Mammalian colour patterns are among the most recognizable characteristics found in nature and can have a profound impact on fitness. However, little is known about the mechanisms underlying the formation and subsequent evolution of these patterns. Here we show that, in the African striped mouse (Rhabdomys pumilio), periodic dorsal stripes result from underlying differences in melanocyte maturation, which give rise to spatial variation in hair colour. We identify the transcription factor ALX3 as a regulator of this process. In embryonic dorsal skin, patterned expression of Alx3 precedes pigment stripes and acts to directly repress Mitf, a master regulator of melanocyte differentiation, thereby giving rise to light-coloured hair. Moreover, Alx3 is upregulated in the light stripes of chipmunks, which have independently evolved a similar dorsal pattern. Our results show a previously undescribed mechanism for modulating spatial variation in hair colour and provide insights into how phenotypic novelty evolves.
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Barsh, G. S. The genetics of pigmentation: from fancy genes to complex traits. Trends Genet. 12, 299–305 (1996)
Jackson, I. J. Molecular and developmental genetics of mouse coat color. Annu. Rev. Genet. 28, 189–217 (1994)
Mills, M. G. & Patterson, L. B. Not just black and white: pigment pattern development and evolution in vertebrates. Semin. Cell Dev. Biol. 20, 72–81 (2009)
Candille, S. I. et al. Dorsoventral patterning of the mouse coat by Tbx15. PLoS Biol. 2, E3 (2004)
Morris, D. Animal Watching. A Field Guide to Animal Behavior. (Jonathan Cape Ltd, 1990)
Cloudsley-Thompson, J. L. Multiple factors in the evolution of animal coloration. Naturwissenschaften 86, 123–132 (1999)
Caro, T., Izzo, A., Reiner, R. C. Jr, Walker, H. & Stankowich, T. The function of zebra stripes. Nat. Commun. 5, 3535 (2014)
Brodie, E. D. III . Correlational selection for color pattern and antipredator behavior in the garter snake Thamnophis ordinoides. Evolution 46, 1284–1298 (1992)
King, R. B. Color pattern polymorphism in the Lake Erie water snake, Nerodia sipedon insularum. Evolution 41, 241–255 (1987)
Caro, T. The adaptive significance of coloration in mammals. Bioscience 55, 125–136 (2005)
Schradin, C. et al. Social flexibility and social evolution in mammals: a case study of the African striped mouse (Rhabdomys pumilio). Mol. Ecol. 21, 541–553 (2012)
Steingrímsson, E., Copeland, N. G. & Jenkins, N. A. Melanocytes and the microphthalmia transcription factor network. Annu. Rev. Genet. 38, 365–411 (2004)
ten Berge, D. et al. Mouse Alx3: an aristaless-like homeobox gene expressed during embryogenesis in ectomesenchyme and lateral plate mesoderm. Dev. Biol. 199, 11–25 (1998)
Beverdam, A., Brouwer, A., Reijnen, M., Korving, J. & Meijlink, F. Severe nasal clefting and abnormal embryonic apoptosis in Alx3/Alx4 double mutant mice. Development 128, 3975–3986 (2001)
Twigg, S. R. F. et al. Frontorhiny, a distinctive presentation of frontonasal dysplasia caused by recessive mutations in the ALX3 homeobox gene. Am. J. Hum. Genet. 84, 698–705 (2009)
Lakhwani, S., García-Sanz, P. & Vallejo, M. Alx3-deficient mice exhibit folic acid-resistant craniofacial midline and neural tube closure defects. Dev. Biol. 344, 869–880 (2010)
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, 751–766 (2002)
Beronja, S., Livshits, G., Williams, S. & Fuchs, E. Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos. Nat. Med. 16, 821–827 (2010)
Lee, M., Goodall, J., Verastegui, C., Ballotti, R. & Goding, C. R. Direct regulation of the Microphthalmia promoter by Sox10 links Waardenburg-Shah syndrome (WS4)-associated hypopigmentation and deafness to WS2. J. Biol. Chem. 275, 37978–37983 (2000)
Potterf, S. B., Furumura, M., Dunn, K. J., Arnheiter, H. & Pavan, W. J. Transcription factor hierarchy in Waardenburg syndrome: regulation of MITF expression by SOX10 and PAX3. Hum. Genet. 107, 1–6 (2000)
Elworthy, S., Lister, J. A., Carney, T. J., Raible, D. W. & Kelsh, R. N. Transcriptional regulation of mitfa accounts for the sox10 requirement in zebrafish melanophore development. Development 130, 2809–2818 (2003)
Pérez-Villamil, B., Mirasierra, M. & Vallejo, M. The homeoprotein Alx3 contains discrete functional domains and exhibits cell-specific and selective monomeric binding and transactivation. J. Biol. Chem. 279, 38062–38071 (2004)
Levy, C., Khaled, M. & Fisher, D. E. MITF: master regulator of melanocyte development and melanoma oncogene. Trends Mol. Med. 12, 406–414 (2006)
García-Sanz, P., Fernández-Pérez, A. & Vallejo, M. Differential configurations involving binding of USF transcription factors and Twist1 regulate Alx3 promoter activity in mesenchymal and pancreatic cells. Biochem. J. 450, 199–208 (2013)
Meredith, R. W. et al. Impacts of the Cretaceous Terrestrial Revolution and KPg extinction on mammal diversification. Science 334, 521–524 (2011)
dos Reis, M. et al. Phylogenomic datasets provide both precision and accuracy in estimating the timescale of placental mammal phylogeny. Proc. R. Soc. B 279, 3491–3500 (2012)
Huchon, D. et al. Multiple molecular evidences for a living mammalian fossil. Proc. Natl Acad. Sci. USA 104, 7495–7499 (2007)
Rawls, J. F., Mellgren, E. M. & Johnson, S. L. How the zebrafish gets its stripes. Dev. Biol. 240, 301–314 (2001)
Parichy, D. M. Pigment patterns: fish in stripes and spots. Curr. Biol. 13, R947–R950 (2003)
Singh, A. P. & Nüsslein-Volhard, C. Zebrafish stripes as a model for vertebrate colour pattern formation. Curr. Biol. 25, R81–R92 (2015)
Jackson, I. J. et al. Genetics and molecular biology of mouse pigmentation. Pigment Cell Res. 7, 73–80 (1994)
Kaelin, C. B. et al. Specifying and sustaining pigmentation patterns in domestic and wild cats. Science 337, 1536–1541 (2012)
Vrieling, H., Duhl, D. M., Millar, S. E., Miller, K. A. & Barsh, G. S. Differences in dorsal and ventral pigmentation result from regional expression of the mouse agouti gene. Proc. Natl Acad. Sci. USA 91, 5667–5671 (1994)
Manceau, M., Domingues, V. S., Mallarino, R. & Hoekstra, H. E. The developmental role of Agouti in color pattern evolution. Science 331, 1062–1065 (2011)
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012)
McCloy, R. A. et al. Partial inhibition of Cdk1 in G 2 phase overrides the SAC and decouples mitotic events. Cell Cycle 13, 1400–1412 (2014)
Spandidos, A., Wang, X., Wang, H. & Seed, B. PrimerBank: a resource of human and mouse PCR primer pairs for gene expression detection and quantification. Nucleic Acids Res. 38, D792–D799 (2010)
Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402–408 (2001)
Henrique, D. et al. Expression of a Delta homologue in prospective neurons in the chick. Nature 375, 787–790 (1995)
Mallarino, R. et al. Two developmental modules establish 3D beak-shape variation in Darwin’s finches. Proc. Natl Acad. Sci. USA 108, 4057–4062 (2011)
Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124, 1283–1298 (2006)
Kent, W. J. et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002)
Schwartz, S. et al. PipMaker—a web server for aligning two genomic DNA sequences. Genome Res. 10, 577–586 (2000)
Brudno, M. et al. LAGAN and Multi-LAGAN: efficient tools for large-scale multiple alignment of genomic DNA. Genome Res. 13, 721–731 (2003)
Schreiber, E., Matthias, P., Müller, M. M. & Schaffner, W. Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells. Nucleic Acids Res. 17, 6419 (1989)
Mirasierra, M. & Vallejo, M. Glucose-dependent downregulation of glucagon gene expression mediated by selective interactions between ALX3 and PAX6 in mouse alpha cells. Diabetologia 59, 766–775 (2016)
de la Serna, I. L. et al. The microphthalmia-associated transcription factor requires SWI/SNF enzymes to activate melanocyte-specific genes. J. Biol. Chem. 281, 20233–20241 (2006)
We thank D. Mishkind, M. Omura, J. Chupasko, P. Walsh, T. Capellini, T. Linden, K. Turner and N. Hughes for providing technical and logistical support, and C. Perdomo, A. Bendesky, C. K. Hu, D. M. Kingsley and J. M. Lassance for discussions. M.Mi. and M.V. are supported by the Spanish Ministry of Economy and Competitiveness (MINECO grants BFU2011-24245 and BFU2014-52149-R) and Instituto de Salud Carlos III. CIBERDEM is an initiative of the Instituto de Salud Carlos III. H.E.H. is an Investigator of the Howard Hughes Medical Institute.
The authors declare no competing financial interests.
Reviewer Information Nature thanks H. Arnheiter, T. Caro, M. Levine and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Striped mice have three different phenotypic categories of hair (light, black and banded) based on individual pigment pattern. All hair types have a black tip, which corresponds to structural hair features (not pigment). b, Relative proportion of light, black and banded guard and awl hair along the striped mouse dorsoventral axis (n = 5; error bars represent s.e.m.). a, Scale bar, 100 μm.
Extended Data Figure 2 Stripe-like differences in hair length along the dorsum in striped mouse embryos and pups.
a–c, Flat-mount skin preparations (dermis up) of embryos at E16 (a) and E19 (b) and P2 pups (c). Middle axis is indicated in all cases (midline). White dashed lines mark regions differing in hair length at E19 (b) and regions differing in pigmentation at P2 (c). Incipient pigmentation stripes are shown in (b). d, Skin punches (1 mm) and length measurements show differences between hair in the dark and light stripe of P2 individuals. Hair length differences in (b) (incipient stripes) correlate with those seen when pigment differences arise (c, d). Differences among dorsal regions were evaluated by ANOVA followed by a Tukey–Kramer test; n = 15 per region; **P < 0.01; ***P < 0.001. Red lines depict the mean. e, Hair length measurements taken from guard, awl and zigzag hair found along the dorsum of adults. Differences among dorsal regions were evaluated by ANOVA followed by a Tukey–Kramer test; n = 24 (guard), 12 (awl) and 12 (zigzag) per region; P = 0.1736 (guard hair), P = 0.8006 (awl hair), P = 0.1038 (zigzag hair). Red lines depict the mean. f, Predicted probabilities of the observed stripe-like phenotypes, as inferred by supervised learning models built and trained to recognize time-point-specific gene expression signatures of the stripes. Bars reflect average probabilities ± s.e.m. computed from 30 consecutive iterations of the predictive model in each analysis. Labels indicate, as a ratio, the time point at which the randomForests model was applied to predict the stripe-like phenotype (the left term of the ratio), and the time point at which the model was built and trained (the right term of the ratio). The dotted line indicates the prior probability of either stripe phenotype (that is, 50% in case of only two distinct phenotypes). See Methods for details.
a, Counts of proliferating cells, as determined by EdU labelling, in the epidermis and inside hair follicles (epidermal cells, dark stripe 1 versus light stripe, two-tailed t-test; n = 15 images per region; P = 0.5417; cells counted: 402 (dark stripe 1) and 444 (light stripe); intrafollicular cells, dark stripe 1 versus light stripe, two-tailed t-test; n = 12 images per region; P = 0.7537; cells counted: 724 (dark stripe 1) and 680 (light stripe)). Dotted lines delineate the epidermal compartment and the hair bulb. b, Number of hair follicles per surface area along the dorsoventral axis. Differences among dorsal regions were evaluated by ANOVA; n = 10 images per region; P = 0.4391; number of hair follicles counted: 139 (light stripe), 141 (dark stripe 1), 132 (dark stripe 2) and 128 (flank). Bright field images in a depict pigment. Boxes represent the 25th to 75th percentile, whiskers show the minimum and maximum value, and the horizontal line indicates the median. Red bars indicate the mean. Scale bars in a 100 μm, b 200 μm.
a–d, Venn diagrams showing numbers of differentially expressed genes identified using either the M. musculus reference or the striped mouse de novo transcriptome assembly in light versus dark stripes (a), light versus flank (b), and flank versus dark (c), or differentially expressed genes in light or dark stripes versus the other skin region (light or dark stripes and the flank) (d). Genes that are specifically upregulated only in the dark or light stripes are highlighted in red. e, f, RNA-seq transcript levels (normalized gene counts) plotted as a function of differential expression (log2 fold-change). e, The 1,777 genes demonstrating significant (FDR < 0.1) differential expression in the light stripe versus the flank are shown in yellow (higher expression in the light stripe) or blue (higher expression in the flank). Four differentially expressed pigmentation-related genes are highlighted (dark yellow or dark blue), while 11 additional pigmentation-related genes that are not differentially expressed are shown in pink (Supplementary Table 1b). f, The 1,148 genes demonstrating significant (FDR < 0.1) differential expression in the flank versus the dark stripe are shown in yellow (higher expression in the flank) or blue (higher expression in the dark stripe). Eleven differentially expressed pigmentation-related genes are highlighted (dark yellow or dark blue), while six additional pigmentation-related genes that are not differentially expressed are shown in pink (Supplementary Table 1c).
a–c, Quantitative PCR of the relative mRNA levels of pigment-type switching genes Asip (a), Edn3 (b), and melanin synthesis genes Tyr and Tyrp1 (c) in different regions of the striped mouse skin and at different time points. Differences among stripes within each time point were evaluated by ANOVA followed by a Tukey–Kramer test; n = 4 per time point; *P < 0.05; **P < 0.01; ***P < 0.001. Red bars indicate the mean.
a, Lentiviral constructs were modified from PLKO.1, a generic vector for expressing human RNU6-1 promoter-driven short hairpin RNAs (red loop). LTR, long terminal repeat; ψ, retroviral packaging element, RRE, Rev response element; cPPT, central polypurine tract; PGK, phosphoglycerate kinase promoter; H2B–GFP, Hist2h2be fused to GFP cDNA; P2A, 2A peptide. b, Western blot for expression of ALX3 in nuclear extracts of B16-F1 cells. Positive controls were extracts from mouse embryonic mesenchyme (MEM) or COS cells transfected with a pcDNA–ALX3 expression vector. COS cells transfected with an empty pcDNA vector served as negative controls. Actin immunoreactivity is shown below for the same extracts as a control. For gel source images, see Supplementary Fig. 1. c, d, Quantitative PCR of Alx3 (white), Mitf (black) and Silver (grey) mRNA levels in cells transduced with LV–Alx3:GFP (c), relative to cells transduced with the LV–GFP control (LV–Alx3:GFP versus LV–GFP, two-tailed t-test; n = 3) or shRNA lentiviral constructs (d), relative to cells transduced with a scrambled control (shRNA1, 2, 3 or 4 versus shRNA scrambled, two-tailed t-test; n = 3). **P < 0.01; ***P < 0.001. Red bars indicate the mean.
a–d, Wild-type B16 melanocytes (B16 wild-type (WT)) were exposed to keratinocytes (a) or melanocytes (c) stably transduced with either LV–Alx3:GFP (LV–Alx3 in graphs) or LV–GFP. b, d, Quantitative PCR of levels of Alx3 mRNA in cells carrying the lentiviral constructs (grey panel) and of Mitf, Tyr and Silver mRNA in B16 wild-type melanocytes exposed to keratinocytes (b) or melanocytes (d) transduced with LV–Alx3:GFP (blue panels) or LV–GFP (red panels) (LV–Alx3:GFP versus LV–GFP, two-tailed t-test; n = 3, ***P < 0.001; NS, not significant). Red bars in b and d indicate the mean.
a–f, Hair follicles from embryos injected at E8.5 with lentiviruses stained for SOX10 (a, d), virus transduced cells (b, e) and merged images with arrowheads showing SOX+ GFP+ cells (c, f). Dotted lines (a–f) delineate the hair bulb. g, Number of detectable SOX10+ cells (LV–Alx3:GFP versus LV–GFP, two-tailed t-test; n = 60; P = 0.1173; cells counted: 426 cells (LV–Alx3:GFP) and 398 cells (LV–GFP)). h–o, Effect of Alx3 on skin. Hair follicles from samples injected with LV–GFP control and LV–Alx3:GFP depicting immunohistochemistry for K14 (h, k), virus transduced cells (i, l), and merged images showing K14+ GFP+ cells (j, m). n, Number of detectable K14+ GFP+ cells per follicular area (LV–Alx3:GFP versus LV–GFP, two-tailed t-test; n = 40, P = 0.275; average: 52.809 cells per hair follicle area (LV–Alx3:GFP) and 55.123 cells per hair follicle area (LV–GFP)). o, Hair follicle density in samples injected with viruses (LV–Alx3:GFP versus LV–GFP, two-tailed t-test; n = 30; P = 0.103; average: 0.84 hair follicles per surface area (LV–Alx3:GFP) and 0.794 hair follicles per surface area (LV–GFP)). Boxes represent the 25th to 75th percentile, whiskers show the minimum and maximum value, and the horizontal line indicates the median. Scale bars in a–f and h–m are 50 μM.
Extended Data Figure 9 Alignment of a ~1.5-kb region of the Mitf M promoter in M. musculus and striped mouse.
Black boxes represent conserved sequences. Mapped onto the sequences are evolutionary conserved regions of the mammalian Mitf M promoter identified in silico (http://genome.ucsc.edu) (yellow), regions from which the EMSA probes were designed (red), and the transcription start site (green). The ten TAAT binding sites, conserved between M. musculus and striped mice (blue), which were tested are labelled sequentially.
a, EMSAs show the binding of nuclear proteins from B16-F1 cells to candidate-binding sites 3, 5 and 10. The absence (−) or presence of non-specific (NSC; 500-fold molar excess) or specific (SC; indicated fold molar excess) competing oligonucleotides, or the addition of ALX3 antibodies or control (NRS or IgG) is indicated. Arrows indicate complexes containing ALX3, arrowhead shows supershift for site 3 and asterisk highlights an artefact in the gel. b, EMSAs showing the binding of recombinant Alx3 synthesized using a rabbit reticulocyte lysate system to the indicated sites (sites 4–9). The absence (−) or presence of non-specific (NSC) or specific (SC) competing oligonucleotides (500-fold molar excess) is indicated. Note that site 5 is the only one showing sequence-specific binding. For gel source images, see Supplementary Fig. 1. c, Relative levels of luciferase activity in B16-F1 cells stably expressing GFP (dark circles) or Alx3 (light circles) for indicated binding sites. Luciferase activity was normalized relative to cells transfected with the empty reporter vector and values are given as a fraction of luminescence for GFP-transfected cells. Differences between cells transfected with LV–Alx3:GFP and LV–GFP for each plasmid were evaluated using two-tailed t-tests; n = 5; *P < 0.05; NS, not significant. Red lines depict the mean. Labels of mutated binding sites correspond to those described in Fig. 4a.
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Mallarino, R., Henegar, C., Mirasierra, M. et al. Developmental mechanisms of stripe patterns in rodents. Nature 539, 518–523 (2016). https://doi.org/10.1038/nature20109
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