Stanford University-School of Medicine-Department of Genetics
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Genomics of mammalian pigmentation patterning
Alternating patches of black and yellow pigment are a ubiquitous feature of pigment patterns on furry mammals that contribute to camouflage, species recognition, and morphologic diversity. These patterns arise from the dynamic regulation of pigment-type switching, a phenomenon in which melanocytes choose between synthesizing eumelanin (black or brown pigment) or pheomelanin (red or yellow pigment), depending on the phase of the hair growth cycle and position on the body.
Most of the work on coat color genetics has been based on forward genetics in laboratory mice, where more than 100 different loci have been identified. Candidate gene approaches to study coat color variation in other animals have shown that the genetic basis of adaptive pigmentary variation is largely conserved in natural populations. Nonetheless, comparative zoologic studies suggest that some components of mammalian pigment-type switching have not been identified. Some examples of these include Sex-linked yellow in Syrian hamsters and Sex-linked Orange in domestic cats that cause black-yellow variegation in heterozygous females and constitutive yellow pigment production in hemizygous males and homozygous females. In addition, the appearance of regular black and yellow dorsal-ventral stripes found in many carnivores (also found in the domestic cat) has often been attributed to the Tabby gene. None of these phenomena have been described in the laboratory mouse, and understanding the molecular genetic basis of these pigment patterns will provide valuable insight into the biology and evolution of pigment variation in nature.
Studying how these pigment patterns arise will require a systematic effort to investigate novel pathways that regulate pigment-type switching in non-laboratory animals. The availability of multiple mammalian genome sequences and the development of ultra high-throughput sequencing platforms have made this possible. We are currently adopting a genomics approach to sequence and compare the skin transcriptome of alternating pigment-type regions dissected from animals that have striking pigment patterns.
Genetic Variation in Regulatory Regions of Human Pigmentation Genes We are interested in identifying the genes and polymorphisms that contribute to normal variation in human eye, hair, and skin color. Much of our understanding of human pigmentation come from studies of mutations resulting in oculocutaneous albinism (OCA). Many polymorphisms in genes associated with OCA are likely to affect gene function in a subtle or quantitative way, and are expected to have similar effects on pigmentary phenotypes. To investigate this hypothesis, we have surveyed a collection of human genomic DNA samples from individuals of diverse continental ancestries for single nucleotide polymorphisms (SNPs) in evolutionarily conserved regions of 6 candidate genes: TYR, TYRP1, MATP, MC1R, OCA2, and SLC24A5.
We resequenced a total of 70 amplicons (420-650 bp each) in 230 individuals and identified 44 to 100 polymorphisms for each gene. Frequencies of these polymorphisms range from 0.4% to 48.6%. The genetic variants identified through resequencing will be used for a large-scale association study based on 1000 DNA samples collected from subjects at the University of Arizona. To control for the effects of population substructure, I used Ancestry Informative Markers (AIMs) to measure the extent of admixture present in each group. We will evaluate which of these SNPs might contribute to phenotypic pigmentary variation. The results will provide a genetic architectural glimpse of one of the most visible characteristics that contributes to human diversity.
Genetic basis for adaptive melanism in North American wolves
In most mammals, two genes, Agouti and Mc1r, regulate the type, amount, and distribution patterns of these pigments, but in domestic dogs, there exists a third gene, Black (K), in which different alleles account for the black, brindled or fawn coat colors. Most wolves and coyotes have gray coats, but melanic morphs are found at high frequency in certain regions of North America. We found no mutations in Agouti or Mc1r that associated with melanistic coat color in wild canids. However, the ΔG23 mutation at the K locus (the same mutation responsible for black in dogs) is perfectly associated with melanism in both coyotes and wolves. In order to establish whether the K mutation occurred before the wolf/coyote/dog divergence or was introgressed from one species into another we studied the haplotype structure around the K locus. Our results indicate that Black (K) was introgressed multiple times from dogs into coyotes within the last 100 years. An introgression event from dogs also introduced the K allele into the North American wolf population where it was exposed to strong selective pressures, resulting in high frequencies of melanistic morphs in some regions. These results have important implications for the fields of molecular evolution and conservation biology, and lead to a better understanding of the molecular and historical basis for mutations that confer a selective advantage.
The melanocortin signaling pathway
The spectrum of color and diversity of patterns that we see in mammals arises, unexpectedly, from variation in the quantity, quality, and regional distribution of just two types of pigment—black eumelanin and yellow pheomelanin. Switching between eumelanin and pheomelanin production—a process commonly known as pigment “type-switching”— is controlled primarily by the melanocortin system, in which a family of G protein–coupled receptors has been implicated not only in pigmentation but also in cortisol production, body weight regulation, and exocrine gland secretion.
Our current understanding of melanocortin biology stems from the identification in laboratory mice of Mc1r mutations as the cause of recessive yellow and Agouti mutations as the cause of lethal yellow. Pigment type-switching is controlled primarily by the Melanocortin 1 receptor (Mc1r) and Agouti, which encode a seven transmembrane–domain receptor and its extracellular ligand, respectively. We are interested in using the melanocortin system to understand the general process whereby a hormone-receptor interaction triggers a cell biological switch.
Pigment-type switching provides a tractable genetic model for identifying additional components of the melanocortin system through genetic mapping of coat color traits in mammalian species. Positional cloning of mouse coat color mutations has revealed that two accessory proteins, Attractin and Mahogunin, are required for Agouti signaling. Also, mapping of the genetic basis for black coat color in domestic dogs has recently led to the discovery a previously unrecognized melanocortin receptor ligand, β-defensin 103 (CBD103). Studies with other β-defensins and additional melanocortin receptors reveal the potential for extensive cross-talk between β-defensins and the melanocortin system. We are currently using molecular genetic, biochemical, and cell-based approaches to study the role of these components in melanocortin signaling.
Genetics of dark skin
Forward genetics in mice is a robust approach for studying fundamental developmental and cell biologic mechanisms that are pertinent to human physiology and disease. The pigmentation system has played a prominent role in this effort, since small changes in gene expression are detected by simple visual inspection and many of the pathways used by pigment cells have parallels in other organs.
During the course of a genetics screen in mice for dark skin color, we recently identified missense mutations in Ribosomal protein S19 and Ribosomal protein S20 in the dominantly-inherited mutants, Dark skin 3 (Dsk3) and Dark skin 4 (Dsk4), respectively. Our work shows that these mutations act through a common pathophysiologic program in which the transcription factor p53 is both necessary and sufficient for the dark skin phenotype. Based on these observations, we are using the dark skin mutants to learn more about the control of p53 in an in vivo setting, and are studying the role of p53 in ribosomal protein mediated diseases, such as Diamond Blackfan anemia and myelodysplastic syndrome.
Molecular basis of neural circuits controlling movement
Grace Zhao has an interest determine the computational properties of the neural circuits that allow for adaptive changes either from the outside world or from within ourselves to make accurate movement. Essentially the neuronal basis of: practice makes perfect. Her approach combines system neural science with molecular-genetic tools to precisely manipulate specific circuit elements. To this end, she has generated a number of transgenic mouse lines which allows for the functional analysis of particular cell types in the cerebellum and thalamus.
Alternating patches of black and yellow pigment are a ubiquitous feature of pigment patterns on furry mammals that contribute to camouflage, species recognition, and morphologic diversity. These patterns arise from the dynamic regulation of pigment-type switching, a phenomenon in which melanocytes choose between synthesizing eumelanin (black or brown pigment) or pheomelanin (red or yellow pigment), depending on the phase of the hair growth cycle and position on the body. 
In addition, the appearance of regular black and yellow dorsal-ventral stripes found in many carnivores (also found in the domestic cat) has often been attributed to the Tabby gene. None of these phenomena have been described in the laboratory mouse, and understanding the molecular genetic basis of these pigment patterns will provide valuable insight into the biology and evolution of pigment variation in nature.
We are interested in identifying the genes and polymorphisms that contribute to normal variation in human eye, hair, and skin color. Much of our understanding of human pigmentation come from studies of mutations resulting in oculocutaneous albinism (OCA). Many polymorphisms in genes associated with OCA are likely to affect gene function in a subtle or quantitative way, and are expected to have similar effects on pigmentary phenotypes. To investigate this hypothesis, we have surveyed a collection of human genomic DNA samples from individuals of diverse continental ancestries for single nucleotide polymorphisms (SNPs) in evolutionarily conserved regions of 6 candidate genes: TYR, TYRP1, MATP, MC1R, OCA2, and SLC24A5. 
In most mammals, two genes, Agouti and Mc1r, regulate the type, amount, and distribution patterns of these pigments, but in domestic dogs, there exists a third gene, Black (K), in which different alleles account for the black, brindled or fawn coat colors. Most wolves and coyotes have gray coats, but melanic morphs are found at high frequency in certain regions of North America. We found no mutations in Agouti or Mc1r that associated with melanistic coat color in wild canids. However, the ΔG23 mutation at the K locus (the same mutation responsible for black in dogs) is perfectly associated with melanism in both coyotes and wolves. In order to establish whether the K mutation occurred before the wolf/coyote/dog divergence or was introgressed from one species into another we studied the haplotype structure around the K locus. Our results indicate that Black (K) was introgressed multiple times from dogs into coyotes within the last 100 years. An introgression event from dogs also introduced the K allele into the North American wolf population where it was exposed to strong selective pressures, resulting in high frequencies of melanistic morphs in some regions. These results have important implications for the fields of molecular evolution and conservation biology, and lead to a better understanding of the molecular and historical basis for mutations that confer a selective advantage.
and disease. The pigmentation system has played a prominent role in this effort, since small changes in gene expression are detected by simple visual inspection and many of the pathways used by pigment cells have parallels in other organs.
Grace Zhao has an interest determine the computational properties of the neural circuits that allow for adaptive changes either from the outside world or from within ourselves to make accurate movement. Essentially the neuronal basis of: practice makes perfect. Her approach combines system neural science with molecular-genetic tools to precisely manipulate specific circuit elements. To this end, she has generated a number of transgenic mouse lines which allows for the functional analysis of particular cell types in the cerebellum and thalamus.