for an effect on adipose tissue deposition by

administering the compound to the transgenic animal of claim 14 and monitoring

the animal for a change in adipose tissue, wherein a change in the adipose tissue

of the animal is indicative of the effect of the compound on adipose tissue.

21. A method for screening compounds for an effect on pigmentation by

administering the compound to the transgenic animal of claim 16 and

monitoring the animal for a change in pigmentation, wherein a change in

pigmentation is indicative of the effect of the compound on pigmentation.

21. An expression construct for conferring follicle specific expression which

comprises a regulatory sequence of Hoxc 13 contained within GC13.

22. An expression construct for conferring keratinocyte specific expression which

comprises a regulatory sequence of Hoxc 13 contained within GC13.

23. The expression construct of 18 or 19 further comprising a promoter operably

linked to the regulatory sequence.

24. The expression construct of claim 18 or 19 further comprising a coding sequence

for a heterologous gene operably linked to the promoter.

5. A method of identifying genes associated with the pathological changes observed

in the skin of GC13 mice comprising:

a) isolating a gene differentially expressed in skin of GC13 mice by

suppressive subtractive hybridization;

b) obtaining a probe or immunohistochemical reagent for the

differentially expressed gene of step (a);

c) utilizing the probe to analyze the expression pattern of the differentially

expressed gene in GC13 wherein altered expression of the gene as

compared to a normal mouse indicates the gene is associated with the

overexpression of Hoxcl 3.