复旦大学：《人类进化 Human Evolution》经典论文_携带受选择EDAR变异在小鼠的表达建模揭示近期人类进化 2013 Cell Modeling Recent Human Evolution in Mice by Expression of a Selected EDAR Variant

Cell Modeling recent Human Evolution in Mice by Expression of a selected EDAR Variant Yana G. Kamberov, 1, 2,3,5,6,7, 16 Sijia Wang, 5, 7, 16, 18 Jingze Tan, 9 Pascale Gerbault, 10 Abigail Wark, 1 Longzhi Tan, 5 unhao Mao 8.21 Asa Schachar, 5, 7 Madeline Paymer, 5,7 Elizabeth Hostetter, Elizabeth byrne, Melissa Burnett, 24.S20 Yajun Yang, 9 Shilin Li,Kun Tang, 13 Hua Chen, 14 Adam Powell, 1 Yuval Itan, 10, 19 Dorian Fuller, 12 Jason Lohmueller ndrew P. McMahon 8,22 Mark G. Thomas 10 Daniel E. Lieberman 6,17 Li Jin 9, 13, 17, Clifford J. Tabin 1, 17 Bruce A. Morgan 2,317, and Pardis C. Sabeti5, 7, 15,17,* 1Department of Genetics Harvard Medical School, Boston, MA 02115, USA cUtaneous Biology Research Center 4Department of Dermatology Massachusetts General Hospital, Boston, MA 02114, USA SThe Broad Institute of Harvard and MIT, cambridge, MA 02142, USA 6Department of Human Evolutionary Biology 7Center for Systems Biology, Department of Organismic and Evolutionary Biology aDepartment of Molecular and cellular Biology ity, Cambridge, MA 02138, US 9MOE Key Laboratory of Contemporary Anthropology, Fudan University, Shanghai 200433, China 10Department of Genetics, Evolution and Environment 11UCL Genetics Institute(UGI ste of University College London, London WC1H OPY, UK 13CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, 14Department of Epidemiology Department of Immunology and Infectious Diseases Harvard School of Public Health, Boston, MA 02115, USA 1 These authors contributed equally to this work 17These authors contributed equally to this work and are cosenior authors 18Present address: Max Planck-CAS Paul Gerson Unna Research Group on Dermatogenomics, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 19Present address: St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY, USA 20Present address: Department of Systems Biology Harvard Medical School, Boston, MA 02115, USA 2Present address: Department of Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA 22Present address: Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School Of Medicine University of Southern Califomia, CA 90089, USA Correspondence:lijinfudan@gmail.com(L.J.)bruce.morgan@cbrc2.mgh.harvard.edu(BA.M),pardis@broadinstituteorg(PC..) ttp/ dx. doi. oro/10.1016/ce.2013.01.016 SUMMARY its direct biological significance and potential adaptive role remain unclear. We generated a An adaptive variant of the human Ectodysplasin knockin mouse model and find that, as in humans receptor, EDARV370A, is one of the strongest candi- hair thickness is increased in EDAR370A mice We lates of recent positive selection from genome- identify new biological targets affected by the muta- wide scans. We have modeled EDAR370A in mice tion, including mammary and eccrine glands and characterized its phenotype and evolutionary Building on these results, we find that EDAR370A origins in humans. Our computational analysis is associated with an increased number of active suggests the allele arose in central China approxi- eccrine glands in the Han Chinese. This interdisci mately 30,000 years ago. Although EDAR370A plinary approach yields unique insight into the has been associated with increased scalp hair thick generation of adaptive variation among modern ness and changed tooth morphology in humans, humans Cell 152, 691-702, February 14, 2013@2013 Elsevier Inc. 691

Modeling Recent Human Evolution in Mice by Expression of a Selected EDAR Variant Yana G. Kamberov,1,2,3,5,6,7,16 Sijia Wang,5,7,16,18 Jingze Tan,9 Pascale Gerbault,10 Abigail Wark,1 Longzhi Tan,5 Yajun Yang,9 Shilin Li,9 Kun Tang,13 Hua Chen,14 Adam Powell,11 Yuval Itan,10,19 Dorian Fuller,12 Jason Lohmueller,5,20 Junhao Mao,8,21 Asa Schachar,5,7 Madeline Paymer,5,7 Elizabeth Hostetter,5 Elizabeth Byrne,5 Melissa Burnett,2,4 Andrew P. McMahon,8,22 Mark G. Thomas,10 Daniel E. Lieberman,6,17 Li Jin,9,13,17, * Clifford J. Tabin,1,17 Bruce A. Morgan,2,3,17, * and Pardis C. Sabeti5,7,15,17, * 1Department of Genetics 2Department of Dermatology Harvard Medical School, Boston, MA 02115, USA 3Cutaneous Biology Research Center 4Department of Dermatology Massachusetts General Hospital, Boston, MA 02114, USA 5The Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA 6Department of Human Evolutionary Biology 7Center for Systems Biology, Department of Organismic and Evolutionary Biology 8Department of Molecular and Cellular Biology Harvard University, Cambridge, MA 02138, USA 9MOE Key Laboratory of Contemporary Anthropology, Fudan University, Shanghai 200433, China 10Department of Genetics, Evolution and Environment 11UCL Genetics Institute (UGI) 12Institute of Archaeology University College London, London WC1H 0PY, UK 13CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Science, Shanghai 200031, China 14Department of Epidemiology 15Department of Immunology and Infectious Diseases Harvard School of Public Health, Boston, MA 02115, USA 16These authors contributed equally to this work 17These authors contributed equally to this work and are cosenior authors 18Present address: Max Planck-CAS Paul Gerson Unna Research Group on Dermatogenomics, CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 19Present address: St. Giles Laboratory of Human Genetics of Infectious Diseases, Rockefeller Branch, The Rockefeller University, New York, NY, USA 20Present address: Department of Systems Biology Harvard Medical School, Boston, MA 02115, USA 21Present address: Department of Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA 22Present address: Department of Stem Cell Biology and Regenerative Medicine, Broad-CIRM Center, Keck School Of Medicine, University of Southern California, CA 90089, USA *Correspondence: lijin.fudan@gmail.com (L.J.), bruce.morgan@cbrc2.mgh.harvard.edu (B.A.M.), pardis@broadinstitute.org (P.C.S.) http://dx.doi.org/10.1016/j.cell.2013.01.016 SUMMARY An adaptive variant of the human Ectodysplasin receptor, EDARV370A, is one of the strongest candi￾dates of recent positive selection from genome￾wide scans. We have modeled EDAR370A in mice and characterized its phenotype and evolutionary origins in humans. Our computational analysis suggests the allele arose in central China approxi￾mately 30,000 years ago. Although EDAR370A has been associated with increased scalp hair thick￾ness and changed tooth morphology in humans, its direct biological significance and potential adaptive role remain unclear. We generated a knockin mouse model and find that, as in humans, hair thickness is increased in EDAR370A mice. We identify new biological targets affected by the muta￾tion, including mammary and eccrine glands. Building on these results, we find that EDAR370A is associated with an increased number of active eccrine glands in the Han Chinese. This interdisci￾plinary approach yields unique insight into the generation of adaptive variation among modern humans. Cell 152, 691–702, February 14, 2013 ª2013 Elsevier Inc. 691

Cell INTRODUCTION et al., 2008). This finding suggested that a pre-existing mouse overexpressed, Humans are unique among primates in having colonized nearly might provide insight into 370A's phenotypic consequences every corner of the world; consequently, niche 008). Indeed, trans- pressures likely helped shape the phenotypic varia present-day g, enlarged globin-B a P. falcipa 2005);muta digest milk (Enattah genes dr 2005) Althor s.We tated the chara tional e ces. affect a by exp ther of car ghlights the variation in idat g other candidate those w S persist unknowr difficu native to t date adap A SNP in 51 worldwide particularl of 370A. Haplotype hum subtle p ype adapti g plasin A et al., 200 of the se East Asian ar scalp hair th 200 2009;Park quantify tained whe phenotypes. The bioch that the variant Structural models Domain(DD) required transducer EDARADD we performed a pression of 370A has bee NFkB signaling in vitro re sent-day Han Chinese 62e,69-7 February 1420120e

INTRODUCTION Humans are unique among primates in having colonized nearly every corner of the world; consequently, niche-specific selective pressures likely helped shape the phenotypic variation currently evident in Homo sapiens. Identifying the genetic variants that underlie regional adaptations is thus central to understanding present-day human diversity, yet only a few adaptive traits have been elucidated. These include mutations in the Hemo￾globin-B and Duffy antigen genes, driving resistance to P. falciparum and P. vivax malaria, respectively (Kwiatkowski, 2005); mutations in lactase allowing some adult humans to digest milk after the domestication of milk-producing livestock (Enattah et al., 2002); and mutations in SLC24A5 and other genes driving variation in skin pigmentation (Lamason et al., 2005). Although breakthroughs in genomic technology have facili￾tated the identification of hundreds of candidate genetic variants with evidence of recent positive natural selection, validation and characterization of putative genetic adaptations requires func￾tional evidence linking genotypes to phenotypes that could affect an organism’s fitness (Akey, 2009). This is made difficult by experimental challenges in isolating the phenotypic effects of candidate loci and by methodological limitations on the phenotypes that can be readily assessed in humans. Accord￾ingly, the best-characterized human adaptive alleles are typically those whose phenotypic outcomes are easily measured and strongly related to known genetic variation, such as lactase persistence or skin pigmentation. Many genes, however, have unknown or pleiotropic effects, making their adaptive advantage difficult to uncover (Sivakumaran et al., 2011). A promising alter￾native to tackle these difficulties is to study the effects of candi￾date adaptive alleles in animal models. Although such models, particularly using mice, have been used extensively to study human disease alleles, they have not been used to model the subtle phenotypic changes expected to result from human adaptive variation. A compelling candidate human adaptive allele to emerge from genome-wide scans is a derived coding variant of the Ectodys￾plasin A (EDA) receptor (EDAR), EDARV370A (370A) (Sabeti et al., 2007; Grossman et al., 2010). Computational fine-mapping of the selection signal and the restricted occurrence of 370A in East Asian and Native American populations have led to sugges￾tions that 370A was selected in Asia (Bryk et al., 2008). In support of this hypothesis, 370A was shown to associate with increased scalp hair thickness and incisor tooth shoveling in multiple East Asian populations (Fujimoto et al., 2008a, 2008b; Kimura et al., 2009; Park et al., 2012). However, because association studies quantify correlation rather than causation, it remains to be ascer￾tained whether 370A is the genetic change driving the observed phenotypes. The biochemical properties of 370A support the possibility that the variant directly causes the associated phenotypes. Structural models predict that V370A lies in the EDAR Death Domain (DD) required for interaction with the downstream signal transducer EDARADD (Sabeti et al., 2007). Moreover, overex￾pression of 370A has been reported to upregulate downstream NFkB signaling in vitro relative to 370V (Bryk et al., 2008; Mou et al., 2008). This finding suggested that a pre-existing mouse model, in which the ancestral 370V allele is overexpressed, might provide insight into 370A’s phenotypic consequences (Headon and Overbeek, 1999; Mou et al., 2008). Indeed, trans￾genic mice expressing multiple copies of 370V have thicker hair shafts as seen in humans with the 370A allele (Fujimoto et al., 2008a, 2008b; Mou et al., 2008). In addition, these animals exhibit increased mammary gland branching, enlarged mammary glands and hyperplastic sebaceous and Meibomian glands that secrete hydrophobic films as a barrier to water loss in the skin and eyes, respectively (Chang et al., 2009). These latter phenotypes led to the proposal that the 370A variant may have been selected in response to cold and arid environ￾mental conditions (Chang et al., 2009). Evaluating which forces may have contributed to the spread of 370A requires knowledge of both the environmental context in which this variant was selected and its phenotypic effects. We therefore employed a multi-disciplinary approach to test the role of 370A in recent human evolution. This included modeling to reconstruct the evolutionary history of 370A, and a knockin mouse model to examine its direct phenotypic consequences. Analysis of the mouse knockin revealed phenotypes not previously reported in human genetic studies, which we further characterized in a Han Chinese cohort. This work highlights the utility of modeling nonpathological human genetic variation in mice, providing a framework for assessing other candidate adaptive human alleles. RESULTS Single Origin of 370A in Central China Using both newly generated and publicly available data, we examined 280 SNPs flanking the 370A SNP in 51 worldwide populations in order to assess the origin of 370A. Haplotype analysis supports a single origin of the derived allele (Figure 1A), with the mutation lying on a unique, nearly unbroken haplotype extending more than 100 kb among both East Asians and Native Americans (Figure S1 available online). To estimate the allele’s geographic and temporal origin, we performed more than one million spatially explicit demic forward simulations modeling the appearance and spread of 370A in Asia (Itan et al., 2009) (Modeling the Origins and Spread of 370A in an Approximate Bayesian Computation Framework). We used approximate Bayesian computation (ABC) (Beaumont et al., 2002) to compare simulated to observed allele frequencies and to estimate key evolutionary and demographic parameters (Fagundes et al., 2007; Itan et al., 2009; Ray et al., 2010). This analysis estimated the 370A allele originated in central China (Figure 1B) between 13,175 and 39,575 years BP (95% credible interval), with a mode of 35,300 years BP and a median of 30,925 years BP. The estimated selection coefficient has a 95% credible interval between 0.030 and 0.186, with a mode of 0.122 and a median of 0.114 (Figures S2, S3, and S4, and Tables S1, S2, S3, and S4, and Modeling the Origins and Spread of 370A in an Approximate Bayesian Computation Framework). As a separate calculation of the age of 370A, we performed a maximum likelihood inference analysis using the allele frequency spectrum of 1,677 nearby SNPs in present-day Han Chinese 692 Cell 152, 691–702, February 14, 2013 ª2013 Elsevier Inc

Cell Figure 1. Origins of 370A (A) Haplotype distribution of the genomic region surrounding V370A, based on 24 SNPs covering 139 kb. The six most common haplotypes are shown, and the haining low-frequency haplotypes grouped as"other" The chimpanzee allele was assumed to be ancestral Derived alleles are in dark blue, except for the ariant which is red (B) The approximate posterior probability density for the geographic origin of 370A obtained by ABC simulation. The heat map was generated usil kemel density estimation of the latitude and longitude co tes from the top 5,000(0. 46%)of 1, 083, 966 simulations. Red color represents the highest probability, and lue the lowest See also Figures S1, S2, s3, S4, S5, and S7 and Tables s1, S2, s3, and S4 stimating Selection Time of 370A using the Coalescent-Based this change and concomitant rough coat phenotype suggest Allele Frequency Spectrum)(Chen, 2012). This method provided this model has limited utility(Mou et al., 2008). In contrast imilar estimates of the allele age (95% confidence interval: 370A knockin mice exhibit a smooth hair coat with all four 34, 775-38, 208 years BP; maximum likelihood estimation hair types that are normally found in the mouse pelage(Sund [MLE]: 36, 490 years BP)and selection intensity(95%confidence berg, 1994; Mikkola, 2011)(Figure 2C and data not shown) interval: 0.0657 7-0.0831;MLE:0.0744; Figure S5) We evaluated the sizes of both the awl and achene hair types in the mouse coat by scoring medulla cell number across the Generation of 370A Mouse Mode hair shafts(Sundberg, 1994; Enshell-Seijffers et aL., 2010)(Fig o test the biological consequence of 370A, we evaluated its ure 3A). Our analysis revealed that Edar genotype was signifi efficiency to drive a phenotypic change in vivo. In humans cantly associated with hair size(MANOVA, p=0.034 and p and mice, loss-of-function mutations in the genes coding for 0.027 for awl and achene hairs respectively, Table S5). 370A the ligand EDA-A1, EDAR, and EDARADD lead to strikingly homozygous mutant mice had more of the thickest, four-cell imilar phenotypes characterized by defective hair development, awl hairs and fewer three-cell hairs than 370V homozygotes absence of eccrine glands, and missing or misshapen teeth(Mik- (p=0.007 and p= 0.005 for four- and three-cell hairs respec- ola, 2008, 2011: Cluzeau et aL., 2011). The conserved role of the tively)(Figure 3B and Table S5). Similarly, 370A homozygotes Ectodysplasin pathway in the development of ectodermally had a higher proportion of thicker achenes than 370V and derived organs(Gruneberg, 1971; Kondo et aL., 2001; Colosimo 370v/370A animals(p=0.007 and p=0.007, respectively, et aL., 2005: Mikkola, 2008, 2011)suggested that a 370A mouse Table S5 and Figure 3C). The 370A mouse thus recapitulates knockin model would be an accurate system in which to isolate the associated human phenotype of increased hair thickness, and examine the effects of the derived allele confirming that the mutation is causal, and demonstrating the The DDs of mouse and human EDAR are identical in sequence, model's utility for accurately characterizing the allele's biolog- with mice natively expressing the 370V allele(Figure 2A). To ical effects construct 370A knockin mice, we used homologous recombina- tion in embryonic stem cells to introduce the T1326C point 370A Does Not Increase Meibomian Gland Size mutation into the endogenous murine Edar locus resulting in aA previous study overexpressing 370V in mice found an V370A substitution in the encoded protein(Figures 2B and S6). increase in Meibomian gland size, leading to speculation on 370A mice were born at expected Mendelian ratios, appeared the adaptive benefit of 370A(Chang et al., 2009). To evaluate healthy, and did not exhibit altered fertility or longevity compared the effect of 370A on Meibomian gland size, we measured the to wild-type littermates(Figure 2C and data not shown) total gland area of the upper and lower eyelids of 370V, 370V/ 370A, and 370A mice. No significant difference in gland area 370A Increases Hair Thickness in Mice yas observed between the different genotypes(MANOVA, p In humans, 370A is associated with increased scalp hair thick- 0.244: Figure 4; Table S5). Similarly, we found no detectable ness(Fujimoto et aL., 2008a, 2008b). Mice that overexpress the change in the size of the related sebaceous glands of the skin 370V allele also have thicker hairs, but the larger magnitude of (data not shown). Cell 152, 691-702, February 14, 2013@2013 Elsevier Inc. 693

(Estimating Selection Time of 370A using the Coalescent-Based Allele Frequency Spectrum) (Chen, 2012). This method provided similar estimates of the allele age (95% confidence interval: 34,775–38,208 years BP; maximum likelihood estimation [MLE]: 36,490 years BP) and selection intensity (95% confidence interval: 0.0657–0.0831; MLE: 0.0744; Figure S5). Generation of 370A Mouse Model To test the biological consequence of 370A, we evaluated its sufficiency to drive a phenotypic change in vivo. In humans and mice, loss-of-function mutations in the genes coding for the ligand EDA-A1, EDAR, and EDARADD lead to strikingly similar phenotypes characterized by defective hair development, absence of eccrine glands, and missing or misshapen teeth (Mik￾kola, 2008, 2011; Cluzeau et al., 2011). The conserved role of the Ectodysplasin pathway in the development of ectodermally derived organs (Gru¨ neberg, 1971; Kondo et al., 2001; Colosimo et al., 2005; Mikkola, 2008, 2011) suggested that a 370A mouse knockin model would be an accurate system in which to isolate and examine the effects of the derived allele. The DDs of mouse and human EDAR are identical in sequence, with mice natively expressing the 370V allele (Figure 2A). To construct 370A knockin mice, we used homologous recombina￾tion in embryonic stem cells to introduce the T1326C point mutation into the endogenous murine Edar locus resulting in a V370A substitution in the encoded protein (Figures 2B and S6). 370A mice were born at expected Mendelian ratios, appeared healthy, and did not exhibit altered fertility or longevity compared to wild-type littermates (Figure 2C and data not shown). 370A Increases Hair Thickness in Mice In humans, 370A is associated with increased scalp hair thick￾ness (Fujimoto et al., 2008a, 2008b). Mice that overexpress the 370V allele also have thicker hairs, but the larger magnitude of this change and concomitant rough coat phenotype suggest this model has limited utility (Mou et al., 2008). In contrast 370A knockin mice exhibit a smooth hair coat with all four hair types that are normally found in the mouse pelage (Sund￾berg, 1994; Mikkola, 2011) (Figure 2C and data not shown). We evaluated the sizes of both the awl and auchene hair types in the mouse coat by scoring medulla cell number across the hair shafts (Sundberg, 1994; Enshell-Seijffers et al., 2010) (Fig￾ure 3A). Our analysis revealed that Edar genotype was signifi- cantly associated with hair size (MANOVA, p = 0.034 and p = 0.027 for awl and auchene hairs respectively, Table S5). 370A homozygous mutant mice had more of the thickest, four-cell awl hairs and fewer three-cell hairs than 370V homozygotes (p = 0.007 and p = 0.005 for four- and three-cell hairs respec￾tively) (Figure 3B and Table S5). Similarly, 370A homozygotes had a higher proportion of thicker auchenes than 370V and 370V/370A animals (p = 0.007 and p = 0.007, respectively, Table S5 and Figure 3C). The 370A mouse thus recapitulates the associated human phenotype of increased hair thickness, confirming that the mutation is causal, and demonstrating the model’s utility for accurately characterizing the allele’s biolog￾ical effects. 370A Does Not Increase Meibomian Gland Size A previous study overexpressing 370V in mice found an increase in Meibomian gland size, leading to speculation on the adaptive benefit of 370A (Chang et al., 2009). To evaluate the effect of 370A on Meibomian gland size, we measured the total gland area of the upper and lower eyelids of 370V, 370V/ 370A, and 370A mice. No significant difference in gland area was observed between the different genotypes (MANOVA, p = 0.244; Figure 4; Table S5). Similarly, we found no detectable change in the size of the related sebaceous glands of the skin (data not shown). Figure 1. Origins of 370A (A) Haplotype distribution of the genomic region surrounding V370A, based on 24 SNPs covering
139 kb. The six most common haplotypes are shown, and the remaining low-frequency haplotypes grouped as ‘‘Other.’’ The chimpanzee allele was assumed to be ancestral. Derived alleles are in dark blue, except for the 370A variant which is red. (B) The approximate posterior probability density for the geographic origin of 370A obtained by ABC simulation. The heat map was generated using 2D kernel density estimation of the latitude and longitude coordinates from the top 5,000 (0.46%) of 1,083,966 simulations. Red color represents the highest probability, and blue the lowest. See also Figures S1, S2, S3, S4, S5, and S7 and Tables S1, S2, S3, and S4. Cell 152, 691–702, February 14, 2013 ª2013 Elsevier Inc. 693

Cel GGCGGTNCG TG A GGCGGCCG TG Human RMLSSTYNSEKAVKTWRHLAESFGLKRDEIGGMTD louse RMLSSTYNSEKAVKTWRHLAESFGLKRDEIGGMTD Human GMQLFDRISTAGYSIPELLTKLVQIERLDAVESLCADIL Mouse GMQLFDRISTAGYSIPELLTKLVQIERLDAVESLCADIL Targeting Vector ecombination 370V 70V/370A370A Figure 2. Generation of the 370A Mouse EDAR DD. 370V is in bold with superscript asterisk. xons 11 and 12 and contains the T1326C mutation (red line). The targeting vector also contained a neomycin (Neo) reseda.dar geno TArgeting strategy for the introduction of the 370A mutation into the mouse Edar locus. The construct spans part of the edar ge cassette flanked by LoxP sites purple arrows)inserted into the EDAR 3 untranslated region (UTR. Neo was excised by breeding to a ubiquitously expressing B-Actin Cre line The final genomic tructure of the knockin Edar locus is shown with exons as boxes, coding sequence in dark gray, UTR in black and all other genomic sequence as a black line Diphtheria toxin A selection cassette(DTA) is shown in blue and vector sequence in light gray. (C) Appearance of 370V, 370v/370A and 370A animals and confirmation of the T1326C (underlined)substitution. See also Figure Mammary Gland Branching and Fat Pad Size Are Altered icantly affected by genotype (ANCOVA, p=0.045) in 370A Mice gland area was significantly affected by body weight The mammary gland and surrounding stromal tissue, the by genotype(Generation and Statistical Analysis of fat are of interest given their importance in Knockin Mouse eproduction(Neville et aL, 1998; Lefevre et aL, 2010). A role for Ectodysplasin signaling in mammary gland development is 370A Increases Eccrine Gland Number in Mice suggested by loss-of-function mutants in which glands are Eccrine sweat glands in humans are widespread throughout the present and functional, but gland branching and size of the skin, reflecting their critical role in heat dissipation, but in mice mammary tree are reduced(Chang et aL., 2009; Mikkola, 2011; and most other mammals, they are restricted to the plantar Voutilainen et al., 2012). In contrast, overexpression of Edar surfaces where they serve in traction. In spite of this difference. nd its ligand Eda-A1 lead to the converse phenotypes(Chang loss-of-function mutants have demonstrated the conserved et aL., 2009; Voutilainen et al. 2012 role of Ectodysplasin signaling in eccrine gland formation across We assessed five aspects of the 4th and 9n mammary glands mammals(Gruneberg, 1971; Mikkola, 2011). Because eccrine in pre-estrus mice: branch number, branch density, gland length, glands are absent in Edar loss-of-function mutants, we evaluated gland area, and mammary fat pad area. Only branch density and the effect of 370A on eccrine gland number in our mouse model mammary fat pad size were affected by the 370A genotype We scored eccrine gland number in four of the six hindlimb MANOVA, univariate main effects: p= 0.044 and p= 0.018, footpads(Figures 6A-6F. Edar genotype was significantly respectively, Figure 5 and Table S5). 370A homozygotes had associated with eccrine gland number in all footpads(MANOV higher branch density than either 370V or 370V/370A mice(p= p=4.3 x 10-7, see Generation and Statistical Analysis of the 0.018 and p=0.047, respectively, Figure 5E) and smaller fat 370A Knockin Mouse, Table S5). 370A homozygous animals pads(p=0.007 and p=0.030, respectively, Figure 5F). Although, had more sweat glands per footpad than wild-type 370Vhomo- body weight was not affected by Edar genotype(ANOVA, p= zygotes(p< 0.01 for all footpads, Figure 6G), and in most 0. 459), linear regression revealed a small effect of body footpads 370V/370A heterozygotes showed an intermediate on gland and fat pad size(Generation and Statistical increase(p <0.01 for footpads FP-3, FP-4, FP-5, Figure 6G) of the 370A Knockin Mouse). To control for this effect, Because a single copy of 370A was sufficient to increase eo alyzed the effect of Edar genotype on these traits using body crine gland number in our model, we directly tested whether weight as a covariate. In this analysis, fat pad area was still signif- 370A is a gain-of-function allele by analyzing its ability to rescue 694ce152,691-702, February14,2013e2013Es

Mammary Gland Branching and Fat Pad Size Are Altered in 370A Mice The mammary gland and surrounding stromal tissue, the mammary fat pad, are of interest given their importance in reproduction (Neville et al., 1998; Lefe` vre et al., 2010). A role for Ectodysplasin signaling in mammary gland development is suggested by loss-of-function mutants in which glands are present and functional, but gland branching and size of the mammary tree are reduced (Chang et al., 2009; Mikkola, 2011; Voutilainen et al., 2012). In contrast, overexpression of Edar and its ligand Eda-A1 lead to the converse phenotypes (Chang et al., 2009; Voutilainen et al., 2012). We assessed five aspects of the 4th and 9th mammary glands in pre-estrus mice: branch number, branch density, gland length, gland area, and mammary fat pad area. Only branch density and mammary fat pad size were affected by the 370A genotype (MANOVA, univariate main effects: p = 0.044 and p = 0.018, respectively, Figure 5 and Table S5). 370A homozygotes had higher branch density than either 370V or 370V/370A mice (p = 0.018 and p = 0.047, respectively, Figure 5E) and smaller fat pads (p = 0.007 and p = 0.030, respectively, Figure 5F). Although, body weight was not affected by Edar genotype (ANOVA, p = 0.459), linear regression revealed a small effect of body weight on gland and fat pad size (Generation and Statistical Analysis of the 370A Knockin Mouse). To control for this effect, we rean￾alyzed the effect of Edar genotype on these traits using body weight as a covariate. In this analysis, fat pad area was still signif￾icantly affected by genotype (ANCOVA, p = 0.045), whereas gland area was significantly affected by body weight but not by genotype (Generation and Statistical Analysis of the 370A Knockin Mouse). 370A Increases Eccrine Gland Number in Mice Eccrine sweat glands in humans are widespread throughout the skin, reflecting their critical role in heat dissipation, but in mice and most other mammals, they are restricted to the plantar surfaces where they serve in traction. In spite of this difference, loss-of-function mutants have demonstrated the conserved role of Ectodysplasin signaling in eccrine gland formation across mammals (Gru¨ neberg, 1971; Mikkola, 2011). Because eccrine glands are absent in Edar loss-of-function mutants, we evaluated the effect of 370A on eccrine gland number in our mouse model. We scored eccrine gland number in four of the six hindlimb footpads (Figures 6A–6F). Edar genotype was significantly associated with eccrine gland number in all footpads (MANOVA, p = 4.3 3 107 , see Generation and Statistical Analysis of the 370A Knockin Mouse, Table S5). 370A homozygous animals had more sweat glands per footpad than wild-type 370V homo￾zygotes (p < 0.01 for all footpads, Figure 6G), and in most footpads 370V/370A heterozygotes showed an intermediate increase (p < 0.01 for footpads FP-3, FP-4, FP-5, Figure 6G). Because a single copy of 370A was sufficient to increase ec￾crine gland number in our model, we directly tested whether 370A is a gain-of-function allele by analyzing its ability to rescue Figure 2. Generation of the 370A Mouse (A) Conservation of human and mouse EDAR DD. 370V is in bold with superscript asterisk. (B) Targeting strategy for the introduction of the 370A mutation into the mouse Edar locus. The construct spans part of the Edar genomic sequence including exons 11 and 12 and contains the T1326C mutation (red line). The targeting vector also contained a neomycin (Neo) resistance cassette flanked by LoxP sites (purple arrows) inserted into the EDAR 30 untranslated region (UTR). Neo was excised by breeding to a ubiquitously expressing b-Actin Cre line. The final genomic structure of the knockin Edar locus is shown with exons as boxes, coding sequence in dark gray, UTR in black and all other genomic sequence as a black line. Diphtheria toxin A selection cassette (DTA) is shown in blue and vector sequence in light gray. (C) Appearance of 370V, 370V/370A and 370A animals and confirmation of the T1326C (underlined) substitution. See also Figure S6. 694 Cell 152, 691–702, February 14, 2013 ª2013 Elsevier Inc.

Cell zygotes(MANOVA post hoc tests, p=0.002, Figure 6H). In agreement with a gain-of-function model, 370A/379K heterozy (p < 0.05 for all footpads, Figure 6H and Table S5) 370A Is Associated with More Eccrine Glands and other Pleiotropic Effects in Humans 4 cell 3 cell 2 cell The change in eccrine gland number we observed in the 370A mouse has important implications for the distribution of variation in this trait in human populations. However, association studies of sweat gland density with nonpathological variation at the EDAR locus have not been reported in humans. To examine whether 370A is associated with altered eccrine gland number in humans, we carried out an association study 82 in individuals of han descent from an established cohort in taiz- 370V hou, China Wang et al., 2009). To sample a sufficient number of 370v/370A the rare 370V alleles, we first genotyped the 370A SNP and found 2, 226 370A homozygotes, 340 370V/370A heterozygotes, and 6 370V homozygotes. We then contacted all individuals with at least one copy of the 370V allele and enrolled 187 of them(184 370V/370A and 3 3701, along with 436 370A individuals and collected phenotypes related to ectodermal appendages ( Table S6 and Association Study of 370A in a Han Chinese Population) Because only three individuals were homozygous for the 370V Number of cells for 370A in statistical analysis of the collected data Consistent with previous reports(Kimura et al., 2009: Park et aL., 2012), 370A was associated with single and doub shoveling of the upper incisors (Wald test, p= 0.0077 and p=0.0004, respectively; Table S6). Additionally, 370A was significantly associated with the presence of a protostylid cusp and the absence of lower third molars (Wald test, p=0.0079 ■370V and p=0.0123, respectively; Association Study of 370A in a 370V/370A Han Chinese Population and Table S6) We tested for an association between 370A and eccrine sweat gland number using the starch-iodine method to measure the number of activated glands in digit pads of the thumb and inde finger(Juniper et aL., 1964: Randall, 1946 ). In agreement with our mouse findings, 370A homozygous individuals had significantly more active eccrine glands than 370V/370A individuals(two- ailed t test, p=0.011, Figure 7). Testing all three genotypes Number of cells sing linear-regression in an additive model revealed a strong Figure 3. 370A Allele Increases Hair Size in the Mouse Coat association between 370A and eccrine gland density ( Wald (A) Representative images of hairs show medulla cell number serves test, p=0.0047; Table S6). This association remained significant a proxy for hair shaft thicknes when we controlled for age, sex, and potential population (B and C)370A mice have a larger proportion of thicker hairs than mice substructure(Association Study of 370A in a Han Chinese pressing the ancestral allele the average frequency (+SEM)of awl( B)and Population and Table S6). DISCUSSION post hoc tests: p<0.05(),p<0.01("). See also Table $5. study integrated population genetic analyses, a humanized the eccrine gland phenotype of mice heterozygous for the mouse model, and human association study to characterize downless(dl,E379K@Edarloss-of-functionmutation(Headonanaturalhumangenevariant.Combiningtheseapproache and Overbeek, 1999). 379K is classically considered a recessive allowed us to determine the direct biological effects of 370A mutation, and animals heterozygous for the 379K allele are and cast new light on their evolutionary uences Extend- described as wild-type(Headon and Overbeek, 1999). However, ing this strategy to other candidate adaptive alleles stands to our quantitative method for scoring eccrine glands revealed advance our understanding of the effects of recent selection a subtle decrease in eccrine gland number in 379E/379K hetero- on the diversification of modern humans. Cell 152, 691-702, February 14, 2013(2013 Elsevier Inc. 695

the eccrine gland phenotype of mice heterozygous for the downless (dlj , E379K) Edar loss-of-function mutation (Headon and Overbeek, 1999). 379K is classically considered a recessive mutation, and animals heterozygous for the 379K allele are described as wild-type (Headon and Overbeek, 1999). However, our quantitative method for scoring eccrine glands revealed a subtle decrease in eccrine gland number in 379E/379K hetero￾zygotes (MANOVA post hoc tests, p = 0.002, Figure 6H). In agreement with a gain-of-function model, 370A/379K heterozy￾gous animals had more eccrine glands than 370V/379K animals (p < 0.05 for all footpads, Figure 6H and Table S5). 370A Is Associated with More Eccrine Glands and Other Pleiotropic Effects in Humans The change in eccrine gland number we observed in the 370A mouse has important implications for the distribution of variation in this trait in human populations. However, association studies of sweat gland density with nonpathological variation at the EDAR locus have not been reported in humans. To examine whether 370A is associated with altered eccrine gland number in humans, we carried out an association study in individuals of Han descent from an established cohort in Taiz￾hou, China (Wang et al., 2009). To sample a sufficient number of the rare 370V alleles, we first genotyped the 370A SNP and found 2,226 370A homozygotes, 340 370V/370A heterozygotes, and 6 370V homozygotes. We then contacted all individuals with at least one copy of the 370V allele and enrolled 187 of them (184 370V/370A and 3 370V), along with 436 370A individuals and collected phenotypes related to ectodermal appendages (Table S6 and Association Study of 370A in a Han Chinese Population). Because only three individuals were homozygous for the 370V allele, we focused on individuals homozygous and heterozygous for 370A in statistical analysis of the collected data. Consistent with previous reports (Kimura et al., 2009; Park et al., 2012), 370A was associated with single and double shoveling of the upper incisors (Wald test, p = 0.0077 and p = 0.0004, respectively; Table S6). Additionally, 370A was significantly associated with the presence of a protostylid cusp and the absence of lower third molars (Wald test, p = 0.0079 and p = 0.0123, respectively; Association Study of 370A in a Han Chinese Population and Table S6). We tested for an association between 370A and eccrine sweat gland number using the starch-iodine method to measure the number of activated glands in digit pads of the thumb and index finger (Juniper et al., 1964; Randall, 1946). In agreement with our mouse findings, 370A homozygous individuals had significantly more active eccrine glands than 370V/370A individuals (two￾tailed t test, p = 0.011, Figure 7). Testing all three genotypes using linear-regression in an additive model revealed a strong association between 370A and eccrine gland density (Wald test, p = 0.0047; Table S6). This association remained significant when we controlled for age, sex, and potential population substructure (Association Study of 370A in a Han Chinese Population and Table S6). DISCUSSION This study integrated population genetic analyses, a humanized mouse model, and human association study to characterize a natural human gene variant. Combining these approaches allowed us to determine the direct biological effects of 370A and cast new light on their evolutionary consequences. Extend￾ing this strategy to other candidate adaptive alleles stands to advance our understanding of the effects of recent selection on the diversification of modern humans. Figure 3. 370A Allele Increases Hair Size in the Mouse Coat (A) Representative images of mouse hairs show medulla cell number serves as a proxy for hair shaft thickness. (B and C) 370A mice have a larger proportion of thicker hairs than mice expressing the ancestral allele. The average frequency (±SEM) of awl (B) and auchene (C) hairs of each size is shown. Significance levels of differences between 370V and 370A animals by ANOVA post hoc tests: p < 0.05 (*), p < 0.01 (**). See also Table S5. Cell 152, 691–702, February 14, 2013 ª2013 Elsevier Inc. 695