A molecular basis for classic blond hair color in Europeans

Journal name:
Nature Genetics
Volume:
46,
Pages:
748–752
Year published:
DOI:
doi:10.1038/ng.2991
Received
Accepted
Published online

Hair color differences are among the most obvious examples of phenotypic variation in humans. Although genome-wide association studies (GWAS) have implicated multiple loci in human pigment variation, the causative base-pair changes are still largely unknown1. Here we dissect a regulatory region of the KITLG gene (encoding KIT ligand) that is significantly associated with common blond hair color in northern Europeans2. Functional tests demonstrate that the region contains a regulatory enhancer that drives expression in developing hair follicles. This enhancer contains a common SNP (rs12821256) that alters a binding site for the lymphoid enhancer-binding factor 1 (LEF1) transcription factor, reducing LEF1 responsiveness and enhancer activity in cultured human keratinocytes. Mice carrying ancestral or derived variants of the human KITLG enhancer exhibit significant differences in hair pigmentation, confirming that altered regulation of an essential growth factor contributes to the classic blond hair phenotype found in northern Europeans.

At a glance

Figures

  1. A distant regulatory region upstream of the KITLG gene controls hair pigmentation in humans and mice.
    Figure 1: A distant regulatory region upstream of the KITLG gene controls hair pigmentation in humans and mice.

    (a) SNPs on human chromosome 12 are associated with blond hair in Europeans (modified from ref. 2). The peak association is found at rs12821256 (red), which is 355 kb upstream of the KITLG transcription start site. (b) Frequency distribution of rs12821256 in different populations. The G allele associated with blond hair (yellow) is most prevalent in northern Europe. Green color represents the frequency of the ancestral A allele. (c) The Slpan allele at the mouse Kitl locus consists of a 65.6-Mb chromosome inversion (GRCm38/mm10, chr. 10 breakpoints: 34.301–99.916 Mb) that displaces upstream sequences orthologous to rs12821256 (red triangle). Mice heterozygous (Slpan/+) and homozygous (Slpan/Slpan) for this allele have lighter-colored coats than control (+/+) mice, demonstrating that alteration of even a single copy of the region upstream of Kitl can reduce hair pigmentation.

  2. The human blond-associated region contains a functional hair follicle enhancer.
    Figure 2: The human blond-associated region contains a functional hair follicle enhancer.

    (a) A 17.1-kb region bounded by SNPs rs444647 and rs661114 defines the candidate interval for blondness2. Within this region, a large block of mammalian sequence conservation (blue peaks) overlaps peak marker rs12821256. Five human fragments were cloned upstream of a lacZ reporter gene and tested for in vivo enhancer activity in transgenic mice. (bf) Representative transgenic embryos generated by pronuclear microinjection with the different lacZ constructs, processed at embryonic day (E) 16.5 to show lacZ gene activity (blue staining). Scale bar, 1 mm. (b) H1. (c) H2. (d) H3. (e) HFE (for hair follicle enhancer). (f) H2b. Pictures in bf are representative of 17, 15, 11, 11 and 13 independent transgenic embryos, respectively. Of the three clones spanning the entire interval, only the 6.7-kb clone, H2, produced consistent lacZ expression in skin and kidney (arrow). Analysis of two subclones of H2 separated HFE skin (e) and H2b kidney (f, arrow) enhancers. (g,h) Cross-sections (6 μm) through E16.5 dorsal skin from H2 (g) and HFE (h) transgenic embryos counterstained with nuclear fast red. Strong lacZ expression is visible in the basal epithelium and developing hair follicles. Scale bar, 30 μm.

  3. Variant hair follicle enhancers produce altered levels of gene expression.
    Figure 3: Variant hair follicle enhancers produce altered levels of gene expression.

    (ac) Representative E16.5 transgenic embryos, generated by pronuclear injection with different 6.7-kb H2-lacZ constructs (shown below), processed for lacZ gene activity (blue). The full H2 region was used for these experiments, as expression in kidney provided a control for successful integration and expression of constructs, even if expression in hair was disrupted. The clones tested were H2-ANC with the A allele at rs12821256 (a), H2-BLD with the G allele at rs12821256 (b) and H2-DEL with an 11-bp deletion that removes the rs12821256 position (c). lacZ gene activity was observed in developing hair follicles and kidney (arrows) in all transgenic embryos. Although no consistent difference was noted between H2-ANC (n = 15) and H2-BLD (n = 9) embryos, H2-DEL embryos (n = 8) showed reduced lacZ activity in skin but normal kidney expression. Scale bar, 1 mm. (d) Expression analysis of different 1.9-kb HFE-luciferase reporters in the human HaCaT keratinocyte cell line. Bars represent the mean increase in luciferase gene activity relative to an empty vector control measured 48 h after transfection for a typical experiment. The enhancers tested differed only by the following: HFE-ANC, A at rs12821256; HFE-BLD, G at rs12821256; HFE-DEL, 11-bp deletion removing rs12821256. Both the HFE-BLD and HFE-DEL constructs exhibited significantly reduced activity in HaCaT keratinocytes compared to the HFE-ANC plasmid. Error bars represent s.e.m. *P < 0.05, ***P < 5 × 10−4, unpaired t test.

  4. The blond-associated allele at rs12821256 alters a TCF/LEF binding site and reduces LEF responsiveness in keratinocytes.
    Figure 4: The blond-associated allele at rs12821256 alters a TCF/LEF binding site and reduces LEF responsiveness in keratinocytes.

    (a) A 4-kb window centered on the blond-associated SNP shows that TCF7L2 ChIP-seq reads from the ENCODE Project11 accumulate over rs12821256 in the 1.9-kb HFE (NCBI36/hg18, chr. 12: 87,852,100–87,853,992; HCT116 cells, TCF7L2 Sg data set). (b) The sequence surrounding rs12821256 resembles a consensus TCF/LEF binding motif39. The blond-associated allele in humans changes a highly conserved, consensus-matching A nucleotide to a non-consensus G nucleotide within the predicted TCF/LEF binding motif. (c) Response of mini-promoters to increasing levels of LEF1 protein 48 h after cotransfection into HaCaT keratinocytes. The three luciferase reporter constructs tested contained seven tandem copies of an artificial consensus LEF binding site (7× LEF) (SuperTOPFlash43), seven copies of the human ancestral binding site (7× ANC) or seven copies of the blond-associated sequence variant (7× BLD). All three mini-promoters demonstrated elevated activity in response to increased amounts of LEF1 protein. The magnitude of the response to moderate LEF1 levels corresponds with the predicted binding capabilities of the variant LEF sites, with 7× LEF >> 7× ANC >> 7× BLD. Note that the 7× BLD human variant shows significantly lower activation than the 7× ANC human sequence at every level of LEF1 tested (5 ng, P < 0.0001; 10 ng, P < 0.0001; 28 ng, P < 0.0001; 50 ng, P < 0.001, Mann-Whitney test). A representative experiment is shown of n = 2 biological replicates. Error bars, s.e.m. **P < 0.005, ***P < 5 × 10−4.

  5. Mouse lines differing at a single base-pair position in the KITLG hair enhancer (HE) show obvious differences in hair color.
    Figure 5: Mouse lines differing at a single base-pair position in the KITLG hair enhancer (HE) show obvious differences in hair color.

    (a) Schematic of the site-specific integration (SSI) strategy used to create matched BLD-Kitl and ANC-Kitl insertions in mice. The blond-associated or ancestral HE was cloned upstream of an Hspa1b minimal promoter–Kitl transgene (Online Methods). Blue arrows denote flanking attB sites that recombine with tandem attP sites (black arrows) in the mouse chromosome 11 H11P3 locus upon pronuclear injection of a mix containing each SSI plasmid with φC31 mRNA. (b) Box plots representing quantitative RT-PCR analysis of Kitl RNA expression in P8 dorsal skin. Both BLD-Kitl/+ and ANC-Kitl/+ heterozygotes exhibit significantly higher levels of epidermal Kitl than control mice. However, mice carrying the blond-associated variant transgene produce 21% less Kitl than mice with the matched ancestral transgene. Mann-Whitney P values: BLD versus +/+ = 5 × 10−9; ANC versus +/+ = 7 × 10−10; BLD versus ANC = 0.03146. *P < 0.05, ***P < 5 × 10–4. (c) Representative 2-month-old mice exhibiting the hair color phenotypes associated with a single copy of each SSI transgene. The mice pictured from left to right are wild type (FVB/C57BL/6J F1 hybrid) and BLD-Kitl/+ and ANC-Kitl/+ heterozygotes. Mice carrying the blond-associated allele at rs12821256 are notably lighter than mice carrying the ancestral allele at the KITLG HE. Representative picture of four matched replicates, with additional quantification in Supplementary Figure 7.

  6. H2 transgenic embryos.
    Supplementary Fig. 1: H2 transgenic embryos.

    Fifteen transgenic embryos produced by pronuclear injection with the 6.7-kb H2 plasmid are shown. Each embryo represents an independent genomic integration event. Embryos were collected at E16.5, stained for lacZ activity and bisected before imaging to show both external lateral (l) and internal medial (m) expression patterns. The skin (n = 13) and kidney (n = 14) were consistent sites of expression. The asterisk denotes the position of the kidney in the internal images. Scale bar, 1 mm.

  7. H2b transgenic embryos.
    Supplementary Fig. 2: H2b transgenic embryos.

    Thirteen transgenic embryos produced by pronuclear injection with the 1.5-kb H2b plasmid are shown. Each embryo represents an independent genomic integration event. Embryos were collected at E16.5, stained for lacZ activity and bisected before imaging to show both external lateral (l) and internal medial (m) expression patterns. The kidney (n = 12) was the only consistent site of expression. The asterisk denotes the position of the kidney in the internal images. Scale bar, 1 mm.

  8. HFE transgenic embryos.
    Supplementary Fig. 3: HFE transgenic embryos.

    Eleven transgenic embryos produced by pronuclear injection with the 1.9-kb HFE clone are pictured. Each embryo represents an independent genomic integration event. Embryos were collected at E16.5, stained for lacZ activity and bisected before imaging to show both external lateral (l) and internal medial (m) expression patterns. Hair/skin expression was visible in 8 of the 11 embryos. The asterisk denotes the position of the kidney in the internal images. Scale bar, 1 mm.

  9. H2-BLD transgenic embryos.
    Supplementary Fig. 4: H2-BLD transgenic embryos.

    Nine transgenic embryos produced by pronuclear injection with the 6.7-kb H2-BLD plasmid are shown. Each embryo represents an independent genomic integration event. Embryos were collected at E16.5, stained for lacZ activity and bisected before imaging to show both external lateral (l) and internal medial (m) expression patterns. Hair/skin (n = 7) and kidney (n = 7) were consistent sites of expression. No clear difference in expression compared to the complete set of H2 (H2-ANC) transgenic embryos was evident (see Supplementary Fig. 1). The asterisk denotes the position of the kidney in the internal images. Scale bar, 1 mm.

  10. H2-DEL transgenic embryos.
    Supplementary Fig. 5: H2-DEL transgenic embryos.

    Eight transgenic embryos produced by pronuclear injection with the 6.7-kb H2-DEL plasmid are shown. Each embryo represents an independent genomic integration event. Embryos were collected at E16.5, stained for lacZ activity and bisected before imaging to show both external lateral (l) and internal medial (m) expression patterns. Seven of the embryos show hair/skin expression. However the strength of this staining appeared reduced compared to H2-ANC and H2-BLD embryos, particularly in embryos that showed comparably strong kidney expression (such as embryos 2 and 5). The asterisk denotes the position of the kidney in the internal images. Scale bar, 1 mm.

  11. Additional pigmentation phenotypes seen in hair enhancer-Kitl mice.
    Supplementary Fig. 6: Additional pigmentation phenotypes seen in hair enhancer-Kitl mice.

    BLD-Kitl/+ and ANC-Kitl/+ heterozygotes exhibit several altered pigmentation patterns compared to wild-type (FVB/C57BL/6J F1 hybrid) littermates. At 2 months, ectopic pigmentation is seen on the muzzles (arrowheads in a) and the epithelium of the antitragus and ear canal (arrows in b–d). In contrast, BLD-Kitl/+ and ANC-Kitl/+ heterozygotes show reduced pigmentation in the whiskers (arrows in a) and the hair on the digits (arrows in e), perhaps because of competitive interactions between body sites for melanocyte colonization and development48 or premature differentiation of migrating melanocytes49. (f–h) Cross-sections (6 μm) through dorsal skin from 2-month-old (f) wild-type, (g) BLD-Kitl/+ and (h) ANC-Kitl/+ heterozygotes counterstained with nuclear fast red. Elevated Kitl expression controlled by both the BLD and ANC hair enhancers leads to ectopic pigmentation of the bulge region of hair follicles (arrows) and the basal epidermis (asterisks). DP, dermal papilla. Scale bars, 30 μm.

  12. Analysis of pigment levels in zigzag hairs from site-specific transgenic mice.
    Supplementary Fig. 7: Analysis of pigment levels in zigzag hairs from site-specific transgenic mice.

    (a) Photographs of zigzag hairs from wild-type (+/+; FVB/C57BL/6J F1 hybrid), BLD (BLD-Kitl/+) line 2 and ANC (ANC-Kitl/+) line 2 heterozygotes at P21. Fifteen hairs per mouse were analyzed to determine the fraction of pigmented pixels per hair shaft. (b) Mean pigmentation density in different genotypes. Both BLD-Kitl/+ and ANC-Kitl/+ heterozygotes exhibit significantly higher levels of pigmentation than wild-type controls. Notably, the amount of pigment in BLD-Kitl/+ heterozygotes is also significantly less than is found in ANC-Kitl/+ heterozygotes (P = 0.0278). Error bars indicate s.e.m. Unpaired t-test values; *P < 0.05, **P < 5 ×10–3.

References

  1. Sturm, R.A. Molecular genetics of human pigmentation diversity. Hum. Mol. Genet. 18, R9R17 (2009).
  2. Sulem, P. et al. Genetic determinants of hair, eye and skin pigmentation in Europeans. Nat. Genet. 39, 14431452 (2007).
  3. Cruz-Inigo, A.E., Ladizinski, B. & Sethi, A. Albinism in Africa: stigma, slaughter and awareness campaigns. Dermatol. Clin. 29, 7987 (2011).
  4. Pitman, J. On Blondes (Bloomsbury Publishing, New York, 2003).
  5. Homer. The Iliad of Homer (University of Chicago Press, Chicago, 2011).
  6. Han, J. et al. A genome-wide association study identifies novel alleles associated with hair color and skin pigmentation. PLoS Genet. 4, e1000074 (2008).
  7. Sulem, P. et al. Two newly identified genetic determinants of pigmentation in Europeans. Nat. Genet. 40, 835837 (2008).
  8. Zhang, M. et al. Genome-wide association studies identify several new loci associated with pigmentation traits and skin cancer risk in European Americans. Hum. Mol. Genet. 22, 29482959 (2013).
  9. Kenny, E.E. et al. Melanesian blond hair is caused by an amino acid change in TYRP1. Science 336, 554 (2012).
  10. Hindorff, L.A. et al. Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc. Natl. Acad. Sci. USA 106, 93629367 (2009).
  11. ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 5774 (2012).
  12. Praetorius, C. et al. A polymorphism in IRF4 affects human pigmentation through a tyrosinase-dependent MITF/TFAP2A pathway. Cell 155, 10221033 (2013).
  13. Yang, J. et al. Common SNPs explain a large proportion of the heritability for human height. Nat. Genet. 42, 565569 (2010).
  14. Olalde, I. et al. Derived immune and ancestral pigmentation alleles in a 7,000-year-old Mesolithic European. Nature 507, 225228 (2014).
  15. Morrison-Graham, K. & Takahashi, Y. Steel factor and c-kit receptor: from mutants to a growth factor system. Bioessays 15, 7783 (1993).
  16. Russell, E.S. Hereditary anemias of the mouse: a review for geneticists. Adv. Genet. 20, 357459 (1979).
  17. Nocka, K. et al. Molecular bases of dominant negative and loss of function mutations at the murine c-kit/white spotting locus: W37, Wv, W41 and W. EMBO J. 9, 18051813 (1990).
  18. Bedell, M.A., Copeland, N.G. & Jenkins, N.A. Multiple pathways for Steel regulation suggested by genomic and sequence analysis of the murine Steel gene. Genetics 142, 927934 (1996).
  19. Walsh, S. et al. The HIrisPlex system for simultaneous prediction of hair and eye colour from DNA. Forensic Sci. Int. Genet. 7, 98115 (2013).
  20. Rajeevan, H. et al. ALFRED: the ALelle FREquency Database. Update. Nucleic Acids Res. 31, 270271 (2003).
  21. Abecasis, G.R. et al. An integrated map of genetic variation from 1,092 human genomes. Nature 491, 5665 (2012).
  22. Beechey, C.V., Loutit, J.F. & Searle, A.G. Panda, a new Steel allele. Mouse News Lett. 74, 92 (1986).
  23. Bedell, M.A. et al. DNA rearrangements located over 100 kb 5′ of the Steel (Sl)-coding region in Steel-panda and Steel-contrasted mice deregulate Sl expression and cause female sterility by disrupting ovarian follicle development. Genes Dev. 9, 455470 (1995).
  24. Peters, E.M.J., Tobin, D.J., Botchkareva, N., Maurer, M. & Paus, R. Migration of melanoblasts into the developing murine hair follicle is accompanied by transient c-Kit expression. J. Histochem. Cytochem. 50, 751766 (2002).
  25. Jordan, S.A. & Jackson, I.J. MGF (KIT ligand) is a chemokinetic factor for melanoblast migration into hair follicles. Dev. Biol. 225, 424436 (2000).
  26. Boukamp, P. et al. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 106, 761771 (1988).
  27. Giese, K., Amsterdam, A. & Grosschedl, R. DNA-binding properties of the HMG domain of the lymphoid-specific transcriptional regulator LEF-1. Genes Dev. 5, 25672578 (1991).
  28. Travis, A., Amsterdam, A., Belanger, C. & Grosschedl, R. LEF-1, a gene encoding a lymphoid-specific protein with an HMG domain, regulates T-cell receptor α enhancer function. Genes Dev. 5, 880894 (1991).
  29. Waterman, M.L., Fischer, W.H. & Jones, K.A. A thymus-specific member of the HMG protein family regulates the human T cell receptor Cα enhancer. Genes Dev. 5, 656669 (1991).
  30. Zhou, P., Byrne, C., Jacobs, J. & Fuchs, E. Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate. Genes Dev. 9, 700713 (1995).
  31. DasGupta, R. & Fuchs, E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 126, 45574568 (1999).
  32. van Genderen, C. et al. Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1–deficient mice. Genes Dev. 8, 26912703 (1994).
  33. Zhang, Y. et al. Activation of β-catenin signaling programs embryonic epidermis to hair follicle fate. Development 135, 21612172 (2008).
  34. Love, J.J. et al. Structural basis for DNA bending by the architectural transcription factor LEF-1. Nature 376, 791795 (1995).
  35. Yun, K., So, J.S., Jash, A. & Im, S.H. Lymphoid enhancer binding factor 1 regulates transcription through gene looping. J. Immunol. 183, 51295137 (2009).
  36. Jash, A., Yun, K., Sahoo, A., So, J.S. & Im, S.H. Looping mediated interaction between the promoter and 3′ UTR regulates type II collagen expression in chondrocytes. PLoS ONE 7, e40828 (2012).
  37. Visser, M., Kayser, M. & Palstra, R.J. HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter. Genome Res. 22, 446455 (2012).
  38. Berger, M.F. et al. Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities. Nat. Biotechnol. 24, 14291435 (2006).
  39. Newburger, D.E. & Bulyk, M.L. UniPROBE: an online database of protein binding microarray data on protein-DNA interactions. Nucleic Acids Res. 37, D77D82 (2009).
  40. Tasic, B. et al. Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc. Natl. Acad. Sci. USA 108, 79027907 (2011).
  41. Grichnik, J.M., Burch, J.A., Burchette, J. & Shea, C.R. The SCF/KIT pathway plays a critical role in the control of normal human melanocyte homeostasis. J. Invest. Dermatol. 111, 233238 (1998).
  42. Kamberov, Y.G. et al. Modeling recent human evolution in mice by expression of a selected EDAR variant. Cell 152, 691702 (2013).
  43. Veeman, M.T., Slusarski, D.C., Kaykas, A., Louie, S.H. & Moon, R.T. Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements. Curr. Biol. 13, 680685 (2003).
  44. DiLeone, R.J., Russell, L.B. & Kingsley, D.M. An extensive 3′ regulatory region controls expression of Bmp5 in specific anatomical structures of the mouse embryo. Genetics 148, 401408 (1998).
  45. Mortlock, D.P., Guenther, C. & Kingsley, D.M. A general approach for identifying distant regulatory elements applied to the Gdf6 gene. Genome Res. 13, 20692081 (2003).
  46. Whitlock, M.C. Combining probability from independent tests: the weighted Z-method is superior to Fisher's approach. J. Evol. Biol. 18, 13681373 (2005).
  47. Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45 (2001).

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Author information

Affiliations

  1. Department of Developmental Biology, Stanford University School of Medicine, Stanford, California, USA.

    • Catherine A Guenther &
    • David M Kingsley
  2. Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California, USA.

    • Catherine A Guenther,
    • Bosiljka Tasic,
    • Liqun Luo &
    • David M Kingsley
  3. Department of Biology, Stanford University, Stanford, California, USA.

    • Bosiljka Tasic &
    • Liqun Luo
  4. Department of Genetics, University of Georgia, Athens, Georgia, USA.

    • Mary A Bedell
  5. Present address: Allen Institute for Brain Science, Seattle, Washington, USA.

    • Bosiljka Tasic

Contributions

C.A.G. and D.M.K. conceived and oversaw the project. M.A.B. isolated and sequenced the Slpan breakpoint. B.T. and L.L. provided advice, reagents and mice for generating site-specific integrants. C.A.G. performed the gene expression analysis in Slpan mutants, carried out the transgenic analysis of the blond-associated GWAS interval, identified the hair follicle enhancer and performed in vitro and in vivo tests of the effects of the rs12821256 polymorphism. C.A.G. and D.M.K. wrote the manuscript with input from all authors.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: H2 transgenic embryos. (1,097 KB)

    Fifteen transgenic embryos produced by pronuclear injection with the 6.7-kb H2 plasmid are shown. Each embryo represents an independent genomic integration event. Embryos were collected at E16.5, stained for lacZ activity and bisected before imaging to show both external lateral (l) and internal medial (m) expression patterns. The skin (n = 13) and kidney (n = 14) were consistent sites of expression. The asterisk denotes the position of the kidney in the internal images. Scale bar, 1 mm.

  2. Supplementary Figure 2: H2b transgenic embryos. (861 KB)

    Thirteen transgenic embryos produced by pronuclear injection with the 1.5-kb H2b plasmid are shown. Each embryo represents an independent genomic integration event. Embryos were collected at E16.5, stained for lacZ activity and bisected before imaging to show both external lateral (l) and internal medial (m) expression patterns. The kidney (n = 12) was the only consistent site of expression. The asterisk denotes the position of the kidney in the internal images. Scale bar, 1 mm.

  3. Supplementary Figure 3: HFE transgenic embryos. (779 KB)

    Eleven transgenic embryos produced by pronuclear injection with the 1.9-kb HFE clone are pictured. Each embryo represents an independent genomic integration event. Embryos were collected at E16.5, stained for lacZ activity and bisected before imaging to show both external lateral (l) and internal medial (m) expression patterns. Hair/skin expression was visible in 8 of the 11 embryos. The asterisk denotes the position of the kidney in the internal images. Scale bar, 1 mm.

  4. Supplementary Figure 4: H2-BLD transgenic embryos. (605 KB)

    Nine transgenic embryos produced by pronuclear injection with the 6.7-kb H2-BLD plasmid are shown. Each embryo represents an independent genomic integration event. Embryos were collected at E16.5, stained for lacZ activity and bisected before imaging to show both external lateral (l) and internal medial (m) expression patterns. Hair/skin (n = 7) and kidney (n = 7) were consistent sites of expression. No clear difference in expression compared to the complete set of H2 (H2-ANC) transgenic embryos was evident (see Supplementary Fig. 1). The asterisk denotes the position of the kidney in the internal images. Scale bar, 1 mm.

  5. Supplementary Figure 5: H2-DEL transgenic embryos. (534 KB)

    Eight transgenic embryos produced by pronuclear injection with the 6.7-kb H2-DEL plasmid are shown. Each embryo represents an independent genomic integration event. Embryos were collected at E16.5, stained for lacZ activity and bisected before imaging to show both external lateral (l) and internal medial (m) expression patterns. Seven of the embryos show hair/skin expression. However the strength of this staining appeared reduced compared to H2-ANC and H2-BLD embryos, particularly in embryos that showed comparably strong kidney expression (such as embryos 2 and 5). The asterisk denotes the position of the kidney in the internal images. Scale bar, 1 mm.

  6. Supplementary Figure 6: Additional pigmentation phenotypes seen in hair enhancer-Kitl mice. (1,035 KB)

    BLD-Kitl/+ and ANC-Kitl/+ heterozygotes exhibit several altered pigmentation patterns compared to wild-type (FVB/C57BL/6J F1 hybrid) littermates. At 2 months, ectopic pigmentation is seen on the muzzles (arrowheads in a) and the epithelium of the antitragus and ear canal (arrows in b–d). In contrast, BLD-Kitl/+ and ANC-Kitl/+ heterozygotes show reduced pigmentation in the whiskers (arrows in a) and the hair on the digits (arrows in e), perhaps because of competitive interactions between body sites for melanocyte colonization and development48 or premature differentiation of migrating melanocytes49. (f–h) Cross-sections (6 μm) through dorsal skin from 2-month-old (f) wild-type, (g) BLD-Kitl/+ and (h) ANC-Kitl/+ heterozygotes counterstained with nuclear fast red. Elevated Kitl expression controlled by both the BLD and ANC hair enhancers leads to ectopic pigmentation of the bulge region of hair follicles (arrows) and the basal epidermis (asterisks). DP, dermal papilla. Scale bars, 30 μm.

  7. Supplementary Figure 7: Analysis of pigment levels in zigzag hairs from site-specific transgenic mice. (208 KB)

    (a) Photographs of zigzag hairs from wild-type (+/+; FVB/C57BL/6J F1 hybrid), BLD (BLD-Kitl/+) line 2 and ANC (ANC-Kitl/+) line 2 heterozygotes at P21. Fifteen hairs per mouse were analyzed to determine the fraction of pigmented pixels per hair shaft. (b) Mean pigmentation density in different genotypes. Both BLD-Kitl/+ and ANC-Kitl/+ heterozygotes exhibit significantly higher levels of pigmentation than wild-type controls. Notably, the amount of pigment in BLD-Kitl/+ heterozygotes is also significantly less than is found in ANC-Kitl/+ heterozygotes (P = 0.0278). Error bars indicate s.e.m. Unpaired t-test values; *P < 0.05, **P < 5 ×10–3.

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