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Independent Inheritance of Genes Regulating Two Subpopulations of Mouse Clonogenic Keratinocyte Stem Cells

  • Author Footnotes
    1 Current address: Department of Dermatology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, VC-15, New York, New York 10032, USA.
    Natalia V. Popova
    Correspondence
    Department of Dermatology, Columbia University College of Physicians and Surgeons, New York, New York, USA
    Footnotes
    1 Current address: Department of Dermatology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, VC-15, New York, New York 10032, USA.
    Affiliations
    Department of Dermatology, Columbia University College of Physicians and Surgeons, New York, New York, USA
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  • Author Footnotes
    1 Current address: Department of Dermatology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, VC-15, New York, New York 10032, USA.
    Naira E. Suleimanian
    Footnotes
    1 Current address: Department of Dermatology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, VC-15, New York, New York 10032, USA.
    Affiliations
    Department of Dermatology, Columbia University College of Physicians and Surgeons, New York, New York, USA
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  • Author Footnotes
    1 Current address: Department of Dermatology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, VC-15, New York, New York 10032, USA.
    Ekaterina A. Stepanova
    Footnotes
    1 Current address: Department of Dermatology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, VC-15, New York, New York 10032, USA.
    Affiliations
    Department of Dermatology, Columbia University College of Physicians and Surgeons, New York, New York, USA
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  • Kimberly A. Teti
    Affiliations
    Department of Dermatology, Columbia University College of Physicians and Surgeons, New York, New York, USA
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  • Kai Q. Wu
    Affiliations
    Department of Dermatology, Columbia University College of Physicians and Surgeons, New York, New York, USA
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  • Author Footnotes
    1 Current address: Department of Dermatology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, VC-15, New York, New York 10032, USA.
    Rebecca J. Morris
    Footnotes
    1 Current address: Department of Dermatology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, VC-15, New York, New York 10032, USA.
    Affiliations
    Department of Dermatology, Columbia University College of Physicians and Surgeons, New York, New York, USA
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  • Author Footnotes
    1 Current address: Department of Dermatology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, VC-15, New York, New York 10032, USA.
      Mouse keratinocyte stem cells originate from the bulge of hair follicle, and, according o definition, possess a clonogenic activity in vitro. We have investigated seven inbred (C57BL/6, C3H, DBA/2, BALB/c, FVB) and outbred (SENCAR, CD-1) mouse strains and found that three genetically distinct subsets of mouse strains differ significantly in the frequency of clonogenic activity in vitro. The analysis of keratinocyte colonies in two reciprocal backcross [C57BL/6 × (BALB/c × C57BL/6); BALB/c × (BALB/c × C57BL/6)] and intercross [(BALB/c × C57BL/6)F2] of BALB/c and C57BL/6 mice allowed us to identify two subpopulations of clonogenic keratinocytes able to produce small (less than 2 mm2) and large (more than 2 mm2) colonies. We conducted linkage analysis and found that small colonies associated with mouse chromosomes 1, 6, 7, 8, and 9; but large colonies—with the chromosome 4. We defined locus on the chromosome 9 that associated with small colonies as keratinocyte stem cell locus 1 (Ksc1), and locus on the mouse chromosome 4 associated with large colonies-keratinocyte stem cell locus 2 (Ksc2). Ksc1 and loci on chromosomes 6 and 7 are close if not equal to loci associated with sensitivity to skin carcinogenesis. We conclude that two subpopulations of stem cells able to produce small and large colonies regulated by different genes and genes regulating small colonies might be responsible for sensitivity to skin carcinogenesis.

      Keywords

      Abbreviations:

      DMBA
      7,12-dimethylbenz(a)anthracene
      KC
      keratinocyte colony
      TPA
      12-O-tetradecanoylphorbol-13-acetate
      The epidermis is a continuously renewing tissue in which keratinocytes are organized into multiple layers and undergo terminal differentiation as they move through the suprabasal layers toward the skin surface. The basal layer in adult mice consists primarily of keratinocytes, Langerhans cells (∼10%), melanocytes (∼3%), and Merkel cells (rare) (
      • Leblond C.P.
      Classification of cell populations on the basis of their proliferative behavior.
      ;
      • Potten C.S.
      Stem cells in epidermis from the back of the mouse.
      ). Hair follicles are highly specialized cutaneous appendages contiguous with both the interfollicular epidermis and the sebaceous gland. A small population of self-renewing stem cells produces proliferative cells that undergo a series of amplification division prior to terminal differentiation. Stem cells are responsible for epidermal homeostasis and are required throughout life to maintain the self-renewing epithelium.
      The traditional approach to the recognition of mouse keratinocyte stem cells has been the identification of slow-cycling label-retaining cells. Such cells in the bulge region of hair follicles have been demonstrated in adult mice after neonatal [3H]-thymidine injection (
      • Cotsarelis G.
      • Sun T.T.
      • Lavker R.M.
      Label-retaining cells reside in the bulge area of pilosebaceous unit: Implication for follicular stem cells, hair cycle, and skin carcinogenesis.
      ;
      • Morris R.
      • Potten C.
      Highly persistent label-retaining cells in the hair follicles of mice and their fate following induction of anagen.
      ). Hence, the hair follicle bulge region might function as a “niche” for most mouse keratinocyte stem cells.
      Keratinocyte colony-forming cells are closely related or identical to multipotential stem cells. Most (95%) clonogenic keratinocytes originate from the bulge region of hair follicles (
      • Kobayashi K.
      • Rochat A.
      • Barrandon Y.
      Segregation of keratinocyte colony–forming cells in the bulge of the rat vibrissa.
      ;
      • Rochat A.
      • Kobayashi K.
      • Barrandon Y.
      Location of stem cells of human hair follicles by clonal analysis.
      ;
      • Oshima H.
      • Rochat A.
      • Kedzia C.
      • Kobayashi K.
      • Barradon Y.
      Morphogenesis and renewal of hair follicles from adult multipotent stem cells.
      ). Moreover, slow-cycling, label-retaining cells possess clonogenic activity in vitro (
      • Morris R.
      • Potten C.
      Slow cycling (label-retaining) epidermal cells behave like clonogenic stem cells in vitro.
      ). Thus, most visible mouse keratinocyte colonies are thought to originate from keratinocyte stem cells (
      • Oshima H.
      • Rochat A.
      • Kedzia C.
      • Kobayashi K.
      • Barradon Y.
      Morphogenesis and renewal of hair follicles from adult multipotent stem cells.
      ), and clonogenic activity in vitro is one characteristic of mouse keratinocyte stem cells (
      • Oshima H.
      • Rochat A.
      • Kedzia C.
      • Kobayashi K.
      • Barradon Y.
      Morphogenesis and renewal of hair follicles from adult multipotent stem cells.
      ).
      Watt and colleagues distinguished human keratinocyte stem cells from transit-amplifying cells based on the type of colony they form (
      • Zhu A.J.
      • Hoose I.
      • Watt F.
      Signalling via β1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro.
      ). Stem cells form self-renewing colonies (
      • Zhu A.J.
      • Hoose I.
      • Watt F.
      Signalling via β1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro.
      ; Morris, unpublished), whereas transit-amplifying colonies containing fewer then 30–40 cells, all of which undergo terminal differentiation (
      • Zhu A.J.
      • Hoose I.
      • Watt F.
      Signalling via β1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro.
      ), and can be identified by microscopy at low magnification. Therefore, most visible colonies probably originate from keratinocyte stem cells.
      We have identified several factors that might influence the number of keratinocyte colonies. Although colony number remains virtually constant throughout most of adult life in mice (
      • Morris R.J.
      • Tacker K.C.
      • Fischer S.M.
      • Slaga T.J.
      Quantitation of primary in vitro clonogenic keratinocytes from normal adult murine epidermis following initiation and during promotion of epidermal tumors.
      ), the frequency of colonies increases during tumor promotion of initiated skin (
      • Morris R.J.
      • Tacker K.C.
      • Fischer S.M.
      • Slaga T.J.
      Quantitation of primary in vitro clonogenic keratinocytes from normal adult murine epidermis following initiation and during promotion of epidermal tumors.
      ). Thus, amplification of keratinocyte stem cells may play a role in skin carcinogenesis, and genes regulating keratinocyte colony number might be involved in regulating of skin carcinogenesis. The identification of regulatory genes involved in stem cell amplification would enhance the understanding of normal skin homeostasis and skin carcinogenesis. To address this issue, we carried out genetic analyses of keratinocyte colony formation in different mouse strains, and their genetic crosses.

      Results

      Mouse keratinocyte colony number is a quantitative multigenic trait

      Recently we demonstrated that five inbred (C57BL/6, C3H, DBA/2, BALB/c, FVB) and two outbred (CD-1, SENCAR) mouse strains differ significantly in the number of keratinocyte colony forming cells per 1000 viable cells isolated from the back of mouse skin (
      • Popova N.V.
      • Tryson K.A.
      • Wu K.Q.
      • Morris R.J.
      Evidence that keratinocyte colony number is quantitatively controlled.
      ) (Figure 1). Keratinocyte colony frequency did not depend on numbers of hair follicles, basal or suprabasal cells, mitotic cells per cm2 of skin, or percentage of viable cells per cm2 of skin. Furthermore, there was no significant difference between high and low colony-expressing mice in the number of attached cells (
      • Popova N.V.
      • Tryson K.A.
      • Wu K.Q.
      • Morris R.J.
      Evidence that keratinocyte colony number is quantitatively controlled.
      ). This finding suggested that the different colony numbers reflected differences in the genetic background of these mouse strains.
      Figure thumbnail gr1
      Figure 1Keratinocyte colony-forming ability in seven mouse strains (mean±SEM/1000 viable cells). C57BL/6, 84.3±3.2; C3H, 39.7±4.5; DBA/2, 36.8±2.1; SENCAR, 36.5±2.7; BALB/c, 36.4±1.7; FVB, 24.4±2.9, and CD-1, 16.8±1.5
      We identified three subsets of mice that differed significantly in the frequency of clonogenic stem cells: C57BL/6>SENCAR=C3H=DBA/2=BALB/c>FVB=CD-1. C57BL/6 mice have high number of colonies per 1000 viable cells. SENCAR, C3H, DBA/2, BALB/c have intermediate colony numbers, whereas FVB, and CD-1 mice have low colony frequency (Figure 1). We found that the mouse strains from each subset had different genetic origins: based on the genealogies of mouse inbred strains (
      • Beck J.A.
      • Lloyd S.
      • Hefezparasr M.
      • Lennon-Pierce M.
      • Eppig J.T.
      • Festing M.F.W.
      • Fisher E.M.C.
      Genealogies of mouse inbred strains.
      ). C57BL/6 mice are in category E; BALB/c, C3H, DBA/2 mice are in category B; and CD-1 and FVB mice are in category A (Figure 2). The finding of significant differences in keratinocyte colony numbers in three subsets of inbred, genetically distinct mouse strains further suggests that colony number is genetically regulated.
      Figure thumbnail gr2
      Figure 2Genealogies of C57BL/6, BALB/c, DBA, C3H, CD-1, FVB mouse strains (
      • Beck J.A.
      • Lloyd S.
      • Hefezparasr M.
      • Lennon-Pierce M.
      • Eppig J.T.
      • Festing M.F.W.
      • Fisher E.M.C.
      Genealogies of mouse inbred strains.
      )
      The biological significance of our observation that genetically distinct mouse strains have difference in the frequency of keratinocyte stem cells continue to be a mystery. But it also had been demonstrated for the blood (
      • Haan G.
      • Nijhof W.
      • Van Zant G.
      Mouse strain-dependent changes in frequency and proliferation of hematopoietic stem cells during aging: Correlation between lifespan and cycling activity.
      ) and liver stem cells (
      • Kolesnichenko T.S.
      • Popova N.V.
      Growth-stimulating effect of some nitroso-compounds on organ cultures of embryonic liver of mice and rats.
      ), and appears to be associated with the difference in life span of the investigated mouse strains. It is interesting that high keratinocyte colony number is associated with long-lived mouse strain (C57BL/6) (
      • Storer J.B.
      Longevity and gross pathology and death in 22 inbred strains of mice.
      ;
      • Goodrick C.L.
      Life span and the inheritance of longevity of inbred mice.
      ), and all other investigated mouse strains have shorter life span (
      • Festing M.F.W.
      • Blackmore D.K.
      Life span of specified-pathogen-free (MRC category 4) mice and rats.
      ). Therefore, we suggest that high frequency of clonogenic stem cells associated with long living mouse strains. Finally, our finding of the genetic regulation of keratinocyte colony frequency, and significant differences between different strains enabled us to map the gene(s) that regulate the high and low frequency of keratinocyte stem cells.

      Frequencies of stem cells able to produce small and large colonies are quantitative multigenic traits

      It is well documented that inheritance of quantitative characters is generally under the control of more than one gene (
      • Falcone D.S.
      Quantitative inheritance.
      ). Recently, we have demonstrated that clonogenic epidermal stem cells are a heterogeneous population (
      • Popova N.V.
      • Teti K.A.
      • Wu K.
      • Morris R.J.
      Identification of two keratinocyte stem cell regulatory loci implicated in skin carcinogenesis.
      ). We have identified at least two subpopulations of clonogenic keratinocytes able to produce small (less than 2 mm2) and large (more than 2 mm2) colonies, and determined that they are regulated by the different genes (
      • Popova N.V.
      • Teti K.A.
      • Wu K.
      • Morris R.J.
      Identification of two keratinocyte stem cell regulatory loci implicated in skin carcinogenesis.
      ). The frequencies of small and large colonies differed significantly between C57BL/6 and BALB/c mice (Figure 3). Moreover, they have a quantitative inheritance in these two strains of mice. Therefore, we concluded that the numbers of small and large colonies are new quantitative multigenic traits.
      Figure thumbnail gr3
      Figure 3(A) Rank order of small colonies (within area of 2 mm2) from smallest to largest area in parental C57BL/6 and BALB/c mice after 2 wk of cultivation: 543 small colonies of C57BL/6 mice were found in 988 all colonies, which were measured from 12 dishes from seven mice; 863 small colonies of BALB/c mice were found in 993 all colonies, which were measured from 18 dishes from 12 mice. Frequency of small colonies in BALB/c mice was higher then in C57BL/6 mice with p<0.0001. (B) Rank order of large colonies (within area >2 mm2) from smallest to largest area in parental C57BL/6 and BALB/c mice after 2 wk of cultivation: 445 large colonies of C57BL/6 mice were found in 988 all colonies, which were measured from 12 dishes from seven mice; 130 large colonies of BALB/c mice were found in 993 all colonies, which were measured from 18 dishes from 12 mice. Frequency of large colonies in C57BL/6 mice was higher then in BALB/c mice with p<0.0001. Analysis Wilcoxon's rank sum test procedure
      To determine the heritability of these two quantitative traits (frequency of small and large colonies), we investigated several genetic crosses between BALB/c and C57BL/6 mice (Table I, Figure 4). We determined that the ratio of small and large colonies per 1000 viable cells was 1:1 in C57BL/6 mice, whereas in BALB/c mice, this parameter was 9:1. The small and large colonies were inherited in a 3:1 ratio in the first hybrid (BALB/c × C57BL/6)F1, and this result was equivalent to the backcross (BALB/c × F1) mice (Table I, Figure 4). It is interesting that the ratio of the small and large colony frequency in the (C57BL/6 × F1) backcross was equivalent to parental strain C57BL/6 (1:1), and this trait in F2 generation was almost equivalent to the parental strain BALB/c (8.3:1.6). These results suggest that the frequency of small and large colonies have independent modes of inheritance, and genes regulating number of small colonies are differed from the genes regulating a number of large colonies.
      Table IIndependent inheritance of small and large colonies in several genetic crosses
      Groups of miceNumber of miceAverage
      Average number of small colonies per 1000 viable cells±SD
      number of small colonies per %
      Average
      Average number of large colonies per 1000 viable cells±SD
      number of large colonies per %
      Ratio of the number of small and large colonies
      C57BL/6742±13 (54%)36±12 (46%)1:1
      BALB/c1253±19 (91%)6±3 (9%)9:1
      CBF11041±15 (71%)17±8 (29%)3:1
      C57BL/6 × F14538±24 (49%)39±23 (51%)1:1
      BALB/c × F14431±12 (73%)12±8 (27%)3:1
      F210455±21 (84%)11±7 (16%)8.3:1.6
      a Average number of small colonies per 1000 viable cells±SD
      b Average number of large colonies per 1000 viable cells±SD
      Figure thumbnail gr4
      Figure 4Small and large keratinocyte colony formation after 2 wk of cultivation from parental strains C57BL/6 and BALB/c and their segregating genetic crosses (BALB/c × C57BL/6)F1, C57BL/5 × F1, BALB/c × F1, (F1 × F1) F2. The frequency of small and large colonies were represented in ratio 1:1 for C57Bl/6; 9:1 for BALB/c; 3:1 for (BALB/c × C57BL/6)F1; 1:1 for C57BL/6 × (BALB/c × C57BL/6)F1; 3:1 for BALB/c × (BALB/c × C57BL/6)F1; and 8.3:1.6 for (BALB/c × C57BL/6)F2 mice

      Identification of keratinocyte stem cell regulatory loci

      To map genes regulating numbers of both small and large colonies, we separated all large and small colonies from each individual dish, and monitored loci associated with numbers of small and large colonies in the two backcrosses and intercross mice using essentially as described by
      • Angel J.
      • Popova N.
      • Lanko N.
      • Turusov V.
      • DiGiovanni J.
      A locus that influences susceptibility to 1,2-dimethylhydrazine-induced colon tumors maps to the distal end of mouse Chromosome 3.
      . Clonogenic activity of keratinocytes from 104 intercross mice was initially investigated (
      • Popova N.V.
      • Tryson K.A.
      • Wu K.Q.
      • Morris R.J.
      Evidence that keratinocyte colony number is quantitatively controlled.
      ) and the same protocol was used to determine the number of small and large colonies in intercross and backcross mice. Uses of 149 microsatellite polymorphic markers allowed us to generate a complete genome-wide scan at about 15 cM intervals. In regions suggestive of linkage small and large colony number, the density of markers was increased to obtain more accurate genetic dissection in the area. Overall, genomic coverage was about 95.3 percent across the 19 autosomal chromosomes. The keratinocyte colony number in mice homozygous and heterozygous for each locus was compared using the Qlink program based on the Wilcoxon rank test.
      Recently, we have identified that the number of small colonies was linked to loci on mouse chromosomes 1, 6, 7, 8, 9, whereas the number of large colonies were linked to mouse chromosome 4 (
      • Popova N.V.
      • Teti K.A.
      • Wu K.
      • Morris R.J.
      Identification of two keratinocyte stem cell regulatory loci implicated in skin carcinogenesis.
      ). Moreover, we have established that Ksc1 regulated the frequency of small colonies, whereas Ksc2—the frequency of the large colonies. Hence, different genes are involved in the regulation of the frequency of small and large colonies.
      In order to identify gene(s) regulating frequency of small colonies in the Ksc1 locus we performed genetic mapping of the region on mouse chromosome 9 with higher resolution (Table II) and combined our data of backcross and intercross mice (Table III). Multilocus analysis of the chromosome 9 (Tables III) suggested that the most likely interval surrounding 41 and 49 cM in the mid-portion and distal region of the chromosome 9 contained the gene(s) involved in regulating the number of keratinocyte stem cells. Association of an increased number of small keratinocyte colonies with inheritance of the C57BL/6 allele was detected for the region between D9Mit262 and D9Mit273 loci with combined LOD score=4.18 (genome-wide p value= 0.0072) and 4.23 (p value for whole genome=0.0065), respectively. Hence, according to
      • Lander E.
      • Kruglyak L.
      Genetic dissection of complex traits: Guidelines for interpreting and reporting linkage results.
      our Ksc1 locus in the central–distal region of the mouse chromosome 9 is closed to highly significant linkage and expected to occur 0.007–0.006 times in a genome scan. We propose that genes regulating frequency of small colonies are located between 41 and 49 cM from centromere (Table III).
      Table IILoci associated with small keratinocyte colony number in intercross between BALB/c and C57BL/6 after 2 weeks of cultivation
      Colony
      Mean number of colonies±SD
      number
      Marker
      Microsatellites markers
      Map
      Map location in cM from the centromere
      location (cM)
      aa
      a/a- homozygous genotype with two copies of the BALB/c allele
      a/b
      a/b- heterozygous genotype with one copy of the BALB/c allele and one copy of the C57BL/6 allele
      bb
      b/b-homozygous genotype with two copies of the C57BL/6 alleles
      p value
      Single-point significance levels. Number of colonies was compared between genotypes using the Wilcoxon rank sum test (two sides). p Value was calculated using the program Qlink
      LOD
      LOD equivalent scores. The individual LOD equivalent score were calculated as described by Kruglyak and Lander
      score
      Genome
      Genome wide p value <0.05 significant for whole genome
      wide p value
      D9Mit2471754±2254±2158±190.20.3
      D9Mit1912653±2353±2060±210.090.6
      D9Mit1623054±2353±2160±190.040.85
      D9Mit3033555±1950±2263±190.004
      Single-point combine p value of the colony number associated with one homozygous genotype (two C57BL/6 alleles or two BALB/c alleles) and corresponding heterozygous genotype (one C57BL/6 allele and one BALB/c allele) that have no significant differences (d.f.=1)
      1.711
      D9Mit3363555±1950±2265±180.001
      Single-point combine p value of the colony number associated with one homozygous genotype (two C57BL/6 alleles or two BALB/c alleles) and corresponding heterozygous genotype (one C57BL/6 allele and one BALB/c allele) that have no significant differences (d.f.=1)
      2.090.6
      D9Mit1043555±1950±22.265±18.80.001
      Single-point combine p value of the colony number associated with one homozygous genotype (two C57BL/6 alleles or two BALB/c alleles) and corresponding heterozygous genotype (one C57BL/6 allele and one BALB/c allele) that have no significant differences (d.f.=1)
      2.090.6
      D9Mit2893854±1950±2265±180.001
      Single-point combine p value of the colony number associated with one homozygous genotype (two C57BL/6 alleles or two BALB/c alleles) and corresponding heterozygous genotype (one C57BL/6 allele and one BALB/c allele) that have no significant differences (d.f.=1)
      2.280.4
      D9Mit1664151±1751±2266±180.0002
      Single-point combine p value of the colony number associated with one homozygous genotype (two C57BL/6 alleles or two BALB/c alleles) and corresponding heterozygous genotype (one C57BL/6 allele and one BALB/c allele) that have no significant differences (d.f.=1)
      2.860.1
      D9Mit2624152±1948±1869±210.00002
      Single-point combine p value of the colony number associated with one homozygous genotype (two C57BL/6 alleles or two BALB/c alleles) and corresponding heterozygous genotype (one C57BL/6 allele and one BALB/c allele) that have no significant differences (d.f.=1)
      3.880.0139
      D9Mit84251±1751±2266±180.0002
      Single-point combine p value of the colony number associated with one homozygous genotype (two C57BL/6 alleles or two BALB/c alleles) and corresponding heterozygous genotype (one C57BL/6 allele and one BALB/c allele) that have no significant differences (d.f.=1)
      2.850.1
      D9Mit1564251±1751±2266±180.0002
      Single-point combine p value of the colony number associated with one homozygous genotype (two C57BL/6 alleles or two BALB/c alleles) and corresponding heterozygous genotype (one C57BL/6 allele and one BALB/c allele) that have no significant differences (d.f.=1)
      2.850.1
      D9Mit2364353±19.947±18.469±21.50.00002
      Single-point combine p value of the colony number associated with one homozygous genotype (two C57BL/6 alleles or two BALB/c alleles) and corresponding heterozygous genotype (one C57BL/6 allele and one BALB/c allele) that have no significant differences (d.f.=1)
      3.840.0153
      D9Mit1334355±18.749±22.366±17.90.0003
      Single-point combine p value of the colony number associated with one homozygous genotype (two C57BL/6 alleles or two BALB/c alleles) and corresponding heterozygous genotype (one C57BL/6 allele and one BALB/c allele) that have no significant differences (d.f.=1)
      2.770.1
      D9Mit2694355±1849±2265±170.0006
      Single-point combine p value of the colony number associated with one homozygous genotype (two C57BL/6 alleles or two BALB/c alleles) and corresponding heterozygous genotype (one C57BL/6 allele and one BALB/c allele) that have no significant differences (d.f.=1)
      2.520.2
      D9Mit1964856±18.949±22.365±180.0006
      Single-point combine p value of the colony number associated with one homozygous genotype (two C57BL/6 alleles or two BALB/c alleles) and corresponding heterozygous genotype (one C57BL/6 allele and one BALB/c allele) that have no significant differences (d.f.=1)
      2.540.2
      D9Mit2734956±18.849±22.265±18.20.0006
      Single-point combine p value of the colony number associated with one homozygous genotype (two C57BL/6 alleles or two BALB/c alleles) and corresponding heterozygous genotype (one C57BL/6 allele and one BALB/c allele) that have no significant differences (d.f.=1)
      2.521
      D9Mit1825555±18.350±1963±250.0256
      Single-point combine p value of the colony number associated with one homozygous genotype (two C57BL/6 alleles or two BALB/c alleles) and corresponding heterozygous genotype (one C57BL/6 allele and one BALB/c allele) that have no significant differences (d.f.=1)
      1.081
      D9Mit516154±1852±1962±260.20.25
      a Microsatellites markers
      b Map location in cM from the centromere
      c Mean number of colonies±SD
      d Single-point significance levels. Number of colonies was compared between genotypes using the Wilcoxon rank sum test (two sides). p Value was calculated using the program Qlink
      e LOD equivalent scores. The individual LOD equivalent score were calculated as described by Kruglyak and Lander
      f Genome wide p value <0.05 significant for whole genome
      g a/a- homozygous genotype with two copies of the BALB/c allele
      h a/b- heterozygous genotype with one copy of the BALB/c allele and one copy of the C57BL/6 allele
      i b/b-homozygous genotype with two copies of the C57BL/6 alleles
      j Single-point combine p value of the colony number associated with one homozygous genotype (two C57BL/6 alleles or two BALB/c alleles) and corresponding heterozygous genotype (one C57BL/6 allele and one BALB/c allele) that have no significant differences (d.f.=1)
      Table IIILinkage of small colony frequency with mouse chromosomes 8 and 9
      Backcross C57BL/6 × F1F2Combined
      MarkerMap location (cM)p
      Single-point significant levels
      LOD
      LOD equivalent scores. The individual equivalent scores were calculated as described by Kruglyak and Lander (1995)
      p
      Single-point significant levels
      LOD
      LOD equivalent scores. The individual equivalent scores were calculated as described by Kruglyak and Lander (1995)
      p
      Genome wide p value <0.05 significant for whole genome
      LOD
      The combined LOD equivalent score, is the sum of the individual LOD equivalent scores, was calculating using the program Qlink
      D8Mit5810.040.840.0021.940.12.7
      D8Mit94130.021.060.030.980.62.0
      D8Mit339230.021.10.040.911.4
      D9Mit191260.40.10.090.611.5
      D9Mit262410.040.840.000023.880.0074.18
      D9Mit133430.0041.780.00032.770.0034.46
      D9Mit273490.0041.740.00062.520.0034.22
      D9Mit182550.030.980.021.080.62.06
      D9Mit51610.050.80.20.251.36
      a Single-point significant levels
      b Genome wide p value <0.05 significant for whole genome
      c LOD equivalent scores. The individual equivalent scores were calculated as described by
      • Kruglyak L.
      • Lander E.
      A nonparametric approach for mapping quantitative trait loci.
      d The combined LOD equivalent score, is the sum of the individual LOD equivalent scores, was calculating using the program Qlink
      Suggestive evidence (
      • Lander E.
      • Kruglyak L.
      Genetic dissection of complex traits: Guidelines for interpreting and reporting linkage results.
      ) for linkage with the number of small colonies was found with markers on mouse chromosomes 1, 6, 7, 8 (Figure 5). On chromosome 1, the D1Mit178 locus had a LOD score of 1.65. Analysis of twelve markers on this chromosome suggests that a gene influencing the number of small colonies may reside in a 35-cM interval from D1Mit178 to D1Mit132. A third wide region showing an association with number of small colonies with C57BL/6 alleles was found on chromosome 6 between D6Mit93 and D6Mit323. A peak suggestive LOD score of 2.25 was obtained at D6Mit99. A fourth region with suggestive linkage to a high number of small colonies was identified between D7Mit66 and D7Mit259. A peak LOD score of 2.16 was obtained at D7Mit105 (63.5cM), and a peak LOD score of 2.7 (combined data of backcross and intercross, Table III) was obtained at locus D8Mit58 near the centromere of Chromosome 8.
      Figure thumbnail gr5
      Figure 5Chromosomal map location of mouse microsatellite markers used in association with small and large KC number in the intercross of BALB/c and C57BL/6 mice. Horizontal lines represent all markers were used. Gray horizontal lines represent markers showing significant association with the number of small (Chr. 1, 6, 7, 8, and 9) large (Chr. 4) colonies
      Suggestive linkage to large colony number was obtained for the markers on mouse chromosome 4 (Figure 5). D4Mit264 and D4Mit181 had a LOD score of 3.05. Analysis of eight markers on chromosome 4 suggested that the gene determining a high number of large colonies was situated between D4Mit264 (1.9 cM) and D4Mit181 (2.5 cM), and was associated with BALB/c alleles. Thus, gene(s) regulating the high number of large colonies reside in the D4Mit264 region. We designated this locus keratinocyte stem cell locus 2 (Ksc2).

      Discussion

      The keratinocyte stem cell regulatory mechanism and the involvement of stem cells in skin carcinogenesis continue to be a matter of intense investigation. Recently we have established that the frequency of clonogenc keratinocyte stem cells is a genetically defined and quantitative complex trait (
      • Popova N.V.
      • Tryson K.A.
      • Wu K.Q.
      • Morris R.J.
      Evidence that keratinocyte colony number is quantitatively controlled.
      ). Moreover, we demonstrated that clonogenic epidermal stem cells are a heterogeneous population, and mouse epidermis has at least two subpopulations of keratinocyte stem cells, which were regulated by the different genes (
      • Popova N.V.
      • Teti K.A.
      • Wu K.
      • Morris R.J.
      Identification of two keratinocyte stem cell regulatory loci implicated in skin carcinogenesis.
      ). Hence, we suggested that different types of clonogenic keratinocytes could be involved in the regulation of different process in mouse epidermis.
      We have determined that these two types of keratinocytes able to produce small (>2 mm2) and large (<2 mm2) colonies in vitro have the independent modes of inheritance. We found that the ratio of small and large colony frequency is different in C57BL/6 and in BALB/c mice. Thus, the ratio of small and large colonies in C57BL/6 mice was 1:1, whereas in BALB/c mice this parameter was 9:1. Despite that the number of all colonies was significantly higher in C57BL/6 mice (
      • Popova N.V.
      • Tryson K.A.
      • Wu K.Q.
      • Morris R.J.
      Evidence that keratinocyte colony number is quantitatively controlled.
      ), the number of small colonies was higher BALB/c mice (p<0.0001, Figure 3a), whereas the number of large colonies was higher in C57BL/6 mice (p<0.0001, Figure 3b). Moreover, the frequency of small and large colonies inherited independently in different genetic crosses (Figure 4). This observation suggested that we could find the genes that regulating these two types of clonogenic stem cells.
      The linkage analysis suggested that the gene(s) regulating the frequency of small colonies are situated on the mouse chromosome 1, 6, 7, 8, and 9 (Figure 5). Previously we have established that several factors, including in vivo treatment with the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) and the promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) involved in two-stage skin carcinogenesis, might influence the frequency of clonogenic cells (
      • Morris R.J.
      • Tacker K.C.
      • Fischer S.M.
      • Slaga T.J.
      Quantitation of primary in vitro clonogenic keratinocytes from normal adult murine epidermis following initiation and during promotion of epidermal tumors.
      ). This observation brought us to conclude that keratinocytes able to produce colonies in vitro might be involved in skin carcinogenesis. Comparing of the loci regulating the frequency of small keratinocyte colonies with the loci published in association with resistance or susceptibility to two-stage skin carcinogenesis revealed overlap between a locus involved in skin tumor promotion, Psl1, previously mapped to a region of Chromosome 9 (
      • Angel J.
      • Beltran L.
      • Minda K.
      • Rupp T.
      • DiGiovanni J.
      Association of murine chromosome 9 locus (PsI1) with susceptibility to mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.
      ), and the Ksc1 locus (
      • Popova N.V.
      • Teti K.A.
      • Wu K.
      • Morris R.J.
      Identification of two keratinocyte stem cell regulatory loci implicated in skin carcinogenesis.
      ). Moreover,
      • Mock B.
      • Lowry D.
      • Rehman I.
      • Padlan C.
      • Yuspa S.
      • Hennings H.
      Multigenic control of skin tumor susceptibility in SENKAR/Pt mice.
      reported a higher than expected linkage score for the marker D9Mit271 (48 cM) in association with skin tumor susceptibility. Recently, Skts6, skin tumor susceptibility locus 6, have been mapped on the mouse chromosome 9 (49 cM) (
      • Nagase H.
      • Mao J.H.
      • Balmain A.
      A subset of skin tumor modifier loci determines survival time of tumor-bearing mice.
      ). These loci (D9Mit271, and Skts6) are also closely related to our Ksc1 (Figure 5). Thus, gene(s) on chromosome 9 regulating the number of small keratinocyte colonies might also affect sensitivity/resistance to skin tumor promotion, or skin tumor development.
      • Nagase H.
      • Bryson S.
      • Cordell H.
      • Kemp C.
      • Fee F.
      • Balmain A.
      Distinct genetic loci control development of benign and malignant skin tumors in mice.
      identified locus Skts2 linked to papilloma development on mouse chromosome 7. The Mus spretus allele of Skts2 (64 cM) in the distal region of chromosome 7 is associated with female-specific papilloma resistance, whereas the Mus musculus allele in this locus is associated with susceptibility to papilloma development. In our intercross mice, the BALB/c allele of D7Mit105 (63.5 cM) locus showed suggestive association with the high number of small colonies in almost the same region as Skts2. Thus, a gene(s) regulating small keratinocyte colony number might be involved in the regulation of susceptibility or resistance to papilloma development in the Skts2 locus.
      Skts11 (36.5cM) is situated on mouse Chromosome 6, and linked with susceptibility or resistance to benign and malignant skin tumors (
      • Nagase H.
      • Bryson S.
      • Cordell H.
      • Kemp C.
      • Fee F.
      • Balmain A.
      Distinct genetic loci control development of benign and malignant skin tumors in mice.
      ,
      • Nagase H.
      • Mao J.H.
      • Balmain A.
      A subset of skin tumor modifier loci determines survival time of tumor-bearing mice.
      ). The same region on mouse chromosome 6, D6Mit99 (36cM) with LOD score peak 2.3 linked with a high frequency of small colonies (
      • Popova N.V.
      • Teti K.A.
      • Wu K.
      • Morris R.J.
      Identification of two keratinocyte stem cell regulatory loci implicated in skin carcinogenesis.
      ). Interestingly, the gene for transforming growth factor-α located on chromosome 6 at 35.8 cM from the centromere has been implicated in papilloma development (
      • Vassar R.
      • Hutton M.E.
      • Fuchs E.
      Transgenic overexpression of transforming growth factor alpha bypasses the need for c-Ha-ras mutation in mouse skin tumorigenesis.
      ) and might also influence keratinocyte colony number (Morris, unpublished data). Together, the data suggest that a gene(s) linked with the number of small colonies on chromosome 6, also influences the development of benign and malignant skin tumors. Finally, the Ksc1 locus on Chromosome 9 and two minor suggestive loci on chromosomes 7 and 6 are surprisingly close to loci found to be associated with skin tumor promotion (
      • Angel J.
      • Beltran L.
      • Minda K.
      • Rupp T.
      • DiGiovanni J.
      Association of murine chromosome 9 locus (PsI1) with susceptibility to mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.
      ,
      • Angel J.
      • Caballero M.
      • DiGiovanni J.
      Confirmation of the mapping of a 12-O-tetradecanoylphorbol-13-acetate promotion susceptibility locus, Psl1 to distal mouse chromosome 9.
      )/sensitivity/resistance to skin tumor development (
      • Mock B.
      • Lowry D.
      • Rehman I.
      • Padlan C.
      • Yuspa S.
      • Hennings H.
      Multigenic control of skin tumor susceptibility in SENKAR/Pt mice.
      ,
      • Nagase H.
      • Bryson S.
      • Cordell H.
      • Kemp C.
      • Fee F.
      • Balmain A.
      Distinct genetic loci control development of benign and malignant skin tumors in mice.
      ,
      • Nagase H.
      • Mao J.H.
      • Balmain A.
      A subset of skin tumor modifier loci determines survival time of tumor-bearing mice.
      ). We hypothesize that the population of clonogenic keratinocytes able to produce small colonies represents a specific population of skin stem cells responsible for sensitivity or resistance to skin carcinogenesis. Identification of loci responsible for sensitivity to skin carcinogenesis awaits further studies of other genetic crosses with strains susceptible to skin carcinogenesis.
      The Ksc1 region of mouse chromosome 9 shares linkage homology with human chromosomes 15q22.1 (MAP2K1), 15q21 (TCF12), 15q21-q22 (ADAM10), 6pter-qter (BMP5), 14pter-qter (COX7A3). The region of the Ksc1 locus contains a cluster of genes responsible for normal mouse development (Adam10, 4; Foxb1a; Bmp5; Hnf6; Tcf12; Om2a, 2b) and possibly involved in regulation of keratinocyte stem cell number through their effects on growth and differentiation (
      • Neurman T.
      • Keen A.
      • Knapik E.
      • Shain D.
      • Ross M.
      • Nornes H.O.
      • Zuber M.X.
      ME1 and GE1: Basic helix–loop–helix transcriptional factors expressed at high levels in the developing nervous system and in morphogenitically active regions.
      ;
      • Sasaki H.
      • Hogan B.L.
      Differential expression of multiple fork head related genes during gastration and axial pattern formation in the mouse embryo.
      ;
      • West M.F.
      • Verrotti A.C.
      • Salles F.J.
      • Tsirka S.E.
      • Strickland S.
      Isolation and characterization of two novel, cytoplasmically polyadenylated, oocyte-specific, mouse maternal RNAs.
      ;
      • Wolfsberg T.G.
      • White J.M.
      ADAMs in fertilization and development.
      ;
      • Furuta Y.
      • Piston D.
      • Hogan B.
      Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development.
      ;
      • Yavari R.
      • Adida C.
      • Bray-Ward P.
      • Brines M.
      • Xu T.
      Human metalloprotease-disintegrin Kuzbanian regulates sympathoadrenal cell fate in development and neoplasia.
      ;
      • Jacquemin P.
      • Durviaux S.M.
      • Jensen J.
      • et al.
      Transcription factor hepatocyte nuclear factor 6 regulates pancreatic cell differentiation and control expression of the proendocrine gene ngn3.
      ). Interestingly, that Adam10, plays a role in the Notch signaling pathway which has been implicated in keratinocyte growth arrest and entry into differentiation (
      • Rangarajan A.
      • Talora C.
      • Okuyama R.
      • et al.
      Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation.
      ), and may be expressed in the mouse hair follicle (
      • Kopan R.
      • Weintraub H.
      Mouse Notch: Expression in hair follicles correlated with cell fate determination.
      ). The involvement of that Delta-Notch signaling in stimulating human epidermal stem cell differentiation (
      • Lowell S.
      • Jones P.
      • Le Roux I.
      • Dunne J.
      • Watt F.M.
      Stimulation of human epidermal differentiation by Delta-Notch signaling at the boundaries of stem-cells clusters.
      ), further suggests that Adam 10 as a candidate gene in the Ksc1 locus.
      Another group of genes in the Ksc1 region includes those encoding mitogen-activated protein kinase 1 (Map2k1) (
      • Alessandrini A.
      • Brott B.K.
      • Erikson R.L.
      Differential expression of MEK1 and MEK2 during mouse development.
      ) and Anxa2, a major substrate for growth factor receptor protein-tyrosine kinases and protein kinase C (
      • Saris C.J.
      • Kristensen T.
      • D'Eustachio P.
      • Hicks L.J.
      • Noonan D.J.
      • Hunter T.
      • Tack B.F.
      cDNA sequence and tissue distribution of the m-RNA for bovine and murine p11, the S100-related light chain of the protein-tyrosine kinase substrate p36 (capactin 1).
      ).
      • Morris R.J.
      • Tacker K.C.
      • Fischer S.M.
      • Slaga T.J.
      Quantitation of primary in vitro clonogenic keratinocytes from normal adult murine epidermis following initiation and during promotion of epidermal tumors.
      showed that the number of clonogenic keratinocytes increases in response to TPA treatment, possibly resulting in protein kinase signal transduction. Thus, Map2k1or/and Anaxa2 might be involved in the regulation of the number of clonogenic keratinocytes.
      The Ksc2 locus on mouse Chromosome 4 shares linkage homology with human chromosomes 8q13 (LYN, GWM), 8q11 (MOS), 8q13-q21 (GEM). Possible candidate genes for Ksc2 include Lyn, a member of the Src subfamily encoding protein tyrosine kinase and expressed in skin (
      • Hibbs M.
      • Stanley E.
      • Maglitto R.
      • Dunn A.
      Identification of a duplication of the mouse gene.
      ), and Mos, a post-transcriptionally regulated gene expressed during oocyte growth and maturation (
      • Newman B.
      • Dai Y.
      Transcription of c-mos protooncogene in the pig involves both tissue-specific promoters and alternative polyadenylation sites.
      ). Finally the mitogen-induced gene, Gem, encodes a GTP-binding protein that belongs to a new family within the Ras superfamily (
      • Santoro T.
      • Maguire J.
      • McBrige O.
      • Avraham K.
      • Copeland N.
      • Jenkins N.
      • Kelly K.
      Chromosomal organization and transcriptional regulation of human GEM and localization of the human and mouse GEM loci encoding an inducible Ras-like protein.
      ); the regulated expression pattern of Gem suggests a role for this protein in cellular responses to growth stimulation and may be a candidate gene for the Ksc2 locus.
      The application of different possible models of stem cells hierarchies to the distribution of small and large colonies in parental strains and different genetic crosses brought us to conclude that small colonies most likely are not the progeny of transit amplifying keratinocytes. The data issued from the laboratory of Barrandon (Kobayashi et al, 1993; Rochat et al, 1994; Oshima et al, 2001) suggested that 95% of all clonogenic cells were originated from the bulge of hair follicle. Let assumed that large colonies originate from stem cells. In this case, 85% of small colonies from BALB/c mice, and at least 45% of small colonies in C57BL/6 mice should originate from stem cells. The investigation of some other parameters in addition to the area of colonies will help to answer the question if these two types of clonogenic cells related to the same or different lineages.
      Thus, our data demonstrate that the frequency of clonogenic keratinocyte stem cell is a genetically defined and quantitative complex trait. The clonogenic keratinocyte stem cells are a heterogeneous population. Two major subpopulations of clonogenic keratinocyte stem cells are regulated by different genes, with the locus responsible for small colony number on mouse chromosome 9 designated Ksc1, and the locus associated with large colonies on chromosome 4 designated Ksc2. Correlative data suggest that clonogenic keratinocytes producing small colonies might represent a specific population of keratinocytes stem cells responsible for sensitivity or resistance to skin carcinogenesis. Because these two subpopulations have independent inheritance and are regulated by different genes, we propose that they might be responsible for different processes in mouse epidermis.
      The identification of genes regulating amplification of keratinocyte stem cell awaits the identification and eventual cloning of the Ksc1 and Ksc2 genes. Moreover, the mechanisms regulating the intrinsic number of keratinocyte stem cells, possibly part of a universal regulatory mechanism for maintaining resting numbers of stem cells or their amplification, undoubtedly underlie the cells responses to external manipulation. The ability to manipulate these genes in vivo raises the exciting possibility of a novel treatment for skin disease and cancer. Further studies are need to determine whether the stem cell regulatory genes might be targets in carcinogenesis caused by environmental chemicals or ultraviolet light.

      Materials and Methods

      Mice

      Six-week-old C57BL/6 [C57BL/6NCrIBR], DBA/2 [DBA/2NCrIBR], BALB/c [BALB/cAnNCrIBR], CD-1 [Crl:CD-1 (ICR)], C3H [C3H/HeNCrIBR], FVB [FVB/NCrIBR], (BALB/c × C57BL/6)F1, C57BL/6 × (BALB/c × C57BL/6)F1 and BALB/c × (BALB/c × C57BL/6)F1 backcross, (BALB/c × C57BL/6)F2 female mice were obtained from Charles River Laboratories (Wilmington, Massachusetts), (http://www.criver.com). Six-week-old SENCAR female mice were obtained from NCI Frederick Cancer Research Center (Bethesda, Maryland). Groups of each mouse strains ranged between 24 and 57 animals; 30 F1, 44–45 backcrosses, and 104 F2 mice were used. A total of 496 mice were analyzed. All experiments were carried out with mice at 7 wk of age, when skin was still pink, indicating the resting stage of the hair follicle growth cycle.

      Histology

      Skin of mice was fixed in 10% neutral buffered formalin, embedded in paraffin and prepared by serial sections (5 μm). Slides were stained with hematoxylin and eosin.

      Phenotyping

      Keratinocytes harvested (
      • Morris R.J.
      Procedure for harvesting epidermal cells from the dorsal epidermis of adult mice for primary cell culture in “high calcium” defined medium.
      ) from individual mice were placed into four 60-mm plastic dishes (1000 viable cells per dish) on irradiated 3T3 (Swiss) mouse cells (American Type Culture Collection, Rockville, Maryland) and were grown for intervals of 2 and 4 wk. Dishes were fixed with neutral buffered formalin, and stained with rhodamine B. Keratinocyte colony number and colony size were analyzed with a Fluor-S MultilImager (BIO-RAD, Hercules, California) after 2 wk of cultivation. Colony size ranged from 0.1 to 12 mm2. The number of keratinocyte colonies 0.1–2 mm2 in area in duplicate dishes was designated as a “small” colony phenotype. The number of keratinocyte colonies greater than 2 mm2 area was considered a “large” colony phenotype. Hence, we had two phenotypes. The first of our phenotypes was the number of small colonies from each individual animal of each segregating crosses; the second––large colonies, respectively.

      Statistical analysis

      A balanced incomplete block design experiment (
      • Box G.E.P.
      • Hunter W.G.
      • Hunter J.S.
      Statistics for experimenters.
      ) was carried out with 12 replicates from each mouse strain and for the CB6F1 mice. Differences in the number of keratinocyte colonies between different mouse groups were analyzed using the Wilcoxon rank sum test and extension of the two-way ANOVA.

      Genotype analysis

      DNA was prepared from mouse liver by a standard proteinase K procedure (
      • Wolff R.
      • Gemmill R.
      Purifying and analyzing genomic DNA.
      ). Genotyping was performed by PCR as previously described (
      • Angel J.
      • Popova N.
      • Lanko N.
      • Turusov V.
      • DiGiovanni J.
      A locus that influences susceptibility to 1,2-dimethylhydrazine-induced colon tumors maps to the distal end of mouse Chromosome 3.
      ), using microsatellite primers (Research Genetics, Hunstsville, Alabama). Microsatellite markers (total 149) were chosen to cover all known chromosomal segments of sequence-tagged sites (STS) originating in autosomes from C57BL/6 and BALB/c mice. The maximum distance between two adjacent markers was no more than 15 cM. PCR products were analyzed by electrophoresis on 3% agarose gels, stained with ethidium bromide and visualized under ultraviolet light. Statistical analyses were performed using Qlink software kindly provided by Dr Norman Drinkwater (University of Wisconsin). Loci were ordered using the map locations reported in the 2000 Mouse Chromosome Committee Reports obtained from the Mouse Genome Database (http://www.informatics.jax.org/ccr/searches/index.cgi?year=2000).
      This work was supported in part by NIH grant CA45293 (RJM) and by the Lankenau Foundation. We thank Dr Spencer M. Free for statistical analysis.

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