EP1181352A4 - Versican und epithel-mesenchym wechselwirkung - Google Patents

Versican und epithel-mesenchym wechselwirkung

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Publication number
EP1181352A4
EP1181352A4 EP99945293A EP99945293A EP1181352A4 EP 1181352 A4 EP1181352 A4 EP 1181352A4 EP 99945293 A EP99945293 A EP 99945293A EP 99945293 A EP99945293 A EP 99945293A EP 1181352 A4 EP1181352 A4 EP 1181352A4
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European Patent Office
Prior art keywords
cells
versican
cell
promoter
population
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EP99945293A
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English (en)
French (fr)
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EP1181352A1 (de
Inventor
Robert Burgeson
Jiro Kishimoto
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General Hospital Corp
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General Hospital Corp
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Publication of EP1181352A4 publication Critical patent/EP1181352A4/de
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2517/00—Cells related to new breeds of animals
    • C12N2517/02—Cells from transgenic animals

Definitions

  • the invention relates generally to interactions between epithelium and mesenchyme, e . g. , interactions at hair follicles, and to the processes of hair development and hair cycling.
  • Epithelial -mesenchymal interactions during development are essential for the induction of organogenesis for many tissues (e . g. , kidney, gut, respiratory organ, cutaneous appendage) .
  • the hair follicle provides an excellent model for studying epithelial -mesenchymal interactions because: 1) it is located on the outermost layer of the body, allowing easy access and observation; 2) it has distinct epithelial and mesenchymal components; and 3) a definitive functional assay for in vivo hair induction already exists.
  • Weinberg, . C Goodman, L. V., George, C, Morgan, D. L., Ledbetter, S., Yuspa, S. H.& Lichti, U. (1993) J. Invest . Dermatol . 100, 229-236) .
  • the dermal papilla is located at the bottom of the hair follicle and is the major mesenchymal component.
  • the DP originates from condensed mesenchymal cells that lie beneath the epithelial hair germ cells (placode) in embryonic skin. These specialized mesenchymal cells are believed to be the source of the dermal -derived signaling molecule (s) involved in hair development and embryogenesis and later in postnatal hair cycling. (Hardy, M. H. (1992) Trends Genet . 8, 55-61) .
  • Hair development during embryogenesis requires a series of reciprocal interactions between the epithelium and the underlying mesenchymal cells.
  • the dermal mesenchyme signals the epithelium to form the epidermal placode.
  • the epithelium sends a message to the underlying mesenchyme to initiate mesenchymal condensation.
  • the condensed mesenchyme then sends a message back to the epithelium promoting hair elongation. (Messenger, A. G. (1993) J. Invest . Dermatol . 101, 4S-9S) .
  • the invention is based, in part, on the discovery that versican-expressing cells, e . g. , versican-expressing dermal papilla (DP) cells have hair-induction ability.
  • Versican gene is a specific marker for hair inducibility and for the ability to promote aggregation and condensation of DP cells.
  • the activity of versican gene can be used to identify or select cells e. g. , DP cells, e . g. , cultured DP cells, useful for testing treatments for the ability to modulate hair-induction and/or hair growth process.
  • the promoter of the versican gene can be used for hair follicle, e . g. , DP- specific expression.
  • the invention features, a method of evaluating a cell, e . g. , a DP cell or a cultured DP cell.
  • the method can be used to evaluate the ability of a DP cell to promote aggregation or condensation, the ability of a DP cell to modulate, e . g. , to promote, a mesenchymal interaction, or to modulate e . g. , to promote, hair growth or induction.
  • the method includes: providing a cell; and evaluating the expression of a versican promoter in the cell, thereby evaluating the cell.
  • the cell e . g. , a DP cell, e . g. , a cultured DP cell, is used to evaluate or screen a treatment for the ability to modulate hair induction or growth.
  • the versican promoter is linked to a nucleic acid sequence which encodes a reporter molecule, e . g. , a luminescent or fluorescent product, e . g. , green fluorescent protein (GFP)
  • a reporter molecule e . g. , a luminescent or fluorescent product, e . g. , green fluorescent protein (GFP)
  • the cells are subsequently implanted in an animal to evaluate their potential, e . g. , their potential for inducing hair growth.
  • the cells can be combined with epithelial cells, epithelial hair germ cells or placode, epithelial cell extract, or a fraction of epithelial cell extract, e . g. , cytoplasmic fraction, membrane fraction, or soluble fraction and implanted in an animal .
  • a versican gene promoter is a nucleotide fragment containing about 900bp.
  • the versican gene promoter fragment includes a nucleotide sequence upstream and/or downstream of a transcription start site, e . g. , from position -559 to +280 of versican gene while the major transcription start site is position +1.
  • a cell is a mesenchymal cell, e . g. , a condensed mesenchymal cell, a DP cell, a versican expressing-mesenchymal cell, a DP versican- expressing cell, or a cell selected for versican promoter activity.
  • a cell is a cultured cell, e . g. , cultured DP cell.
  • the method further includes purifying a versican expressing cell from a cell, preferably of the same type, which does not express the versican promoter.
  • the invention features, a method of identifying a treatment capable of modulating hair growth or induction, or capable of modulating an epithelial-mesenchymal interaction or DP aggregation or condensation.
  • the method includes providing a treatment to a cell, e . g. , a DP cell or a cultured DP cell, having versican gene expression, or selected for versican promoter activity.
  • the method can further include monitoring hair growth or induction, e . g. , monitoring hair formation.
  • the versican promoter is linked to a nucleic acid sequence which encodes a reporter molecule, e . g. , a luminescent or fluorescent product, e . g. , green fluorescent protein (GFP)
  • a reporter molecule e . g. , a luminescent or fluorescent product, e . g. , green fluorescent protein (GFP)
  • a versican gene promoter is a nucleotide fragment containing about 900bp.
  • the versican gene promoter fragment includes a nucleotide sequence upstream and/or downstream of a transcription start site, e . g. , from position -559 to +280 of versican gene while the major transcription start site is position +1.
  • a treated cell is a mesenchymal cell, e . g. , a condensed mesenchymal cell, a DP cell, a versican expressing-mesenchymal cell, a DP versican-expressing cell, or a cell selected for versican promoter activity.
  • a treated cell is a cultured cell, e . g. , cultured DP cell.
  • the method further includes purifying a versican expressing cell from a cell, preferably of the same type, which does not express the versican promoter.
  • the invention features, a method of identifying a treatment, e . g. , a treatment that modulates versican gene, or versican promoter-dependent, expression.
  • Modulation of versican gene, or versican promoter-dependent, expression can be predictive of the modulation of an epithelial -mesenchymal interaction or DP aggregation or condensation, and/or the modulation of hair induction and/or hair growth.
  • the method includes providing a treatment to a cell and monitoring versican gene or promoter activity. Modulation of versican gene or promoter activity can be predictive of the ability to modulate an epithelial- mesenchymal interaction or DP aggregation or condensation or modulate hair induction or growth, e . g.
  • a change of versican gene or promoter activity in the treated cell but not in a control cell identifies such a treatment.
  • Versican gene or promoter activity can be monitored by any suitable means, e . g. , measuring the level of transcription or translation of versican gene or a product under the control of a versican promoter, or the level of activity of versican gene promoter.
  • a versican gene promoter is a nucleotide fragment containing about 900bp.
  • the versican gene promoter fragment includes a nucleotide sequence upstream and/or downstream of a transcription start site, e . g. , from position -559 to +280 of versican gene while the major transcription start site is position +1.
  • a treated cell is a mesenchymal cell, e . g. , a condensed mesenchymal cell, a DP cell, a versican expressing-mesenchymal cell, a DP versican-expressing cell, or a cell selected for versican promoter activity.
  • a treated cell is a cultured cell, e . g. , cultured DP cell.
  • the treatment is administered in the presence of epithelial cells, epithelial hair germ cells or placode, epithelial cell extract, or a fraction of epithelial cell extract, e . g. , cytoplasmic fraction, membrane fraction, or soluble fraction.
  • a population of epithelial cells, e . g. , keratinocytes or a sufficient amount of epithelial cell extract can be incubated or in contact with the to be treated cells before, during, or after the treatment, or in combination thereof.
  • the treated cells are implanted in an animal to evaluate their potential, e . g. , their potential for inducing hair growth.
  • the treated cells can be combined with epithelial cells, epithelial hair germ cells or placode, epithelial cell extract, or a fraction of epithelial cell extract, e . g. , cytoplasmic fraction, membrane fraction, or soluble fraction and implanted in an animal .
  • the invention features a method of inducing hair growth.
  • the method includes introducing a first population of cells containing versican expressing cells or cells selected for versican promoter activity, e . g. , DP versican expressing cells into a subject .
  • a second population of cells e . g. , containing epithelial cells, are introduced before, after, or concurrently with the introduction of the first population of cells into a subject.
  • the invention features, a transgenic mammal which contains a promoter of versican gene operably linked to a nucleotide sequence encoding a reporter sequence; wherein the promoter is active in dermal papilla cells of the transgenic mammal.
  • a versican promoter is capable of providing tissue and/or cell specific activity, e . g. , specific activity in hair follicles.
  • a versican promoter directs or drives specific promoter activity in mesenchymal cells, e . g. , condensed mesenchymal cells and dermal papilla cells.
  • the versican gene promoter fragment includes a nucleotide sequence from position -559 to +280 of versican gene while the major transcription start-site is position +1.
  • the invention also features a method of providing dermal papilla-specific expression.
  • the method includes providing a polynucleotide sequence which includes a versican promoter or a functional fragment or analog thereof, operably linked to a desired nucleic acid sequence; wherein the promoter is active in dermal papilla cells, e.g., specifically activated in dermal papilla cells.
  • the polynucleotide sequence encodes a growth factor, e.g., FGF-2 (jSFGF) , FGF- 7(KGF), EGF, IGF-1, NGF, TGF-j ⁇ l, TGF-/32, TGF-/33, HGF, a growth factor receptor, e.g., NGFR, PDGFR, TGF-3IR, TGF-/3lIr, a hormone receptor, e.g., androgen receptor, retinoic acid receptor, a morphogen molecule, e.g., BMP- 2, BMP-4, epimorphin, an adhesion molecule, e.g., collagen, hyaronic acid receptor, syndecan-1, veriscan, a protease, e.g., nexin-1, or a transcription factor, e.g., LEFi, Glil, Gli2, Gli3.
  • a growth factor e.g., FGF-2 (jSFGF) ,
  • the polynucleotide sequence encodes a gene expressed in early hair follicle development, e.g., a homeobox gene, e.g., HoxC8, HOXD9, HoxDll, HoxD13, Msx-1, Msx-2; a transcription factor, e.g., Lefl, hn, Id 1-3, Id 4, M- Twist; a signaling or adhesion molecules, e.g., Amphiregulin, BRCA-1, BMP-2A, BMP-4, BMP-3, BMP-7, E- Caderin, P-Caderin, Clusterin/TRPM-2 , ⁇ l Connexin, o ⁇ 2 Connexin, CD44, I-CAM, Epimorphin, AGFR, FGF-1 ( ⁇ FGF) , FGF-2 ( ⁇ FGF) , GFG-7 (KGF) , FGFR1 , FGFR2 , KGFR, Hedgehog, Pat
  • Still another feature of the invention is a method of separating a first population of cells from a second population of cells.
  • the method includes providing a mixture of a first population of cells containing a luminescent or fluorescent product and a second population of cells which lack the luminescent or fluorescent product, and separating the first population of cells from the second population of cells, e . g. , by virtue of luminescence or fluorescence.
  • a cell in the first (or both) populations is one into which a nucleotide sequence encoding a luminescent or fluorescent protein or polypeptide has been inserted.
  • a cell in the first population is one into which a luminescent or fluorescent protein has been introduced, e . g. , by injection or electroporation.
  • a cell having a luminescent or fluorescent product is: a cell into which a nucleic acid encoding a luminescent or fluorescent product, e . g. , GFP, has been introduced; a cell into which a nucleic acid encoding a promoter, not normally coupled to a luminescent or fluorescent product is coupled to a nucleic acid encoding a luminescent or fluorescent product, e . g. , a skin specific or a DP cell specific promoter, e . g. , a versican promoter coupled to the nucleic acid encoding a luminescent or fluorescent product, e . g. , GFP.
  • nucleic acid encoding a promoter coupled to a luminescent or fluorescent product has been introduced into cells of both populations but is expressed only in the first population.
  • a cell of one or both populations includes a nucleic acid sequence encoding a luminescent or fluorescent product fused to a sequence which is capable of targeting the product to a particular cell destination, e . g. , to the nucleus, cytoplasm, or a membrane, e . g. , the plasma membrane.
  • a luminescent or fluorescent product e . g. , GFP is, conjugated, coupled, or attached to a partial or full- length membrane protein or a targeting sequence which, e . g. , results in incorporation into the plasma membrane.
  • Separating a population of cells containing a fluorescent product from a population of cells that do not contain a fluorescent product can be achieved by various means, e . g. , a cell sorter, a high-speed cell sorter, or a Moflo high-speed cell sorter.
  • the invention also features a method of modulating an epithelial-mesenchymal interaction, DP aggregation or condensation, or hair growth or induction.
  • the method includes administering an effective amount of a treatment to a mammal in need of such treatment; the treatment modulates the level of versican in the mammal, e . g. , in DP cells.
  • the modulation is promotion and an effective amount of partial or full- length versican protein, partial or full-length versican gene is administered.
  • the modulation is inhibition and an antibody which specifically binds to one or more versican epitopes, or a versican antisense oligo is administered.
  • the invention features, a population of cells, e . g. , DP cells, e . g. , cultured DP cells, which have versican promoter activity.
  • the cells have been separated from cells which do not express the versican promoter, e . g.
  • the population is a population of DP cells, e . g. , cultured DP cells, which has been separated from other DP cells, e . g. , other cultured DP cells which lack versican promoter expressions.
  • the cells are from a transgene animal, e . g. , a mouse.
  • the transgene animal can include a luminescent or fluorescent product coupled to a versican promoter.
  • the cells are a population of versican-expressing DP cells or a population of DP cells having a fluorescent product inside the cells.
  • nucleic acid encoding a promoter coupled to a luminescent or fluorescent product has been introduced into cells of both populations but is expressed only in the first population.
  • a treatment can be an exposure to an environment, to a material, or a combination of both, e . g. , contacting the cell with a compound, a small molecule, a polynucleotide, a polypeptide, a composition, or exposing the cell to a physical or chemical condition including heat-shock, freezing, and laser treatment.
  • a versican promoter is a nucleotide fragment capable of providing promoter activity, e . g. , a fragment containing the nucleotide sequence upstream and/or downstream of a transcription start-site, e . g. , position +1.
  • Modulation refers to promotion or inhibition.
  • Introducing a cell population to a subject refers to any means grafting a cell population to a suitable or desired area in a subject, e . g. , fixing, contacting, implanting, exposing, or incubating a cell population to an area.
  • a suitable or desired area for introducing a population of cells includes but not limited to skin, an area adjoining, contacting, or accessible to epithelial cells, e . g. , epithelial hair germ cells, placode, or keratinocytes .
  • a cell population contains, e . g. , contains substantially desired cells, e . g. , epithelial cells or versican expressing cells means that at least 10% of the cells in the cell population are desired cells, preferably 20%, 50%, 70%, and most preferably 90% of the cells in the cell population are desired cells.
  • a reporter sequence as used herein is a polynucleotide which encodes a product capable of providing a detectable signal to indicate the activity of the promoter which is operably linked to the reporter sequence. Preferred reporter sequences are those which encode products which can luminesce or fluoresce.
  • a particularly suitable reporter is green fluorescent protein. Modified variants of green fluorescent protein, e . g. , EGFP, EBFP, EYFP, d2EGFP, ECFP, GFPuv are included within the term green fluorescent protein. EGFP is particularly preferred. These variants of GFP are commercially available by
  • a reporter sequence can be, a nucleotide sequence encoding a reporter gene, a sequence tag, or a chemically modified sequence activable by absorbing or releasing energy.
  • a reporter sequence is a reporter gene encoding a heterologous protein, preferably a fluorescent protein or a enzymatic substrate .
  • a versican expressing cell refers to a cell containing an active versican gene, e . g. , transcription or translation of versican gene is at a level that is at least technically detectable.
  • a versican expressing cell contains a detectable amount of partial or full-length versican mRNA or protein.
  • a versican expressing cell contains a significant amount of full-length versican mRNA or protein.
  • Fig. 1 is the construction of versican-lacZ transgene.
  • the number in parenthesis indicates the human versican sequence position from the major transcription site as +1 (arrow) .
  • Synthetic splice donor and acceptor sites are labeled SD/SA; polyadenylation signal sequence is labeled pA.
  • the black circle denotes EcoRI ; open circle denotes Xhol ; rectangular box denotes Xbal .
  • versican is the first characterized dermal papilla- specific gene marker for hair inductivity.
  • the 839 bp fragment of the functional human versican promoter (-559 to +280) (Naso, M. F., Zimmermann, D. R. &Iozzo, R. V. (1994) " . Biol . Chem. 269, 32999-33008) was obtained from human genomic DNA. This fragment contains the functional promoter region (-559 to +1) and the first untranslated exon that is not interrupted by an intron. The fragment was inserted in front of the lacZ (/3-galactosidase) reporter gene in the PNASS23 vector (Clontech, San Francisco, CA) . Transgene DNA for pronuclear injection was excised as an EcoRI-Xbal, 4732 bp fragment (Fig.
  • the linearized construct was injected into fertilized oocytes of DBA2xC57BL6 (DBFl) mice, and the eggs were implanted into pseudo-pregnant foster mothers.
  • the offspring (F0) were tested for chromosomal integration of the transgene by Southern hybridization or PCR. All transgenic analyses were performed on F1-F3 hemizygous offspring.
  • lacZ was replaced by the EGFP (Enhanced GFP) gene fragment (Clontech, San Francisco, CA) in the same transgene cassette.
  • Sections were either directly mounted or counterstained with eosin after dewaxing .
  • 5 mm strips of back skin were fixed for 15 min, and subjected to the same procedure. Sections were then counterstained with eosin.
  • transgenic embryo E13.5 or newborn skin was fixed in freshly prepared 4% paraformaldehyde, and processed through a standard paraffin embedding protocol under RNase-free conditions.
  • Digoxigenin-labeled in situ hybridization was performed on 8 ⁇ m paraffin sections, as described previously (Kishimoto, J. , Cox, H. , Keverne, E. B.& Emson, P. C.
  • cDNA for lacZ and mouse versican (nucleotides 243-880, corresponding to a portion of the hyaluronan-binding domain that detects all versican isoforms [8] ) were amplified by RT-PCR.
  • Digoxigenin-labeled antisense and sense RNA probes were prepared by in vitro transcription with T7 RNA polymerase using these cDNAs as templates.
  • Intense lacZ staining was observed in developing fore and hind limbs at the E13.5 embryonic stage of the A4681 line in the region of mesenchymal condensation. This region coincides exactly with the area of prominent endogenous versican expression previously identified immunohistochemically (Kimata, K. , Oike, Y., Tani , K. , Shinomura, T., Yamagata, M. , Uritani, M.& Suzuki, S. (1986) J " . Biol . Chem . 261, 13517-13525) . Transient expression in the ectoderm was also observed in limb regions, but was restricted to the tips of limbs. No skin epidermal expression was observed elsewhere in the body.
  • ⁇ - galactosidase histochemistry revealed lacZ-positive cells in the mesenchyme adjacent to the olfactory epithelium, and in the kidney glomeruli.
  • the perichondrocytes surrounding cartilage, the fore- and hindbrain, facial mesenchyme, blood vessels, and muscle cells also exhibited lacZ staining.
  • Versican promoter drives condensed mesenchymal-specific expression in embryonic transgenic skin
  • LacZ expression in developing skin was examined by X-gal histochemistry on sagittal sections of E13.5, E14.5, E15.5, and E17.5 transgenic mouse embryos.
  • E13.5 ectodermal staining was only observed in the hind and fore limbs. Occasional traces of lacZ staining were found in single mesenchymal cells, which may correlate with the earliest stage of condensation.
  • E14.5 condensed mesenchymal cells immediately beneath the ectodermal placodes were clearly lacZ positive, contrasting strongly with the surrounding negative mesenchymal cells.
  • lacZ staining of condensed mesenchyme under hair plugs appeared more intense than at E14.5 and showed an increased number of lacZ-positive cells.
  • E17.5 the number and intensity of lacZ-positive cells were dramatically increased, yet still virtually restricted to the condensed mesenchyme located at the proximal tips of down-growing hair germs. Similar to the pelage follicle, the condensed mesenchyme of whisker follicles also exhibited lacZ staining at E13.5 and later.
  • the Versican promoter is active in the hair dermal papilla of transgenic skin during the anagen phase of the hair cycling LacZ staining was examined on skin sections from newborn transgenic mice through the second hair cycle.
  • strong staining was confined to the DP cells of pre-formed hair follicles at the anagen (growth) phase.
  • some diffuse staining was also observed in the dermis, especially in the upper dermis and surrounding hair follicles.
  • In situ hybridization for the endogenous mouse versican probe revealed DP-specific mRNA expression at this stage) .
  • transient epithelial lacZ staining was observed in the inner root sheath of the hair follicle.
  • lacZ staining was again restricted entirely to DP cells.
  • catagen transitional phase
  • telogen resting phase
  • no lacZ staining was observed in club (resting) hair.
  • Presumptive second germ DP showed trace lacZ staining.
  • Strong DP-specific staining was again observed in the second anagen hair cycle phase.
  • Dissected skins from newborn versican-GFP transgenic mice (1-3 days old) were floated on a 0.25% trypsin solution (Gibco-BRL, Grand Island, NY) for 16-20 hr at 4°C, after which time the epidermis was discarded.
  • the separated dermis was minced and incubated with 0.25% collagenase for 1 hr at 37°C with gentle stirring to dissociate cells. Microscopic observation revealed that this treatment dissociated most dermal cells, pre-formed follicles, and follicle-associated DP cells.
  • Debris and remaining pre-formed follicles that were not dissociated were removed by passing the cell suspension through a 75 ⁇ m filter followed by low-speed centrifugation to avoid clogging the cell sorter.
  • the resultant cell suspensions were mostly single cells, which allowed sorting for GFP- positive selection.
  • Cell sorting was performed with a Moflo high-speed cell sorter (Cytomation, Ft. Collins,
  • GFP-positive and GFP-negative cells were pooled in collection tubes with 20% fetal calf serum solution. These isolated GFP-positive cells were defined as "sorted DP-derived cells" .
  • Sorted DP-derived cells were plated at 2 x 10 6 cells per 100mm dish and cultured either in DMEM (Life Technologies) with 10% fetal calf serum or complete Chang's medium (Irvine Scientific, Santa Ana, CA) . Cells were passaged every 4 days after trypsin treatment. Some cells were cultured on chamber slides (Nunc Inc., Naperville, IL) for microscopic observation. Fluorescent and phase- contrast images were taken by spot-cooled color digital camera (Diagnostic instruments Inc., Sterling Heights, MI ) and merged images were created using the Adobe Photoshop software program.
  • RNAs were prepared from approximately 5 x 10 6 sorted or cultured DP-derived cells in Trizol solution (Life Technologies, Grand Island, NY) , and first-strand cDNAs were synthesized using the Advantage RT-for-PCR Kit (Clontech) .
  • Semi -quantitative RT-PCR amplifications were performed using the following settings: 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min; 25 cycles.
  • the primers were specific to the sequences for mouse versican sequences (Ito, K. , Shinomura, T., Zako, M. , Ujita, M.& Ki ata, K. (1995) J. Biol . Chem . 270, 958-965): 5'- GACGACTGTCTTGGTGG-3' , SEQ ID No. and 5'-
  • GAPDH 5 ' -TGAAGGTCGGAGTCAACGGA -3', SEQ ID No and 5 ' -GATGGCATGGACTGTGGTCA-3 ' .
  • a second transgenic mouse line was generated, in which the same versican promoter fragment (-559 to +280) was fused with green fluorescent protein (GFP) .
  • the new transgenic line exhibited strong DP-specific GFP fluorescence in newborn skin in the same pattern as that seen in versican-lacZ transgenic mouse skin.
  • Partially dissociated dermal cell suspensions further revealed that strong fluorescence originated from DP cells located at the bottom of pre-formed follicles. After complete dissociation of the dermis by collagenase, these follicle-associated GFP-positive DP cells were released into the cell suspension.
  • RT-PCR analysis showed an abundance of both GFP and versican mRNA in sorted GFP- positive cells relative to sorted negative cells, in which both GFP and versican mRNA were not detected. This confirms that versican promoter-driven GFP expression correlated with endogenous versican expression.
  • GFP-positive cells Most of the sorted GFP-positive cells survived in primary culture. These cultured GFP positives were spindle-shaped, small, aggregating, multi layer-forming cells with a short doubling time in Chang's medium (about 2 days) and a longer doubling time in DMEM + FCS medium (about 4 days) . This morphology and behavior is consistent with that of human scalp DP cells reported previously (Warren, R., Chestnut, M. H. , Wong, T. K. , Otte, T. E., Lammers, K. M.& Meili, M. L. (1992) J. Invest . Dermatol . 98, 693-699). However, even after the first passage (4 days) , the relative number of GFP- fluorescent cells was significantly decreased, with approximately less than 20% of the initial GFP-positive cells remaining. This loss coincided with a decrease in versican mRNA.
  • Either sorted GFP- positive cell suspension (5 x 10 6 ) , sorted negative cell suspension (5 x 10 6 ) , or unsorted cell suspension (1 x 10 7 ) was combined with keratinocytes (4 x 10 6 ) , resuspended in lOO ⁇ l medium, and transferred to a grafting chamber, which was implanted onto the dorsal skin of nude mice (nu/nu) . Sorted positive cells without epithelial cells were also grafted. The chamber was removed after 1 week, and hair formation was assessed 3 weeks later and thereafter. For histological observation grafting sites were dissected 3 weeks after the graft.
  • the cultured GFP- positive cells were harvested with trypsin after first passage (4 days) and re-sorted for GFP positive and negative selection.
  • the same number of resorted GFP- positive and negative cultured cells (5 x 10 6 ) , were grafted with keratinocytes. All animal procedures had the approval of the Massachusetts General Hospital Animal Care and Use Committee.
  • this property of the versican promoter is useful for targeting condensed mesenchyme- specific gene expression, e . g. , in the study of epithelial-mesenchymal interactions in general as well as in the study of hair development. Because the 280 bp of 5 'untranslated region after the transcriptional initiation site exhibits the condensing mesenchyme- specific expression, response elements for DP-specific expression may be confined to the remaining 559 bp of the promoter sequence . Cursory examination of the sequence reveals potential AP-2 (Naso, M. F., Zimmermann, D. R. ⁇ clozzo, R. V. (1994) J. Biol . Chem .

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DU CROS DIANA L ET AL: "Association of versican with dermal matrices and its potential role in hair follicle development and cycling.", JOURNAL OF INVESTIGATIVE DERMATOLOGY, vol. 105, no. 3, 1995, pages 426 - 431, XP001079719, ISSN: 0022-202X *
KISHIMOTO J ET AL: "Human versican promoter-driven LACZ activity in transgenic mouse skin and hair dermal papilla.", JOURNAL OF DERMATOLOGICAL SCIENCE, vol. 16, no. SUPPL. 1, March 1998 (1998-03-01), Third Joint Meeting of the European Society for Dermatological Research, Japanese Society for Investigative Dermatology, Society for Investigative Dermatology;Cologne, Germany; May 7-10, 1998, pages S26, XP001080459, ISSN: 0923-1811 *
ROHDE M ET AL: "THE RETINOBLASTOMA PROTEIN MODULATES EXPRESSION OF GENES CODING FORDIVERSE CLASSES OF PROTEINS INCLUDING COMPONENTS OF THE EXTRACELLULAR MATRIX", ONCOGENE, BASINGSTOKE, HANTS, GB, vol. 12, no. 11, 7 March 1996 (1996-03-07), pages 2393 - 2401, XP001023850, ISSN: 0950-9232 *
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