WO2001085964A2 - Over expression of the whn protein - Google Patents

Abstract

The invention relates to a non-human mammal which over expresses the Whn gene wherein the over expression results in stimulation of hair follicle development.

Description

Over Expression of the Whn Protein

The invention relates to a non-human mammal characterised in that the murine winged helix nude gene (Whn or Hfh-11) of said mammal over expresses the Whn protein.

Alopecias are related disorders which result in varying degrees of hair loss. By far the most common of the alopecias is androgenetic alopecia (male pattern baldness) which affects approximately 80% of the male population and results from the inheritance of a dominant allele.

Another form of alopecia is alopecia areata which is thought to be an auto-immune disease resulting in patchy hair loss on the scalp which can progress, in severe forms of the disease, to a complete loss of scalp hair. If the disease progresses to this stage it is referred to alopecia totalis. Alopecia universalis is the most extreme alopecia resulting in total loss of scalp and body hair. Alopecia universalis is also known as alopecia atrichia.

Hair loss can also result from various therapeutic treatments which result in loss of precursor cells which differentiate into the various cells which comprise the hair follicle. These include the use of powerful chemotherapeutic drugs which are used in the treatment of cancer.

The mammalian hair follicle is a self-renewing appendage that undergoes regular cycles of growth, differentiation, regression and quiescence (Hardy, 1992). The specification of hair follicles occurs during embryogenesis and this process is driven by interactions between the follicular epithelium and the underlying dermal mesenchymal fibroblasts. The regenerative potential of adult follicles resides in a population of epithelial stem cells whose proliferation and commitment is also thought to be regulated by interactions with, adjacent dermal fibroblasts (Cotsarelis et al, 1990; Rochat et al, 1994). A number of signalling pathways have been implicated in mediating various steps in the induction and morphogenesis of hair follicles (Bitgood and McMahon, 1995; Carroll et al., 1995; Guo et al, 1993; Hebert et al., 1994; Jones et α ., 1991; Kere et al, 1996; Luettke et al, 1993; Lyons et al, 1990; Mann et al, 1993; Powell et al, 1998; St- Jacques et al, 1998; Wang and Shackleford, 1996). Moreover, several transcription factors have been implicated in control of hair follicle development (Gat et al, 1998; van Genderen et al, 1994; Nehls et al, 1995; Cachon-Gonzalez et al, 1994; Yang et al, 1999). Despite this progress, still relatively little is known about the molecular pathways that control and coordinate hair follicle induction, morphogenesis and cycling.

In humans, alopecia universalis is associated with a mutation in the human hairless gene, see Ahmad et al Science (1998), 279, 720-724; Frank et al Nature (1999), 398, 473-474; and PCT/US99/02128. The mouse and rat homologues of hairless have been cloned and are highly homologous to the human protein (human and mouse sequences are 80% identical and the human and rat sequences are 78% identical). The human, mouse and rat proteins have a single zinc finger domain and 6 conserved cysteine motifs and is therefore thought to be a transcription factor, see Thompson (1996) J. Neuroscience, 16, p7832.

A key gene required for hair follicle development in mice is the Whn gene, mutations in which cause the nude mutant phenotype (Flanagan, 1966; Pantelouris, 1968). Nude mice lack a coat, yet they are not hairless (Kopf-Maier et al, 1990). Hair follicles are specified in nude mutants and their development is initiated correctly, but morphological abnormalities of the hair shaft become apparent within 6 days of birth (Flanagan, 1966). Nascent, wild-type hair follicles typically grow downwards into the adipose layer of the dermis during the anagen phase of the growth cycle, and the hairs within these follicles grow upwards from the hair bulb , eventually penetrating the epidermis by 9 days of post-natal life (Paus et al, 1999). However in nude animals, although each hair follicle begins downward growth normally during embryogenesis, its inner root sheath becomes discontinuous, the hair cuticle fails to develop, and keratinization of the hair itself is impaired (Flanagan, 1996; Kopf-Maier et α/.,..1990).* The resulting hairs in nude follicles are thinner and twisted with much reduced -cortex, and they coil up within the follicles or break up into fragments. Nevertheless, cycling of nude mutant hair follicles initially appears to be unimpaired (Eaton, 1976). Thus, loss of murine Whn function impairs the ability of hair follicles to support normal hair growth and differentiation, but their ability to cycle is retained. However there is a progressive loss of the ability of hair follicles to cycle and properly self renew with time, since adult nude mice possess very few hair follicles (Sundberg, 1994).

The Whn protein is a member of the forkhead family of transcription factors and possesses both a winged-helix DNA-binding domain and a potent transcription activating domain (Nehls et al, 1995; Schuddekopf et al, 1996). Many winged helix-containing transcription factors have been described that are required for the initiation of a variety of cell fate decisions during embryogenesis and organogenesis (Kaufmann and Knochel, 1996). Whn is expressed in proliferating and post-mitotic descendants of the hair bulb matrix cells that give rise to the precursors of the hair cuticle and cortex, in addition to the outer root sheath (ORS) and inner root sheath (IRS) of the hair follicle (Lee et al, 1999). Together with the mutant phenotype, this expression pattern implies that Whn functions at an early stage in the realization of multiple cell fates required for normal hair follicle morphogenesis and differentiation.

The hair follicle phenotype of the murine nude mutation was rescued in genetically modified mice generated from fertilized nu/nu homozygous eggs with a cosmid encompassing the murine Whn gene plus 8.5kb of 5'-flanking and 4kb of 3'-flanking DNA (Kurooka et al, 1996). However gene rescue of the mutation was incomplete, since hair density in rescued animals was lower than that of wild-type animals.

We have increased the level of Whn gene expression within its normal expression domain in the hair follicle, by generating genetically modified mice carrying extra copies of the wild-type Whn gene with all the necessary regulatory elements to determine efficient, tissue-specific expression of the gene. Large BAC and PAC-derived genomic clones were used which comprise the Whn gene together with all of the regulatory elements required to specify faithful Whn expression. We have generated several founder mice carrying these BAC and PAC clones and observed that founder mice and genetically modified progeny exhibited enhanced growth and differentiation of the hair follicles and the hairs within them. These results suggest that the function of Whn is to specify many of the differentiated cell types of the mammalian hair follicle from a pool of proliferating, multipotent progenitor cells. This implies that over expression of the Whn gene causes over expression of genes involved in the growth and differentiation of the hair follicle, some of which may only be weakly expressed in wild-type skin and consequently be difficult to isolate from cDNA libraries made from wild-type skin mRNA. It would therefore be desirable to provide the means by which genes involved in hair follicle growth and differentiation are more readily identifiable. An example of a method used successfully to identify differentially regulated cDNA's is representational difference analysis (RDA). This technique enables^' the detection of nucleic acid sequences present in one population but absent in* another wherein the mRNA between populations are basically similar. These populations are called "tester" and "driver" DNA. Target tester sequences are separated from unwanted tester sequences by providing driver DNA in excess over the tester DNA so that most sequences common to both tester and driver form tester: driver duplexes.

In this technique (adapted from Hubank and Schatz, Nucl. Acids Res., vol 22, pp5640- 5648, 1994), cDNA from Whn-genetically modified skin is designated tester cDNA, whereas cDNA from nude mutant skin is designated driver cDNA. The objective is to isolate specific cDNA fragments present in the. tester cDNA population that are absent or substantially reduced in abundance in the driver cDNA population.

In its broadest aspect the invention relates to a non-human mammal which over expresses the Whn gene which results in the stimulation of hair follicle development.

According to an aspect of the invention there is provided a non-human mammal characterised in that the genome of said mammal is genetically modified to provide at least one Whn gene which is over expressed when compared to a non-genetically modified non-human mammal.

In a preferred embodiment of the invention the genome of said non-human mammal includes additional copies of a Whn gene. Preferably said mammal includes at least 2, 3, 4, 5, 10, 15, 20, 25, 30, additional copies of the Whn gene.

We have over-expressed the Whn gene in skin by generating genetically modified mice with contructs that encompass the wild-type Whn locus.

For clarity, the following definitions will be adhered to in the subsequent description. Reference to Whn is to the endogenous, naturally occuring, murine Whn gene. Reference to Whn+ is to an isolated nucleic acid molecule encompassing the wild- type murine Whn gene comprising sufficient cw-acting regulatory elements to direct faithful transcription of Vhn+ in the hair follicles used to generate genetically modified mice over expressing Whn. Reference to Whn is to the protein product of either Whn or Whn+.

Genetically modified Whn+ mice exhibited a distinctive coat phenotype that was caused by an enhancement in the growth and differentiation of hair follicles. Hair follicles of Whn+ mice were longer and grew more deeply into the dermis, and their spatial orientation, direction of growth and distribution were less regular, than follicles of wild-type, non-genetically modified mice. Hair growth rates were also enhanced in Whn+ animals. However the patterns of hair follicle cell proliferation and differentiation within Whn+ follicles were relatively normal. It will be apparent to one skilled in the art that over expression of Whn can be effected either by providing multiple copies of the gene or by enhancing the activity of the endogenous Whn or Whn+ promoter or by providing a Whn+ gene with a highly active heterologous promoter that functions in the correct cell type. Promoter is an art recognised term which is defined in more detail below.

In yet a further preferred embodiment of the invention said non-human mammal shows enhanced expression of a Whn or Whn+ gene.

In a further preferred embodiment of the invention said non-human mammal shows enhanced hair follicle growth through overexpression of a Whn or Whn+ gene.

In yet still a further preferred embodiment of the invention said non-human mammal shows enhanced hair follicle cell differentiation through over expression of a Whn or Whn+ gene.

In a yet further preferred embodiment of the invention.* said non-human mammal is a rodent, ideally a mouse. In a further preferred embodiment of the invention said non-human mammal is selected from: non-human primate; pig; sheep; cat; dog; goat; cow; camel; horse; llama; alpaca.

In yet a further preferred embodiment of the invention the expression of the Whn + gene is controlled by its cognate promoter.

Promoters are transcription control sequences (promoter sequences) which may mediate cell/tissue specific expression of physically linked genes. Promoter sequences may be cell/tissue specific, inducible or constitutive.

Promoter is an art recognised term and, for the sake, of clarity, includes the following features which are provided by example only, and not by way of limitation. Enhancer elements are cis acting nucleic acid sequences that may be located either upstream or downstream of a gene. Enhancers function to increase the rate of transcription of the gene to which the enhancer is linked. Enhancer activity is responsive to trans acting transcription factors (polypeptides) which have been shown to bind specifically to enhancer elements. The binding/activity of transcription factors (please see Eukaryotic Transcription Factors, by David S Latchman, Academic Press Ltd, San Diego) is responsive to a number of environmental cues which include, by example and not by way of limitation, intermediary metabolites (eg glucose, lipids), environmental effectors (eg light, heat,), and polypeptide growth factors.

Promoter elements also include so called TATA box and RNA polymerase initiation selection (RIS) sequences which function to select a site of transcription initiation. These sequences also bind polypeptides which function, inter alia, to facilitate transcription initiation selection by RNA polymerase.

Expression control sequences also include so-called Locus Control Regions (LCRs). These are regulatory elements which confer position-independent, copy number- dependent expression to linked genes when assayed as transgenic constructs in mice. LCRs include regulatory elements that insulate transgenes from the silencing effects of adjacent heterochromatin, Grosveld et al, Cell (1987), 51: 975-985.

In alternative embodiment of the invention said Whn+ gene is over expressed.

In yet a still further preferred embodiment of the invention the over expression of said Whn+ gene is by a promoter which shows an expression pattern predominantly restricted to those cells in which the Whn gene is expressed. These include, by example and not by way of limitation, proliferating cells the hair bulb matrix and outer root sheath, post-mitotic suprabulbar progenitor cells of the inner root sheath, hair cuticle, hair cortex and hair medulla.

According to a further aspect of the invention there is provided a vector, ideally a vector adapted for expression in a eukaryotic cell, characterised in that said vector includes an open reading frame which encodes the Whn protein and is operably linked to a promoter adapted for over expression of the nucleic acid encoding said Whn protein.

Adaptations include the provision of selectable markers and autonomous replication sequences which both facilitate the maintenance of said vector in either the eukaryotic cell or prokaryotic host. Vectors which are maintained autonomously are referred to as episomal vectors. Episomal vectors are desirable since these molecules can incorporate large DNA fragments (30-200kb DNA). Episomal vectors of this type are described in WO98/07876.

Adaptations which facilitate the expression of vector encoded genes include the provision of transcription termination/polyadenylation sequences. This also includes the provision of internal ribosome entry sites (IRES) which function to maximise expression of vector encoded genes arranged in bicistronic or multi-cistronic expression cassettes. These adaptations are well known in the art, There is a significant amount of published literature with respect to expression vector construction and recombinant DNA techniques in general. Please see, Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, NY and references therein; Marston, F (1987) DNA Cloning Techniques: A Practical Approach Vol III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

According to a further aspect of the invention there is provided an isolated nucleic acid molecule which comprises the Whn+ gene including expression control sequences sufficient to direct over expression of the said gene.

In a preferred embodiment of the invention said isolated nucleic acid molecule includes greater than 8.5 kb of the 5' region of DNA upstream of the initiation codon of the Whn+ gene. Ideally said isolated nucleic acid molecule includes between 8.5 kb and 74 kb of 5' region upstream of the initiation codon of the Whn+ gene.

In a further preferred embodiment of the invention said isolated nucleic acid molecule includes at least 4 kb of the 3' region downstream of the termination codon of the Whn+ gene. Ideally said isolated nucleic acid molecule includes between 4 kb and 21 kb of the 3' region downstream of the termination codon of the Whn+ gene.

More preferably still said vector is a bacterial artificial chromosome (BAC) or PI artificial chromosome (PAC).

PAC and BAC vectors are able to accommodate much larger fragments of genomic DNA than cosmids thereby enabling the inclusion of all the necessary upstream and downstream regulatory elements required for over expression of the Whn+ gene.

According to a further aspect of the invention there is provided a method to isolate/identify genes involved in the growth ahd/όr differentiation of hair follicles using the non-human genetically modified mammal according to any previous aspect or embodiment of the invention.

According to a yet further aspect of the invention there is provided a method to identify agents capable of enhancing the expression of a Whn gene comprising:

i) providing a cell expressing a Whn gene; ii) contacting said cell with at least one agent to be tested; and iii) monitoring the transcriptional activity a Whn gene or Whn gene promoter.

According to a yet further aspect of the invention there is provided a method to identify agents capable of enhancing the expression of a Whn+ gene comprising:

i) ^' providing a cell transfected or transformed with a Whn+ gene, or a Whn + gene promoter; ii) contacting said cell with least one agent to be tested; and iii) monitoring the transcriptional activity of said Whn+ gene or Whn+ gene promoter.

It will be apparent to one skilled in the art that means to monitor agents capable of enhancing Whn or Whn+ expression are readily available. This can been done using immunological methods (eg western blot, ELISA) to detect amounts of Whn protein; nucleic acid based methods (eg hybridisation, polymerase chain reaction); or the use of reporter proteins (eg luciferase, green fluorescent protein, β-galactosidase) wherein the expression of nucleic acid encoding said reporter protein is controlled by the Whn promoter.

According to a yet further aspect of the invention there is provided a cell or cell line transfected/transformed with the Whn+ gene according to the invention wherein said cell/cell-line over expresses the Whn protein. In a preferred embodiment of the invention said cell or cell line is stably transfected/transformed with the Whn+ gene according to the invention.

According to a further aspect of the invention there is provided a composition comprising the nucleic acid encoding the Whn protein. Preferably said nucleic acid is the Whn+ gene according to the invention. More preferably still said nucleic acid is for use in the manufacture of a medicament for use in the treatment or prevention of hair loss.

In a preferred embodiment of the invention said nucleic acid is included in a vector suitable for gene therapy.

In yet a further preferred embodiment of the invention said vector is a viral based vector. Ideally said viral vector is selected from the following: adenovirus; retrovirus; adeno- associated virus; herpesvirus; lentivirus; baculovirus.

Viral based vectors according to the invention may also include hybrid viral vectors which include advantageous features of selected viruses which facilitate, for example and not by way of limitation, viral infectivity, replication or expression of genes carried by said hybrid vector.

According to a further aspect of the invention there is provided a composition comprising the Whn protein, or the effective part thereof. Preferably said Whn protein, or the effective part thereof, is for use in the manufacture of a medicament for use in the treatment of hair loss.

In a preferred embodiment of the invention said composition/medicament comprises a diluent, excipient or carrier.

The use of cationic lipids (eg liposomes, Feigner (1987) Proc.Natl.Acad.Sci USA, 84:p7413) has become a common method to introduce DNA into cells. The cationic head of the lipid associates with the negatively charged nucleic acid backbone of the DNA to be introduced. The lipid/DNA complex associates with the cell membrane and fuses with the cell to introduce the associated DNA into the cell. Liposome mediated DNA transfer has several advantages over existing methods. For example, cells which are recalcitrant to traditional chemical methods are more easily transfected using liposome mediated transfer.

Alternatively, peptide based transfection agents are available, see WO96/41606.

When Whn protein is used in therapeutic compositions liposomes can also be used to deliver said composition to cells. Liposomes are lipid based vesicles which encapsulate a selected therapeutic agent which is then introduced into a patient. The liposome is typically manufactured either from pure phospholipid or a mixture of phospholipid and phosphoglyceride. Typically liposomes can be manufactured with diameters of less than 200nm, this enables them to be intravenously injected and able to pass through the pulmonary capillary bed. Furthermore the biochemical nature of liposomes confers permeability across blood vessel membranes to gain access to selected tissues. Liposomes do have a relatively short half-life. So called STEALTH^R liposomes have been developed which comprise liposomes coated in polyethylene glycol (PEG). The PEG treated liposomes have a significantly increased

■ half-life when administered intravenously to a patient. In addition STEALTH^R liposomes show reduced, uptake in the reticuloendothelial system and enhanced accumulation selected tissues. In addition, so called immuno-liposomes have been develop which combine lipid based vesicles with an antibody or antibodies, to increase the specificity of the delivery of the DNA vector to a selected cell/tissue.

The use of liposomes as delivery means is described in US 5580575 and US 5542935.

It will be apparent to one skilled in the art that the therapeutic compositions/medicaments can be formulated in a variety of ways to facilitate delivery. For example, liposome compositions may be usefully employed to deliver said compositions/medicaments.

It will be apparent to one skilled in the art that the compositions can be provided in the form of an oral or nasal spray, an aerosol, suspension, emulsion, and/or eye drop fluid. Alternatively compositions may be provided in tablet form. Alternative delivery means include inhalers or nebulisers.

Alternatively or preferably the composition/medicament can be delivered by direct injection. It is also envisioned that the compositions be delivered intravenously, intramuscularly, subcutaneously or topically. Further still, the medicament may be taken orally or rectally .

It will also be apparent that compositions/medicaments are effective at preventing and/or alleviating hair loss conditions in animals other than humans, for example and not by way of limitation, family pets, livestock, horses.

According to a yet further aspect of the invention there is provided a method to treat a mammal which would benefit from the administration of the Whn+ gene or Whn protein. Preferably said method is a cosmetic treatment.

Preferably said mammal is human.

More preferably still said mammal is treated for a condition selected from: androgenic alopecia; alopecia areata; alopecia totalis; congenital alopecia universalis; congenital alopecia; hair loss as a result of chemotherapy.

An embodiment of the invention will now be described, by example only and with reference to the following figures:

Figure 1 shows the production and identification of Whn + mice (a) PAC and BAC clones used for production of Whn+ mice.

The PAC clone 436p24 and BAC clone 82G8 were obtained by screening the mouse 129/Sv genomic libraries RPCI-21 and RPCL23,, respectively, with a PCR fragment spanning exon 3 of the mouse Whn gene. Clones, were restriction mapped, typed for STS content by PCR and the intact nature of the Whri+ gene was confirmed by further restriction mapping prior to their use in genetic modification. For genetic modification, a 1 lOkb Not I DNA fragment spanning the Whn locus was isolated from PAC 436p24 by Pulsed Field Gel Electrophoresis, whereas the intact circular BAC clone 82G8, spanning over 180kb encompassing the Whn gene was used for microinjection without linearization.

(b) Identification of Whn+ animals. PCR-based identification of genetically modified animals was carried out by testing for the presence in mouse genomic DNA of sequences at the 5'-end of the HOkb, PAC-derived PJ^n-containing gene. An approximately 270 bp fragment that was diagnostic of the gene was amplified by PCR using primers specific for the end of the PAC to the right of DllSeg29. PAC-derived animals were distinguished by the appearance of an approximately 270 bp fragment. BAC-derived animals were identified by testing for the presence in mouse genomic DNA of a 650bp DNA fragment of the BAC vector gene encoding chloramphenicol resistance by PCR. Lanesl-11: PCR amplification of mouse genomic DNA samples to detect sequences from the 1 lOkb Not I fragment of 436p24. 1 : VCl A founder male; 2, 3, 4, 5, 6, 7, 8, 9: progeny of cross between founder VCl A and CBA/C57B16J Fl hybrid female. (4 out of 8 progeny are J¥hn+ gene positive, demonstrating Mendelian inheritance of the Whn+ gene.) 10: purified 436p24 PAC DNA positive control; 11: CBA/C57B16J Fl hybrid genomic DNA negative control. Lanes 12-15: PCR amplification of mouse genomic DNA samples to detect sequences from the circular BAC clone 82G8. 12: VC3AA-4 founder female; 13: VC3AC-1 founder female; 14: purified 82G8 BAC DNA positive control; 15: CBA/C57B16J Fl hybrid genomic DNA negative control. M: lkb ladder DNA marker.

(c) RNAase protection analysis for. expression of . Whn+ and non-genetically modified animals. Animals were sacrificed at post-natal day*-31 when the hair follicles had entered their second anagen period. Total RNA was extracted from tissues and 3μg was assayed for expression of the Whn and γ-actin mRNAs by Ribonuclease protection assay (Sambrook et al, 1989). Whn mRNA was detected with an antisense probe complementary to exon 3 sequences, whereas γ-actin mRNA was detected with a probe complementary to the 3 '-untranslated region. RNA samples analysed were from the dorsal back-skin of Whn+ (VC1F1-6) and control (VC1F1-3) non-genetically modified progeny of the founder male VCl A, plus heart and kidney RNAs from a CBA/C57 ¥ control non-genetically modified animal;

Figure 2 represents the coat phenotype of Whn+ mice. Whn+ animals exhibited a thicker, more ruffled coat (a) in comparison to the normal, sleek coats of age- and sex-matched non-genetically modified animals (b). Ruffling of the coat was particularly prominent in the upper back and dorsal neck region of Whn+ animals (a).

Whole-mount preparations of non-genetically modified back skin (c, e) and Whn+ back skin (d, f) from age- and sex-matched littermates, taken during early catagen (day 15 of post-natal life), (c, d) longitudinal cross-sections of back skin from behind the neck region, with anterior to the left and dorsal up, showing the angling of the hair follicles and their increased growth in Whn+ skin. (e, f) transverse cross-sections of back skin from behind the neck region, showing hair follicles and hairs in cross-section;

Figure 3 represents longitudinal histological sections of non-genetically modified back skin (a, c) and Whn+ back skin (b, d) from age- and sex-matched littermates, taken during mid-anagen (a, b) and early catagen (c,- d), stained with haematoxylin-eosin. Note waved, highly elongated appearance of hair follicles in Whn+ skin which penetrate the dermis more deeply than those of non-genetically modified skin. The spatial organisation of follicles is also more disordered at anagen and catagen in Whn+ skin than in non- genetically modified skin. The arrow in (d) indicates a typical, highly elongated catagen follicle in Whn+ skin;

Figure 4 represents transverse histological sections of non-genetically modified back skin (a, c) andWhn+ back skin (b, d) from age- and sex-matched littermates, taken during mid-anagen (a, b) and early catagen (c, d), stained with haematoxylin-eosin. Generally, the skin of Whn+ mice was thicker that that of non-genetically modified mice, and hair follicles penetrated to a greater, but variable depth in Whn+ skin than in non-genetically modified skin;

Figure 5 represents higher power magnification of upper (a-d) and lower (e-h) regions of transverse back skin sections from non-genetically modified mice (a, c, e, g) and Whn+ back skin (b, d, f, h) at mid-anagen (a, b, e, f) and early catagen (c, d, g, h), stained with haematoxylin-eosin; Flattened, thickened hair shafts (arrows) can be seen in many Whn+ follicles as can larger diameter outer and inner root sheath epithelia (particularly in the upper regions), in contrast to the narrow hair shafts in non-genetically modified follicles; Figure 6 Hair growth rates are enhanced in Whn+ follicles. Individual hair follicles were dissected from fixed skin samples taken from the same region of dorsal neck upper back in age- and se -matched Whn+ and non-genetically modified mice at 31 days of age. Hair follicles were mounted in DPX and photographed on a compound microscope.

(a, b): Non-genetically modified hair follicles with pigmented hair at (a) low and (b) high power magnification.

(c, d): Whn+ hair follicles with pigmented hair at (c,d) low and (d) high power magnification. Note the hair bulbs are of similar size in hairs from both animals, but the Whn+ hair shaft is much longer and thicker than that from its non-genetically modified littermate.

(e, f): Hair re-growth at 21 days following shaving of 3 month-old anaesthetized (e) non- genetically modified and (f) Whn+ animals on the posterior flank;

Figure 7 is a comparison of cytokeratin expression in differentiated cell types of Whn + and non-genetically modified skin during the second post-natal anagen phase. Transverse sections of skin were immunostained with polyclonal antibody NCL-CKp that recognises epidermal cytokeratins. In normal skin, strong immunoreactivity is detected in basal cells of the epidermis, sebaceous glands, and outer root sheath of hair follicles ,(a, c). These sites were also strongly immunoreactive in Whn+ skin (b, d). Note the longer, more deeply penetrating, sometimes buckled hair follicles in Whn+ skin, containing hairs of broader diameter than those in non-genetically modified follicles. Higher power views (c, d) demonstrate that the cytokeratin-positive basal layer of cells in the epidermis is of normal thickness, demonstrating that there is no epidermal hyperplasia in Whn+ skin;

Figure 8 shows the identification of proliferating cells in the hair follicle matrix and Outer Root Sheath and quiescent cells of the dermal papilla. Transverse sections of (a) non-genetically modified and (b) ΪVhn+ skin taken at the second post-natal anagen phase were immunostained with polyclonal antibody NCL-Ki-

67p raised against the cell proliferation marker Ki-67. Similar patterns of strong immunoreactivity are evident in cells of the matrix region and outer root sheath in both Whn+ and non-genetically modified skin, demonstrating that although growth and morphogenesis of Whn+ follicles is greater that of control non-genetically modified follicles, the proliferating cells are located in the^' same regions within follicles.

Transverse sections of (c) non-genetically modified and (d) Whn+ skin taken at the second post-natal anagen phase were stained with NBT/BCIP for endogenous alkaline phosphatase activity that is specific for cells of the dermal papilla in each hair follicle.

Similar almond-shaped groups of alkaline phosphatase-positive dermal fibroblasts can be distinguished in Whn+ and non-genetically modified skin, surrounded by proliferating cells of the hair follicle matrix (a, b). Distributions of proliferating and quiescent cells in

Whn+ follicles are normal;

Figure 9 shows a Southern analysis of Whn genes in Whn+ transgenic and non- transgenic mice. DNA was cleaved with restriction enzymes Pstl (P), Hindlll (H),

EcoRI (E) and BamHI (B) and electrophoresed in a 1% agarose gel prior to Southern hybridisation with a probe derived from exon 3 of the wild-type Whn gene. Numbers on the left of the figure indicate the positions and sizes (in kb) of the λ/Hindlll DNA molecular weight markers;

Figure 10 shows that a Whn+ transgene rescues both the hair follicle and thymus defects in animals homozygous for the nude mutation at the endogenous Whn locus;

Figure 11 shows that the Foxnl protein is more abundantly expressed in Whn+ transgenic hair follicles than in non-transgenic hair follicles and is localized to the nuclei of differentiating hair cuticle and hair cortex precursors; and Table 1 shows WhnTg+; nu/+ males (derived from the VCl A founder animal) were crossed to WhnTg-; nu + females in order to determine whether the Whn+ transgene could rescue the effects of homozygosity for the nude mutation at the endogenous Whn locus. Progeny were scored for coat phenotype (wild-type or nude), genotyped by PCR to detect Whn+ transgene sequences and classified into the above four categories. If the Whn+ transgene rescued the coat phenotype due to homozygosity for the nude mutation at the endogenous Whn locus, then a maximum of 12.5% of progeny would be expected to be phenotypically nude and none of these nude individuals would be WhnTg+. If the Whn+ transgene does not rescue the nude coat phenotype, then a maximum of 25% _ of progeny would be expected to be phenotypically nude and half of these nude individuals would be WhnTg+. The results demonstrate that 92% of progeny were phenotypically wild-type and none of the 7 phenotypically nude progeny carried the Whn+ transgene, which indicates that the Whn+ transgene rescues the phenotypic effects of homozygosity for the nude mutation at the endogenous Whn locus.

Materials and Methods

Isolation of PAC and BAC clones encompassing the Whn gene, preparation of DNA or oocyte microinjection, and generation of Whn+ mice.

A DNA fragment comprising exon 3 of the Whn gene was amplified from C57B1/6J genomic DNA by the Polymerase Chain Reaction (PCR) using the following primers (PCR): 5*-GCATGCTAACTTCAGCTGCTCGTCGT-3' (exon 3 forward primer), 5'- GAATTTGGTTGTGTTCCTGGCTGGGGTAAG-3' (exon 3 reverse primer). This exon 3 probe was used to screen mouse genomic libraries constructed in the pPAC4 and pBACe3.6 vectors (libraries supplied by MRC Human Genome Mapping Project Resource Centre, Cambridge, UK and Department of Cancer Genetics, Roswell Park Cancer Institute, NY). Positive clones were isolated by alkaline lysis miniprep, typed for Sequence Tagged Site (STS) content by PCR, and restriction mapped using frequent cutter restriction enzymes such as BamHI and EcoRI. Rare cutter restriction maps were generated using Pulsed Field Gel Electrophoresis and Southern blotting. These maps were compared to the restriction maps of the genomic region and clones that were un- rearranged were used for further studies. PAC clone 436p24 was unrearranged and encompassed over 130kb between STS markers DllSegl4 and DllSeg29, which included the Whn gene. PAC and BAC DNA maxipreps were purified using Qiagen columns. A 1 lOkb Not I fragment from 436p24 that contained the entire Whn locus plus 74kb of 5' flanking sequence and 21kb of 3' flanking sequence, was isolated from 436p24 by preparative PFGE and purified using Gelase (Epicentre Technologies, Wisconsin). , The purified linear DNA fragment was resuspended to a concentration of l-2pg/nl in microinjection buffer (IQmM Tris pH 7.5, O.lmM EDTA) and used to generate genetically modified mice by pronuclear microinjection (Hogan et αl, 1994). This fragment also contained two genes that closely flanked the Whn gene, the Na⁺ - dicarboxylate cotransporter gene located l lkb upstream and the retinal 4 gene located 8.5 kb downstream of Whn (Higashide et al, 1996; Sekine et αl, 1997; Chen et αl, 1998). Our own RT-PCR analyses confirm that the Na⁺ -dicarboxylate cotransporter gene is specifically expressed only in the kidney, and the retinal 4 gene is specifically expressed only in the retina. Neither gene is expressed in either genetically modified or non-genetically modified skin and so both can.be discounted as contributing to the skin phenotype described herein.

In addition to the 1 lOkb Not I fragment of 436p24 , a BAC clone, 82G8, which spanned over 180kb between STS markers DllSegl5 and DllSeg36 was used directly for transgenesis as closed circular DNA, after purification on Qiagen columns, at a concentration of 1-2 pg/nl in microinjection buffer. Identification and breeding of Whn+ mice

Genomic DNA was prepared from tail biopsy samples from pups at 3 weeks of age and analyzed for the presence of Whn + gene sequences. The end of the 1 lOkb Not I fragment of PAC 436p24 that lies upstream of the Whn gene terminates in the SP6 promoter sequence derived from the PAC vector. Therefore genomic DNA sequence immediately adjacent to this SP6 sequence in 436p24 was determined and a primer designed against sequence approximately 270bp downstream of the SP6 primer. This primer was then used together with the SP6 primer to test for transgene sequences in genomic DNA samples by PCR amplification. The primer sequences were as follows: Forward (SP6) primer: 5'-TATTTAGGTGACACACTATAG-3'; . Reverse (Whn) primer: 5'- AATCTCATTCCGTTACGCAG-3'. To detect BAC transgenes, PCR amplification of a 638 bp fragment of the Chloramphenicol Resistance gene in the vector backbone was used. Forward Cm^R primer: 5'-ATCACTGGATATACCACCG -3'; Reverse Cm^R primer: 5'-CTGCCACTCATCGCAGTGT-3'. PCR-positive animals were then crossed to C57B1/6J, CBA, (C57B1/6J x CBA)Fi, or BALB/c animals to establish genetically modified lines. Increased copy number of the Whn gene in genetically modified DNA was confirmed by Southern blotting.

Histology, immunocytochemistry and alkaline phosphatase histochemistty Skin tissue was dissected from the dorsal neck / upper back region of appropriately aged animals and fixed in 4% formaldehyde in Phosphate Buffered Saline (PBS) overnight at 4°C. Samples were dehydrated through an ethanol series, cleared in xylene and embedded in paraffin wax. Histological sections of 8μm thickness were taken onto Vectabond-subbed slides, dehydrated and cleared, then stained with haematoxylin and eosin and mounted in DPX. Cell types were identified by reference to histological texts. For immunostaining wax-embedded skin sections, tissues were mounted on Vectabond- subbed slides, then cleared in xylene, dehydrated through an ethanol series and rinsed sequentially in distilled water followed by PBS. Endogenous peroxidase was quenched with 0.3% hydrogen peroxide for 30 minutes at room temperature, followed by rinsing in PBS. To prepare sections for immunostaining with the NCL-CKp polyclonal rabbit antiserum (to visualize cytokeratins; Novocastra Laboratories, UK), slides were treated with 0.1% trypsin in PBS for 15 minutes at 37°C. To prepare sections for immunostaining with NCL-Ki67p polyclonal rabbit antiserum (to visualize Ki-67 antigen; Novocastra Laboratories, UK), slides were boiled for 15 minutes in Citrate Buffer (Vector Laboratories, Burlinga e, CA), which was then cooled to below 60°C over a period of 20 minutes. After rinsing all slides in PBS, tissues were blocked with 3% heat-inactivated goat serum (HIGS) in PBS, for 30 minutes at room temperature in a humidified chamber. Slides were then blocked for non-specific avidin-biotin interactions with the Vector Laboratories Blocking Kit (Vector Laboratories, Burlingame, CA). The first incubation of 15 minutes with Avidin D solution was followed by a PBS rinse, then 15 minutes with biotin solution. Slides were then drained, and primary rabbit antiserum was added after dilution in 3% HIGS/PBS (for NCL-CKp, 1 : 100; for NCLKi67p, 1 :200) and incubated overnight at 4°C in a humidified chamber. Slides were then washed for 10 minutes in PBS, prior to secondary antibody incubation (biotinylated goat anti-rabbit IgG; from Vectastain Elite Kit, added at the dilution specified by the manufacturer (Vector laboratories) in 3% HIGS/PBS. Visualization of staining was carried out with the Vectastain Elite Kit, according to the manufacturer's instructions. Specimens were mounted in Glycergel (DAKO laboratories).

I

Alkaline phosphatase (AP) histochemistry was used to visualize cells of the dermal papilla, after sections were de-waxed and re-hydrated. Following a rinse in PBS, Sections were incubated in AP Buffer (0.1M Tris pH9.5, 50mM MgCl₂, 0.1M NaCl, 0.1% Tween-20, containing 4.5μl of 75mg/ml Nitro-Blue-Tetrazolium in dimethylformamide and 3.5μl of 50mg/ml Bromo-Chloro-Indolyl-Phosphate in dimethylformamide, per ml of AP Buffer). Incubation was at room temperature for 15 minutes, after which sections were re-fixed for 10 minutes, rinsed in PBS and mounted in Glycergel. AP reaction product was visualized as an intense purple stain.

Preparation of skin section whole-mounts and dissection of anagen hair follicles

Dorsal neck / upper back skin was dissected and fixed in 4% formaldehyde/PBS. Whole mount preparations were photographed under a dissecting microscope. Individual follicles with enclosed hairs were dissected with needles and watchmakers forceps, air dried onto glass slides, and then mounted in DPX.

To compare rates of hair re-growth after shaving adult Whn+ and non-genetically modified mice, age- and sex-matched mice at 3 months of age were anaesthetized with Enflurane and shaved on the posterior flank with an electric shaver. Animals were allowed to return to consciousness and then returned to their cages.

Identification of genes whose expression is under the control of the Wh transcription factor by Representational Difference Analysis (RDA).

Oligon ucleotides: The sequences of oligonucleotides used in cDNA RDA are as follows:

R-Bgl-24: 5'-AGCACTCTCCAGCCTCTCACCGCA-3 '

R-Bgl-12: 5'-GATCTGCGGTGA-3'

J-Bgl-12: 5'-GATCTGTTCATG-3'

J-BgI-24: 5'-ACCGACGTCGACTATCCATGAACA-3' N-Bgl-12: 5'-GATCTTCCCTCG-3'

N-Bgl-24: 5'-AGGCAACTGTGCTATCCGAGGGAA-3'

mRNA isolation and cDNA synthesis Poly A+ RNA was isolated from the anterior-dorsal regions of the skin of age- matched Whn-genetically modified and nude mutant mice of between 7 and 15 days of age, using standard procedures. Double-stranded cDNA was then synthesised from each mRNA population using Superscript II reverse transcriptase (GIBCO-BRL), then precipitated with ethanol and resuspended in TE pH 8.0.

cDNA RDA: Generation of Driver and Tester representations

In this technique (adapted from Hubank and Schatz, Nucl. Acids Res., vol 22, pp5640-5648, 1994), cDNA from PF7zn+-genetically modified skin is designated Tester cDNA, whereas cDNA from nude mutant skin is designated Driver cDNA. The objective is to isolate specific cDNA fragments present in the Tester cDNA population that are absent or substantially reduced in abundance in the Driver cDNA population.

Both Tester and Driver cDNA populations were treated as follows: Double stranded cDNA (2μg) was digested with Dpnll, phenol extracted, ethanol precipitated and resuspended in 20μl TE (pH 8.0). 12μl of DpnII-cut cDNA was then ligated to the R-Bgl- 12/24 adapter in a mixture, containing 4μl desalted R-Bgl-24 oligo (2mg/ml), 4ml desalted R-Bgl-12 oligo (lmg/ml), 6μl 10X ligase buffer (New England Biolabs), and 31μl water. Oligonucleotides were annealed to each other and the cDNA by heating the ligation reaction to 50oC in a PCR machine, then cooling to lOoC over a period of 1 hour, and samples were then ligated by adding 3μl T4 DNA ligase (400U/ml), and incubating for 12-14 at i2-16oC. Ligations were diluted to 6μg/ml and multiple PCR reactions were then set up to generate the initial representations. Each 200ul reaction contained 2μl diluted ligation and (final concentrations) 66mM Tris-HCl, ρH8.8, 4mM MgC12, 16mM (NH4)2SO4, 33ug/ml BSA, dATP, dCTP, dGTP, and dTTP (all 0.3mM) and 2mg R-Bgl-24 primer. The R- Bgl-12 primer was melted away at 72oC for 3 minutes, and the 3' ends of the DNA fragments were repaired with 5U Taq Polymerase at 72oC for 5 minutes. 20 cycles of amplification were then performed (1 minute 95oC, 3minutes 72oC. PCR products were then combined, phenol extracted, isopropanol precipitated, and resuspended to 0.5μg/ml in TE, to provide the representations. To form the Driver, the R-Bgl- 12/24 adapters were removed from the nude mutant representations by Dpnll digestion, followed by phenol extraction and ethanol precipitation.

To form the Tester, the R-Bgl- 12/24 adapters were removed from the Whn- genetically modified representations by Dpnll digestion, followed by phenol extraction and ethanol precipitation. A 20μg portion of this digested representation was gel purified on a 1.2% agarose TAE gel, and the products were isolated free of the R-Bgl- 12/24 adapters using QiaEx resin (Qiagen), to form the Tester. 2μg of the Tester were then ligated to the J-Bgl- 12/24 in the manner described above.

Hybridization and selective amplification of Tester-specific cDNA fragments

For the initial subfractive hybridization, 0.4μg (40μl) J-ligated Whn-genetically modified Tester DNA was mixed with 40μg (80μl) nude mutant Driver DNA. The mixture was phenol extracted, ethanol precipitated, and resuspended in 4μl 3xEE buffer [lxEE: 10mMN-(2-hydroxyethyl)piperazine-N'-(2-propanesulfonicacid), pH8.0/lmM EDTA]; (Sigma). The solution was overlaid with mineral oil and DNA was denatured at 98oC for 6 minutes. The salt. concentration was adjusted with lμl 5M NaCl and the sample was allowed to anneal at 67oC, over 20 hours. The hybridized DNA was diluted with 8μl TE containing 5μg/ml yeast tRNA and resuspended in 400μl TE. For each subtraction, 4 x 200μl PCR reactions were set up as before with 20μl diluted hybridization mix, but omitting the primer.

The J-Bgl- 12 oligo was melted away at 72oC, for 3 minutes, ends were filled in with 5U Taq Polymerase at 72oC, 5 minutes, and 2ug J-Bgl -24 primer was added. Ten cycles of PCR amplification were performed and the 4 reactions were then combined, phenol extracted, isopropanol precipitated and resuspended in 40μl 0.2XTE.

20ul of PCR product was digested with 20U (2ml) Mung Bean nuclease (MBN) in IX digestion buffer (New England Biolabs) at 30oC for 35 minutes and the reaction stopped by adding 160μl 50mM Tris-HCl, pH 8.9. The digest was heated to 98oC for 5 minutes, then chilled on ice. Final amplifications (4 per hybridization) were set up on ice: 20ml MBN-treated product were mixed with.2μl J-Bgl-24 oligo (lmg/ml). lμl Taq Polymerase (5U) was added at 80oC, and 18 cycles of PCR carried out (1 minute 95oC; 3 minutes at 70oC). Products were combined, phenol extracted, isopropanol precipitated and resuspended to 0.5μg/ml, to produce the First Difference Product (DP-1).

The J-Bgl- 12/24 adapters on DP-1 were exchanged for N-Bgl- 12/24 adapters (by Dpnll digestion, agarose gel/QiaEx purification and ligation of adapters) and the process of subfractive hybridization and selective amplification repeated to generate the Second Difference Product (DP-2). The second round of hybridization was identical to the first except that only 50ng Tester DNA was mixed with 40μg Driver DNA, and annealing and extension in the amplifications were carried out at 72oC. To generate a Third Difference Product (DP-3), lOOpg J-ligated DP-2 was mixed with 40μg Driver DNA, and the process repeated, except that final amplification was performed for 22 cycles (1 minute 95oC; 3 minutes70oC).

Cloning and sequencing of Difference Products

Difference Products were visualised in samples by gel electrophoresis of an aliquot. Samples containing Difference Products were then .digested with Dpnll and cloned into the BamHI site of pBluescript II KS+ (Stratagene). Double stranded DNA was sequenced using an ABI automatic sequencer. Resulting sequences were compared to the contents of the EMBL database using BLAST and FASTA.

Confirmation of Whn-dependent expression patterns

Differential expression of Tester-specific cDNA fragments was confirmed by northern blot analysis of RNA from Whn+ and nude mutant skin. The expression patterns of cDNA clones with verified Tester-specificity were then analysed in greater detail using in situ hybridization to Whn+, wild-type and nude mutant skin samples, before they were designated as a Candidate Whn Target gene. Examples

Isolation of genomic clones spanning the murine Whn locus and generation of Whn+ mice The objective of our experiments was to generate Whn+ genetically modified mice with a fragment of genomic DNA spanning the Whn gene that included all the transcriptional regulatory elements required to guarantee faithful and physiological levels of Whn+ gene expression in the skin. A previous study had demonstrated that a cosmid genomic clone spanning the Whn gene with 8.5kb of 5' flanking DNA and 4kb of 3' flanking DNA was sufficient to rescue partially the hairless phenotype of nude mice, however rescued mice exhibited only 50% to 80% normal hair density (Kurooka et al, 1996). These results suggested that either some critical regulatory elements were absent from the cosmid used, or dominant effects of adjacent heterochromatin resulted in gene silencing. We therefore employed large PI Artificial Chromosome (PAC) and Bacterial Artificial Chromosome (BAC) clones encompassing the Whn gene to generate genetically modified mice (Yang et al, 1996; Antoch et al, 1997). These PAC and BAC clones comprised much larger fragments of genomic DNA encompassing the JVhn gene than can be accommodated in cosmids, which we expected would increase the probability of including all the necessary regulatory elements to * determine faithful and physiological levels of Whn expression in the skin (Raguz et al. , 1998).

A PCR-amplified DNA probe spanning exon 3. of the murine Whn gene was used to screen the mouse genomic PAC and BAC libraries by hybridization. Genomic clones spanning the Whn locus were isolated and their Sequence Tagged Sites (STS) content was determined by PCR using primer pairs specific for several STSs distributed within and around the Whn locus (Segre et al, 1995). This analysis enabled the amount of genomic DNA flanking the Whn gene in each PAC and BAC clone to be determined, and a contig of clones across the Whn locus was assembled. Selected PACs and BACs were restriction mapped and their structures were compared to that of the corresponding genomic DNA, to confirm that they*, were unrearranged (Figure 1(a)). One PAC clone, 436p24, spanned over 1301cb between STS markers DllSegl4 and DllSeg29. A llOkb Not I fragment containing the entire Whn locus, plμs 74kb of 5' flanking sequence and 21kb of 3' flanking sequence, was isolated from.436p24 and used to generate genetically modified mice. In addition, a BAC clone, 82G8,', which spanned over 180kb between STS markers DllSegl5 and DllSegSό was used directly for transgenesis as circular DNA, without linearization.

Identification and characterization of Whn+ mice.

Founder mice were identified by PCR analysis of tail tip DNA, as in Figure lb and bred where possible to establish genetically modified lines. Figure lc shows that genetically modified mice expressed higher steady state levels of Whn mRNA in skin than non- genetically modified littermates, consistent with the increased gene dosage of Whn in Whn+ in mice. Founder Whn+ mice exhibited similar coat phenotypes that were evident in their external appearance (Figure 2) although the degree of severity appeared to vary in proportion to the number of Whn+ gene copies. The coats of Whn+ animals appeared to be thicker and more ruffled than those of non-genetically modified littermates, and individual hairs were more erect, giving a spiky appearance to the coat which was particularly prominent around the dorsal neck and upper back region. Non-genetically modified animals were much sleeker by comparison, with flatter, more uniform coats. Whn+ animals did not appear to experience any discomfort resulting from the expression of the Whn+ gene, and they did not scratch or groom excessively, retaining a full coat of hair, as did the non-genetically modified animals. One founder male was generated with the PAC-derived l lOkb Not I fragment encompassing the Whn+ gene, and two founder females were generated with the circular BAC clone 82G8. Analysis of whole mount, fixed specimens of skin taken at the early catagen phase of the first post-natal growth cycle revealed that WhnX skin was thicker than that of non- genetically modified animals (Figure 2, c-f). Moreover, Whn+ hair follicles were longer than those of non-genetically modified skin, .penetrating for a greater distance beneath the epidermis, and the hair shafts enclosed within the Whn+ hair follicles were broader in diameter than those of non-genetically modified animals.

Enhanced hair follicle growth in skin of Whn÷ mice In order to investigate further the observation that hair follicles of the Whn+ mice were larger than those of non- genetically modified littermates, haematoxylin-eosin stained histological sections of Whn+ and non-genetically modified skin at anagen and early catagen stages of the hair growth cycle were examined. Longitudinal sections of skin taken from the upper back/dorsal neck region at either anagen or early catagen stages confirmed that hair follicles of Whn+ mice penetrated much further beneath the epidermis than those of non-genetically modified animals (Figure 3). During anagen, growing hair follicles in the skin of non-genetically modified animals penetrated to the base of the adipose layer of the dermis, as did the follicles of Whn+ skin (Figure 3a, b). However the lengths of the follicles within the dermis of Whn+ skin were much greater than those in non-genetically modified skin, which caused an increase in thickness of the adipose layer of the dermis. To accommodate.: further growth within the dermis, some follicles had adopted a waved appearance and no longer grew parallel to one another, which allowed them to continue growth withiri a vertically constrained space. By early catagen, wild-type hair follicles regressed towards the epidermis synchronously (Figure 3c). However many of the catagen hair follicles of Whn+ mice remained extended in the dermis with their tips closer to the base of the adipose layer (Figure 3d). Many catagen- stage Whn+ follicles were at least twice the length of hair follicles from age-matched non-genetically modified animals, and many were relatively disordered, disoriented and had grown into the dermis at a shallower angle than wild-type follicles. Indeed some JVhn+ follicles had continued to grow horizontally in an anteriorward direction on reaching the base of the adipose layer (Figure 3d). . ^■ ,

The enhanced growth and differentiation of hair .follicles within the dermis was also evident when transverse cross-sections of skin were, examined (Figure 4). In all skin sections analysed, many more individual hair. follicle cross-sections could be observed in Whn+ skin than in non-genetically modified skin, reflecting the increased length of each genetically modified follicle embedded in the dermis. Moreover, whilst the follicle cross- sections in non-genetically modified skin were organized into well-stratified horizontal rows of similar morphology (Figure 4a, c), the follicle cross-sections in Whn+ skin were much more disordered and exhibited less well-defined stratification, with different types of cross-sections being found adjacent to one another (Figure 4b, d). This was particularly evident at early catagen, when the tips of the follicles in non-trangenic skin had begun to regress away synchronously from the base of the adipose layer. In Whn+ skin, by contrast, a variety of follicle cross-section types remained close to the base of the adipose layer and had regressed little if at all. As was seen in longitudinal sections, these follicles were of much greater length in Whn+ skin than in non-genetically modified skin and many were observed to have grown horizontally along the base of the adipose layer. Histological sections taken parallel to the skin surface demonstrated that hair follicle density was not increased in genetically modified, Whn+ mice, which implied that the frequency of hair follicle induction is unaffected by Whn over-expression ( data not shown).

Closer examination of transverse sections of both anagen and catagen skin from age- and sex- matched littermates revealed that the follicle Outer Root Sheath and Inner Root Sheath of Whn+ skin were larger in diameter and were comprised of many more cells than those of non-genetically modified follicles (Figure 5). Although significant differences could be observed in the morphogenesis and differentiation of hair follicles of Whn+ skin in comparison to normal skin, the interfollicular epidermis of Whn+ skin was relatively normal, and there was no evidence of altered rates of cell proliferation or differentiation of basal keratinocytes (Figure 5 a-d).

Increased hair growth rates in WIιn+ mice

The above results demonstrated that the growth of the hair follicles in Whn+- genetically modified mice was enhanced in comparison to those of non-genetically modified littermates, and that hair follicle morphogenesis was relatively normal, resulting in the extended but relatively disordered downward growth of an elongated hair follicles.

Further analysis of transverse skin sections demonstrated that many Whn+- genetically modified follicles enclosed much larger, flattened hairs, in sharp contrast to the narrow, circular cross-sections of hairs that could be seen in non-genetically modified follicles (Figure 5). Both cortex and medulla of Whn+-genetically modified hair shafts were enlarged, and this enlargement began at the base of the follicle in the supra-bulbar region and was maintained along the entire length of the hair shaft within the follicle (Figure 5, b, d, f, h). To compare the external morphology and size of growing hairs, individual anagen follicles containing developing awl hairs were dissected from the same region of upper back / dorsal neck skin of age- and sex- matched Whn+-genetically modified and non-genetically modified mice. Figure 6 shows that the hairs within the anagen follicles from Whn-genetically modified mice were much longer and broader in cross-section than those from non-genetically modified littermates. To investigate further the. enhanced hair growth observed in Whn-genetically modified mice, a patch of hair of approximately 2 cm2 was shaved from the posterior flank region of age- and sex-matched 3-month old adults. 21 days later, the non-genetically modified animal exhibited a very limited degree of hair re- growth in the shaven region, if any, since the skin could still be seen through the field of very short hairs. However the Whn-genetically modified animal exhibited almost complete re-growth of thick, agouti hair in the shaven region, demonstrating that the hair growth rate was dramatically enhanced by over-expression of Whn.

The enhanced growth of Whn-genetically modified hair follicles during anagen together with the increased growth rate of differentiated hairs within them indicated that growth and differentiation of several different cell types within the hair follicle are closely coupled through the function of the Whn gene product

The spatial distributions of differentiating and proliferating cells in hair follicles of Whn+ mice are relatively normal.

To examine the differentiation of Whn+ skin further, the expression of cytokeratins was studied using the polyclonal antibody NCL-CKp. This antibody recognises cytokeratins that are most abundant in the epidermis, sebaceous glands and outer root sheath of the hair follicles, and is a good general indicator for differentiated epithelial cells in the skin (Mygind et al, 1988). In Whn+ mice, a strong expression pattern of cytokeratins was observed that was similar to that observed in non-genetically modified skin, being restricted to the same structures (Figure 7). These results demonstrated that although growth and differentiation of hair follicles was enhanced in Whn+ mice, the distribution of cytokeratin-expressing cells in the skin was relatively normal.

Next, the location of proliferating cells in anagen follicles of Whn+ and non-genetically modified mice was determined using an antibody against the proliferation marker Ki-67 (Schluter et al, 1993). In normal hair follicles most of the proliferating cells reside in the matrix of the hair follicle bulb that surrounds the non-proliferating, alkaline phosphatase- positive dermal papilla, and also in the Outer Root Sheath (DasGupta and Fuchs, 1999; Lee et al, 1999). Hair follicle matrix cells are one of the most rapidly dividing populations in the mammalian body, with an average cell cycle time of 12 hours (Potten, 1985). The distribution of proliferating cells in Whn+ skin was similar to that observed in normal follicles (Figure 8), which suggested that the enhanced follicular growth rate and differentiation was likely to be due to an increase in the rate of proliferation of either the matrix cells or their descendants, rather than the forced entry of other, normally quiescent cells into a proliferative state. Whn+ follicles appeared to function normally and gave rise to hairs of relatively normal structure, which suggested that any increased proliferation was matched by co-ordinated, normal differentiation of the resulting cell progeny.

WIιn+ transgenic mice exhibit increased Whn gene dosage

The dosage of the Whn gene in Whn+ transgenic mice was compared to that of non-transgenic animals by Southern blot analysis of genomic DNA samples. The

Whn+ transgenic founder male VCl A was crossed to non-transgenic CBA females and Whn+ transgenic and non-transgenic sibling progeny were identified by PCR analysis of genomic DNA samples. High quality genomic DNA was then isolated from the spleens of Whn+ transgenic and non-transgenic siblings and 15ug samples were digested with the restriction enzymes Pstl, Hindlll, EcoRI and

BamHI, electrophoresed in a 1% agarose IX TAE gel and then transferred to a Hybond N membrane by Southern blotting. The resulting Southern blot was hybridised with a 32P-labelled probe corresponding to exon 3 of the wild-type mouse Whn gene overnight at 65 degrees C in Church buffer. After washing the membrane at 65 degrees C in 0.1X SSC, 0.1% SDS, the filter was autoradiographed. The results in Figure 9 clearly indicate that Whn+ transgenic animals possess extra copies of the wild-type Whn locus and that these copies exhibit an identical restriction pattern in comparison to the wild-type endogenous Whn gene, which demonstrates that they are* intact and not rearranged. Thus, Whn+ transgenic animals have an increased Whn gene dosage in comparison to non-transgenic animals.

A Whn+ transgene fully rescues both the hair and thymus phenotypes of animals homozygous for the nude mutation at the endogenous Whn locus.

In order to test whether the expression and function of Whn+ transgenes were correctly targeted to the hair follicles and thymus, the phenotypes of Whn+ transgenic mice that were also homozygous for the nude mutation at the endogenous Whn locus on chromosome 11 were evaluated. These mice were produced using genotyping and breeding strategies that enabled wild-type and nude mutant alleles at the endogenous locus to be distinguished both from each other and from the wild-type Whn+ transgene.

The genotyping strategy employed the transgene-specific PCR marker that distinguishes Whn+ transgenic from non-transgenic animals (See Figure 1(b) and materials and methods) and a Simple Sequence Length Polymorphic (SSLP) marker, DBhml48, that lies just outside the region of DNA encompassing the Whn gene defined by the l lOkb Notl fragment used for transgenesis, but that is nevertheless tightly linked to the endogenous Whn gene on mouse chromosome 11. DBhml48 maps to <0.5cM of the Whn gene and it exhibits length polymorphisms which are distributed amongst inbred stains in the following way (Nehls et al. (1995), Mamm. Genome 6, 321-331): C57B16J: 97bp PCR fragment CBA: 107bp PCR fragment 129/S v: 107bp PCR fragment Balb/c: 107bp PCR fragment

DBhml48primers and condition used were: Forward: 5 '-AGG GGA AGT CCT GTA TGG ACA -3 ' Reverse: 5'-ACC AAC CTC GAT AGA GCC ATC-3' Step 2

WhnTg+; nu/+ animals were then crossed to Balb/c nu/+ heterozygotes and female progeny identified that were (a) non-transgenic (b) Positive for both 97bρ and 107bp DBhml 8 alleles

(c) By genomic sequencing were heterozygous for the nude mutation at the endogenous Whnlocus.

This identified animals that carried the nude mutation linked to the 107bp DBhml 48 allele and the wild-type Whngene linked to the 97bp DBhml48 allele.

Animals with the above characteristics were designated as WhnTg-; nu/+.

Step 3

WhnTg+; nu/+ males from Step 1 were then crossed to WhnTg-; nu/+ females from Step 2 and all progeny were then genotyped for the presence of the Whn+ transgene and for the presence of the 97bp and 107bp alleles of DBhml48 (Figure 10). Both coat and thymus phenotypes of all progeny individuals were also scored. Phenotypically nude animals were always Whn+ transgene negative and homozygous for the 107bp DBhml 48 allele, which was consistent with homozygosity for the tightly linked nude mutation at the endogenous Whn locus and the observed phenotype. However progeny that were Whn+ transgene-positive and homozygous for the 107bp DBhml48 allele were phenotypically fully rescued with respect to both their hair follicle and thymus phenotypes (Figure 10). Thus, despite being homozygous for the 107bp DBhml 48 allele that is tightly linked to the nudemutant allele of Whn, these individuals were phenotypically wild-type due to the presence of the fully functional Whn+ transgene.

Whn+ transgenic males (descendants of VCl A founder) that were also heterozygous for the nude mutation were crossed to nude heterozygous, non-transgenic females as outlined in the text. Both parents and all progeny were then genotyped for DBhml48

36 SSLPs and the presence of the Whn+ transgene. These data are presented in Figure 10. In each genotyping test, lane 1 represents the* results of PCR analysis for the presence of the VCl A transgene (see Figure.1) and lane 2 represents the products generated by the PCR assay for DBhml48 SSLPs. Markers in lane M are lOObp ladder. A DBhml 48 fragment of 97bp indicates the presence of a wild-type Whn allele at the endogenous locus whereas a 107bp product indicates the presence of a nude mutant allele at the endogenous Whn locus (DBhml 48 fragment sizes are indicated). Progeny were genotyped for the presence of Whn-l- transgenes and for polymorphisms at the DBhml48 locus (upper right hand figure in each panel). Progeny were also scored for coat (upper left figure in each panel) and thymus phenotypes (bottom figures in each panel). Fluorescence Activated Cell Sorting (FACS) analysis of peripheral blood lymphocytes was employed to assay thymus function using standard methods for isolation of peripheral blood leukocytes, labelling with fluorescently conjugated antibodies against the cell surface proteins B220, TCR (T-cell Receptor), CD4 and CD8, and analysis using a Becton Dickinson FACScan machine. The lower left figure in each panel indicates the relative proportion of B-lymphocytes (strongly B220-ρositive cells) and T-lymphocytes (strongly TCR-positive cells) in peripheral blood. The lower right figure in each panel indicates the relative proportion of strongly CD4-positive and CD8-positive T lymphocytes in peripheral blood. Axes of each graph show fluorescence intensity of antibody staining on a logarthmic scale.

Thus, the individual analysed in the upper panel of Figure 10 is non-transgenic but heterozygous for the nude mutation. Accordingly, this animal has wild-type hair follicle and thymus phenotypes. The individual in the middle panel of Figure 10 is non- transgenic and homozygous for the nude mutation. Accordingly, this animal both lacks external hair and detectable T lymphocytes in peripheral blood. However the individual in the lower panel of Figure 10 is Whn+ transgenic and homozygous for the nude mutation at the endogenous Whn locus. This animal has wild-type hair follicle and thymus phenotypes. Therefore the Whn+ transgene in this animal fully

37 rescues the effects of homozygosity for the nude mutation at the endogenous Whn locus.

The frequency of wild-type and nude mutant phenotypes in the progeny of crosses between WhnTg+; nu/+ males and WhnTg-; nu + females was scored (Table 1). These results are further consistent with full rescue of the nude phenotype by the Whn+ transgene derived from the 1 lOkb Notl fragment.

Whn protein is more abundantly expressed inW n+ transgenic hair follicles than in non-transgenic hair follicles and is localized to the nuclei of differentiating, hair cuticle and hair cortex precursors

To further confirm that Whn+ transgenic animals exhibited an increased, functional Whn gene dosage, developing hair follicles from P6.5 Whn+transgenic and non- transgenic littermates were analysed by immunohistochemistry for the expression of nuclearly localised Whn protein.

Figure 11 clearly demonstrates that in both non-transgenic and Whn+ transgenic hair follicles, Whn protein is localised to the newly formed, hair keratin-expressing precursors of the hair cuticle and hair cortex. Moreover, the immunoreactive signal in nuclei of Whn+ transgenic follicles is stronger than that detectable in non-transgenic follicles, consistent with over-expression of Whn protein due to increased Whn gene dosage. ^{' ' "}

Samples of medially located backskin from the anterior saddle region of Whn+ transgenic and non-transgenic littermates (descended from VCl A founder male) were embedded in paraffin wax and 8μm longitudinal sections were taken onto Vectabond-subbed slides. After boiling in Citrate buffer (Vector Labs) for 15 minutes and cooling to <60 degrees C, sections were blocked with 3%BSA in PBS and then immunostained with antibodies recognising the Whn protein (rabbit polyclonal, diluted 1 :250) and hair keratins (mouse monoclonal AE13, diluted 1:20). After washing in PBS, then blocking with 3% BSA in PBS and incubating with

38 fluorescently labelled secondary antisera and washing in PBS, sections were mounted with Vectashield and analysed by confocal microscopy for expression of Whn and hair keratins. Figure 11 shows the following:

(a) Non-transgenic follicles immunostained with anti-Whn antiserum to show localisation of Whn protein in nuclei of nascent hair cuticle and hair cortex (white signal, arrow).

(b) Non-transgenic follicles immunostained with AE13 antibody to show localisation of hair keratins in cytoplasm of differentiating hair cuticle and hair cortex (white signal, arrow). Note how expression of Whn in (a) is restricted to nuclei of newly formed, hair keratin-expressing hair cortex and hair cuticle cells

(c) Whn+ transgenic follicles immunostained with anti-Whn antiserum to show localisation of Whn protein in nuclei of nascent hair cuticle and hair cortex (white signal, arrow). Note how signal in (a) is weaker than signal in (c), due to over- expression of Whn in (c). (d) Whn+ transgenic follicles immunostained with AE13 antibody to show localisation of hair keratins in cytoplasm of differentiating hair cuticle and hair cortex (white signal, arrow). Again, expression of Whn in (c) is restricted to nuclei of newly formed, hair keratin-expressing hair cortex and hair cuticle cells.

Discussion

The effects of inactivating mutations in the Whn gene indicate that this gene is necessary for the growth and differentiation of mammalian hair follicles. We have described the effects in the skin of increasing the level of Whn gene activity by generating genetically modified mice carrying extra copies of the Whn genomic locus. Our most striking finding was that growth and differentiation of the hair follicles were enhanced in all genetically modified animals analyzed. Whilst the hair follicles of genetically modified animals were enlarged and grew more deeply into the dermis, and their spatial orientation and distribution were more disordered in comparison to those of wild-type littermates, Whn+ Hides exhibited the normal pattern of morphogenesis and cellular differentation, suggesting that the balance between growth and differentiation was not.

39 PCR was performed on genomic DNA in a reaction containing 1.5mM MgCl₂ for 30 cycles that include a denaturation step at 95°C for 1 minute, annealing at 55°C for 1 minute and extension at 72 °C for 1 minute. PCR products were then analysed on a 4% Metaphor agarose gel in IX TAE.

The following breeding strategy was then initiated with the VCl A Whn+ transgenic founder male and non-transgenic females that were also heterozygous for the nude mutation at the endogenous Whn locus:

Step 1

Nude heterozygous females are homozygous for the 107bp Balb/c allele at the DBhml48 locus. In contrast, the VC1A founder was a CBA/C57B16J Fl hybrid and therefore contained both the 97bp C57B16J allele and the 107bp CBA allele of DBhml48. VC1A was crossed to females heterozygous for the nude mutation and males were identified amongst the progeny with the following characteristics:

<ι

a) Whn+ transgenic b) Positive for the C57B16/J-derived 97bp allele of DBhml48 that is tightly linked to the C57B16/J-derived wild-type allele of the endogenous Whn gene c) Produced some homozygous nude ^' mutant progeny when mated with heterozygous nude females.

These males were therefore heterozygous for the nude mutation and genotyped positive for the Balb/c-derived 107bp allele of DBhml 48 as well as the C57B16/J- derived 97bρ DBhml48 allele. Thus, the presence of the 107bp Bhml48 allele in these animals^' is a surrogate marker for a nude mutant allele at the endogenous Whn locus and enabled animals to be identified that carried both the nude mutation linked to the 107bp DBhml 48 allele and the wild-type Whngene linked to the 97bp DBhml48 allele.

Progeny animals with the above characteristics were designated as WhnTg+; nuΛh

35 perturbed. These results implied that a function of Whn is to promote growth and differentiation within the hair follicle, resulting in the development of both larger hair follicles and hairs more rapidly than in normal. This role for Whn is consistent with its normal expression domain within the hair follicle, which encompasses proliferating cells of the hair bulb matrix and Outer Root Sheath, in addition to the post-mitotic suprabulbar progenitor cells that are fated to differentiate, into the components of the Inner Root Sheath, hair cuticle, cortex and medulla (Lee et al, 1999). We did not observe any increase in the size of the hair bulb matrix in anagen follicles, nor did we observe any pathological effects, such as hyperplasia or neoplasia, that could result from the ι increased production of proliferating stem cells. Immunohistochemical analysis of cytokeratin and Ki-67 demonstrated that the domains of differentiation and proliferation within the Whn+ follicles were relatively normal, and alkaline phosphatase histochemistry indicated that the dermal papillae were identical to those of wild-type hair follicles. Moreover, we observed that cycling of the hair follicles was not disrupted in Whn+ mice, which suggests that Whn gene action is subordinate to the cues that initiate growth, regression and quiescence. Our observations contrast with the results of Whn mis-expression in terminally differentiating cells of the epidermis and hair follicle (Prowse et al., 1999). In these experiments, Whn expression was targeted to the involucrin-expressing, normally post-mitotic keratinocytes of the epidermis and follicular epithelium, causing dramatic epidermal hyperplasia, disruption of hair formation and the failure of follicles to exit the anagen phase of the hair cycle. These results demonstrated that Whn can cause quiescent keratinocytes that are en route to terminal differentation to enter a proliferative state when this protein is mis-expressed, and that such cells then fail to complete the normal progamme of differentiation in both the hair follicle and the interfoUiclular epidermis. Our findings emphasise that one of the principal functions of Whn is to couple the proliferation of committed progenitors to a subset of developmental fates within the hair follicle, in order to co-ordinate morphogenesis of the hair with that of the hair follicle. This is again consistent with the phenotype of nude mutants, because in the absence of functional Whn protein hairs are produced but they are small and defective with a discontinuous or absent cuticle, and they are enclosed within an imperfect, discontinuous Inner Root Sheath. Loss of Whn function therefore appears to exhaust prematurely the ability of follicles to produce an adequate number of cells for normal hair and hair follicle morphogenesis^ whereas over-expression of Whn amplifies the capacity of hair follicles to grow and differentiate normally.

Intriguingly, the time required to complete follicle growth cycles is significantly reduced in nude mutant mice, which may be due to shortened periods of both growth and quiescence in each follicle cycle (Eaton, 1976). This finding, when reconsidered in the light of our observation that Whn-over-expression enhances normal hair growth induces precocious hair re-growth, implies that Whn functions as a pacemaker that influences the time of onset of anagen, its duration and intensity.

Our results suggest a similarity between hair follicle development and the process of mammalian haemopoiesis, where pluripotent stem cells also produce a balanced set of committed, proliferating, multipotent progenitors each of which has a restricted subset of developmental fates. Where Whn is implicated in the control of proliferation and commitment of multipotent progenitors in the hair follicle, the murine winged helix-turn- helix PU.l transcription factor may perform an analogous function in haemopoiesis (Simon, 1998). Given that different members of transcription factor families often perform similar functions in different developmental contexts, it is tempting to speculate that winged helix transcription factors may function as co-ordinators of proliferation and cell fate selection in multipotent progenitors in a variety of developmental systems.

The signals that induce Whn expression in the hair follicle are unknown, however a variety of signalling molecules have been implicated in the early stages of hair follicle development and so they may act upstream of Whn. Sonic hedgehog (Shh) is initially expressed in the follicular epithelial cells that are fated to form the matrix and it is required in the embryo to promote downgrowth of the follicular epithelium and formation of the inner root sheath (St- Jacques et al., 1998). In post-natal anagen hair follicles Shh is expressed asymmetrically in the hair bulb matrix on the side closest to the epidermis, and so it may play a further role- in determining the angle and orientation of hair follicles in the skin (Bitgood and McMahon, 1995; Gat et al, 1998). Other signalling molecules implicated in the specification and morphogenesis of hair follicles include Bmρ2/4 (Bitgood and McMahon, 1995; Lyons et al, 1990; Jones et al. ,1991), FGF-5 (Hebert et al, 1994) and WntlOb (Wang and Shackleford, 1996). Whether any of these signalling pathways act as inducers of Whn in the precursors of the hair shaft, Inner Root Sheath and Outer Root Sheath is not yet known. The recent description of de novo hair follicle formation in genetically modified mice expressing a stabilized form of beta- catenin lends further support to a role for Wnt-signalling in hair follicle induction (Gat et al, 1998), and the finding that LEFl protein is restricted to the precursors of the hair cortex indicates that Wnt signalling may have later roles in hair differentiation (DasGupta and Fuchs, 1999), raising the possibility that LEFl co-operates with Whn in regulation of hair cortex-specific target genes.

Whn has been demonstrated to encode a DNA-binding transcription activator (Brissette et al, 1996; Schlake et al, 1997; Schuddekopf et al, 1996), however direct interactions of this protein with promoter elements in a relevant target gene remain to be demonstrated. We observed that hair re-growth in shaved Whn+ skin was more rapid than that in shaven, non-genetically modified skin. This may reflect a shortened cycle time in genetically modified Whn+ mice. The identification of genes that are expressed in the skin of Whn+ mice but not in the skin of nu/nu homo∑ygous mutant mice may be a particularly efficient means of discovering other candidate Whn target genes. It is hoped that such studies will shed new light on the mechanism of action of Whn protein during the tightly coupled processes of hair follicle growth and morphogenesis.

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Phenotype; genotype wild-type; WhnTg+ wild-type; WhnTg- nude, WhnTg+ nude, WhnTg-

No. Observed 54 30 0 7

Totals (phenotypes) 84- (92%) 7 (8%)

No. expected, if no rescue (phenotypes) 68 (75%) 23 (25%)

No. expected, if rescue (phenotypes) 80 (87.5%) 11 (12.5%)

Claims

1. A non-human mammal characterised in that the genome of said mammal is genetically modified to provide at least one Whn gene which is over expressed when compared to a non-genetically modified non-human mammal.

2. A mammal according to Claim 1 wherein said mammal includes additional copies of a Whn gene.

3. A mammal according to Claim 1 or 2 wherein said mammal has enhanced expression of Whn gene.

4. A mammal according to any of Claims 1-3 wherein said mammal has enhanced hair follicle growth through overexpression of a Whn gene.

5. A mammal according to any of Claims 1-3 wherein said mammal has enhanced hair follicle cell differentiation through over expression of a Whn gene.

6. A mammal according to any of Claims 1-5 wherein said mammal is a rodent.

7. A mammal according to any of Claims 1-6 wherein said mammal is selected from the following group: non-human primate; pig; sheep; cat; dog; goat; cow; camel; horse; llama; alpaca; mouse; rat.

8. A mammal according to any of Claims 1-7 wherein expression of the Whn gene is controlled by its cognate promoter.

9. A mammal according to any of Claims 1 -8 wherein over expression of the Whn gene is by a promoter which shows an expression pattern predominantly restricted to those cells in which the Whn gene is naturally expressed.

10. A mammal according to Claim 9 wherein said cells are selected from the following group: proliferating cells the hair bulb matrix and outer root sheath; post- mitotic suprabulbar progenitor cells of the inner root sheath; hair cuticle cells; hair cortex cells; and hair medulla cells.

11. An isolated nucleic acid molecule which comprises the Whn gene including expression control sequences sufficient to direct over expression of the said gene.

12. An isolated nucleic acid molecule according to Claim 11 wherein said molecule includes at least 8.5 kb of the 5' region of DNA upstream of the initiation codon of the Whn gene.

13. An isolated nucleic acid molecule according .to Claim 12 wherein said molecule includes between 8.5kb and 74kb of the 5' region upstream of the initiation codon of the Whn gene.

14. An isolated nucleic acid molecule according to Claim 12 or 13 wherein said molecule includes at least 4 kb of the 3' region downstream of the termination codon of the Whn gene.

15. An isolated nucleic acid molecule according to Claim 14 wherein said molecule includes between 4 kb and 21 kb of the 3' region downstream of the termination codon of the Whn gene.

16. A vector characterised in that said vector includes an open reading frame which encodes the Whn protein and is operably linked to a promoter adapted for over expression of the nucleic acid encoding the Whn protein.

17 A vector according to Claim 16 wherein said vector is a bacterial artificial chromosome (BAC) or PI artificial chromosome (PAC).

18. A vector according to Claim 16 or 17 wherein said vector comprises a nucleic acid molecule according to any of Claims 11-15.

19. A method to identify agents capable of enhancing the expression of a Whn gene comprising: i) providing a cell expressing a Whn gene; ii) contacting said cell with at least one agent to be tested; and iii) monitoring the transcriptional activity a Whn gene or Whn gene promoter.

20. A method to identify agents capable of enhancing the expression of a Whn gene comprising: i) providing a cell transfected or transformed with a Whn gene, or a Whn gene promoter; ii) contacting said cell with least one agent to be tested; and iii) monitoring the transcriptional activity of said Whn gene or Whn gene promoter.

21. A cell transfected transformed with the nucleic acid or vector according to any of Claims 11-18.

22. A cell according to Claim 21 wherein said cell is stably transfected/transformed.

23. A therapeutic composition comprising the nucleic acid or vector according to any of Claims 11-18.

24. The use of a nucleic acid or vector according to any of Claims 11-18 for the manufacture of a medicament for use in the treatment or prevention of hair loss.

25. A therapeutic composition comprising the Whn protein, or the effective part thereof.

26. Use of the Whn protein, or an effective part thereof, for the manufacture of a medicament for use in the treatment of hair loss.

27. A composition or medicament according to any of Claims 23-26 which further comprises a diluent, excipient or carrier.

28. A method to treat hair loss in a mammal comprising administering an effective amount of the nucleic acid or vector according to any of Claims 11-18 or the composition/medicament according £o any of Claims 23-27.

29. A method according to Claim 28 which method is a cosmetic treatment.

30. A method according to Claim 28 or 29 wherein said method is to treat a condition selected from the following group: androgenic alopecia; alopecia areata; alopecia totalis; congenital alopecia universalis; congenital alopecia; hair loss as a result of chemotherapy.

31. A non-human mammal characterised in that the genome of said mammal is genetically modified to provide at least one Whn gene which is over expressed when compared to a non-genetically modified non-human mammal characterised in that said mammal is transfected with the nucleic acid or vector according to any of Claims 11-18.

32. An agent identified by the method according to Claim 19 or 20.

33. Use of the transgenic mammal according to any of Claims 1-10 in the identification of genes involved in hair follicle growth and/or hair follicle differentiation.