Cel Figure 4. 370A Does Not Increase Meibo- mian Gland Size rbital (white arrow) and suborbital (red arrow) D Differences between genotypes did not reach statistical significance. See also Table s5 12 370v/370A 370A level that requi priori knowledge of biological t his is especially useful for stu Melic variants of 6 n Ancient Asian Origin for 370A patially explicit simulation, haplotype Superorbital and maximum likelihood analyses sug Gland Type gest that 370A originated once in central China more than 30,000 years BP with a selective coefficient that is one of the Modeling Human Adaptive Variation in Mice highest measured in human populations. Our results are consis- The laboratory mouse is an established tool for understanding tent with previous inferences that 370A must have arisen prior to pathological variants in the human genome(Cox and Brown, 15,000 BP(Bryk et aL., 2008; Peter et al., 2012) and the first 2003). Recent work on an adaptive variant of the FoxP2 gene peopling of the Americas(Goebel et al., 2008: O'Rourke and showed that a mouse model can also enable functional examina- Raff, 2010)but also suggest that the allele likely emerged in tion of nonpathological, hominin-specific alleles(Enard et al., East Asia even earlier. It should be noted that haplotype-based 2009). To our knowledge, our study is the first to demonstrate methods, such as that used by Bryk and colleagues(Bryk that mice can be used to model the phenotypic effects of adap- et al., 2008)assume recombination occurs between distinct tive variation within H. sapiens, rather than between humans and haplotypes. However, in a case of rapid local fixation, as is likely other species. There are several advantages to this approach. for a strongly selected and semidominant allele like 370A, First, the existence of inbred mouse strains allows phenotypes recombination of the selected haplotype with itself would be to be evaluated on a genetically homogeneous background, masked, reducing the observed number of recombinations and making it possible to isolate the effects of a variant and draw leading to underestimation of the time of origin( Figure S7) conclusions about the causal effect of a genetic change. In the Thus, our findings shift the context in which to consider the current case, the 370a knockin mouse shows that the derived selective forces that could have acted on 370A mutation is sufficient to alter multiple traits in vivo. Second, the tractability of an animal model allows us to easily explore novel Phenotypic Consequences of 370A traits, e.g, eccrine gland number, and ones that are not readily A comparison of mice harboring the 370V and 370A alleles on assayed in humans, e. g, mammary gland structure. The results the same genetic background revealed multiple differences from mouse models can thus serve to inform human association including increased hair thickness, increased eccrine gland studies pursuing the identification of traits sensitive to candidate number, reduced mammary fat pad size, and increased mammary gland branch density in mice carrying 370A. With Successfully modeling human adaptive alleles in mice relies the exception of mammary fat pad size, which has not been on the conservation of target organ form and function between analyzed in gain-of-function models, these phenotypes are the two species. There are clear limitations to this approach. expected if 370A confers modestly enhanced signaling activity For example, in this study, the absence of murine dental features on EDAR. This mode of action was previously proposed based homologous to the phenotypes observed in humans, such as on the observation that 370A can potentiate NFKB signaling incisor shoveling or the presence of protostylid cusps, makes in vitro(Bryk et al., 2008; Mou et al., 2008) and a clinical case it difficult to equate any changes in 370A mouse dental report in which 370A was associated with reduced severity of morphology to a specific human dental trait -and in fact we hypohidrotic ectodermal dysplasia caused by an EDA missense observed no gross phenotypic changes in the dentition of mutation(Cluzeau et al., 2012). The hair phenotype of 370A 70A animals. Despite such caveats, when direct homology animals is consistent with this model as is the increase in eccrine exists, modeling an allele's effects in vivo has the advantage of gland number and mammary gland branching. We demon- enabling assessment of phenotypic impact on a whole-organism strated this in vivo for the eccrine gland trait by showing 370A cel152,691-702, February14,2013@2013EModeling Human Adaptive Variation in Mice The laboratory mouse is an established tool for understanding pathological variants in the human genome (Cox and Brown, 2003). Recent work on an adaptive variant of the FoxP2 gene showed that a mouse model can also enable functional examina￾tion of nonpathological, hominin-specific alleles (Enard et al., 2009). To our knowledge, our study is the first to demonstrate that mice can be used to model the phenotypic effects of adap￾tive variation within H. sapiens, rather than between humans and other species. There are several advantages to this approach. First, the existence of inbred mouse strains allows phenotypes to be evaluated on a genetically homogeneous background, making it possible to isolate the effects of a variant and draw conclusions about the causal effect of a genetic change. In the current case, the 370A knockin mouse shows that the derived mutation is sufficient to alter multiple traits in vivo. Second, the tractability of an animal model allows us to easily explore novel traits, e.g., eccrine gland number, and ones that are not readily assayed in humans, e.g., mammary gland structure. The results from mouse models can thus serve to inform human association studies pursuing the identification of traits sensitive to candidate adaptive alleles. Successfully modeling human adaptive alleles in mice relies on the conservation of target organ form and function between the two species. There are clear limitations to this approach. For example, in this study, the absence of murine dental features homologous to the phenotypes observed in humans, such as incisor shoveling or the presence of protostylid cusps, makes it difficult to equate any changes in 370A mouse dental morphology to a specific human dental trait—and in fact we observed no gross phenotypic changes in the dentition of 370A animals. Despite such caveats, when direct homology exists, modeling an allele’s effects in vivo has the advantage of enabling assessment of phenotypic impact on a whole-organism Figure 4. 370A Does Not Increase Meibo￾mian Gland Size (A–C) Representative images from 370V (A), 370V/ 370A (B), and 370A (C) mouse eyelids. Super￾orbital (white arrow) and suborbital (red arrow) Meibomian glands are visible through the connective tissue. (D) Average glandular area is shown (±SEM). Differences between genotypes did not reach statistical significance. See also Table S5. level that requires no a priori knowledge of biological targets. This is especially useful for studying allelic variants of genes with unknown or pleiotropic functions. An Ancient Asian Origin for 370A Spatially explicit simulation, haplotype, and maximum likelihood analyses sug￾gest that 370A originated once in central China more than 30,000 years BP with a selective coefficient that is one of the highest measured in human populations. Our results are consis￾tent with previous inferences that 370A must have arisen prior to 15,000 BP (Bryk et al., 2008; Peter et al., 2012) and the first peopling of the Americas (Goebel et al., 2008; O’Rourke and Raff, 2010) but also suggest that the allele likely emerged in East Asia even earlier. It should be noted that haplotype-based methods, such as that used by Bryk and colleagues (Bryk et al., 2008) assume recombination occurs between distinct haplotypes. However, in a case of rapid local fixation, as is likely for a strongly selected and semidominant allele like 370A, recombination of the selected haplotype with itself would be masked, reducing the observed number of recombinations and leading to underestimation of the time of origin (Figure S7). Thus, our findings shift the context in which to consider the selective forces that could have acted on 370A. Phenotypic Consequences of 370A A comparison of mice harboring the 370V and 370A alleles on the same genetic background revealed multiple differences, including increased hair thickness, increased eccrine gland number, reduced mammary fat pad size, and increased mammary gland branch density in mice carrying 370A. With the exception of mammary fat pad size, which has not been analyzed in gain-of-function models, these phenotypes are expected if 370A confers modestly enhanced signaling activity on EDAR. This mode of action was previously proposed based on the observation that 370A can potentiate NFkB signaling in vitro (Bryk et al., 2008; Mou et al., 2008) and a clinical case report in which 370A was associated with reduced severity of hypohidrotic ectodermal dysplasia caused by an EDA missense mutation (Cluzeau et al., 2012). The hair phenotype of 370A animals is consistent with this model, as is the increase in eccrine gland number and mammary gland branching. We demon￾strated this in vivo for the eccrine gland trait by showing 370A 696 Cell 152, 691–702, February 14, 2013 ª2013 Elsevier Inc