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