How is Coat Color Controlled in Dogs?

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1 How is Coat Color Controlled in Dogs? Coat color is the most challenging physical feature to assess for dogs there are multiple loci interacting (epistasis, polygenetic traits), pleiotropy, and an allelic series. Some simple problems are possible, but it would be best if students learn basic principles of inheritance using simpler autosomal and sex-linked traits, and then move up to coat color later. It is helpful if you teach students the biochemical pathway for melanin production first. After that, genotypes and phenotypes can be explained in terms of actual protein activities The specific proteins are described in more detail as part of the description of each locus. Biochemistry of Dog Coat Color The color of a dog s coat is determined by relative levels of two pigments: eumelanin, which can be either brown or black, and pheomelanin, which is yellow to orange or red. Both pigments are generated by metabolism of tyrosine. A schematic diagram of the pathway is below. Schematic of the melanin synthesis pathways.

2 Schematic showing how MC1R regulates melanin synthesis. In melanocytes, the default pathway produces yellow-red pheomelanin. This partly explains why mixed breed dogs tend to be yellow or red. Eumelanins are only made if melanocytes receive specific signals from the melanocortin receptor (MC1R; the E locus). MC1R has two main ligands: melanocyte stimulating hormone (α-msh) and agouti signaling peptide (ASIP; the A locus). Normally, ASIP and α-msh compete to bind to the receptor. When ASIP binds, melanocytes produce pheomelanin primarily; when A-MSH binds, eumelanin is produced. Animals with agouti coats have individual hairs with alternating bands of black or brown and yellow/red, corresponding to alternating binding of the two ligands. Different alleles for the A locus determine how strongly ASIP binds. Different alleles for the E locus determine how MC1R signals in response to each ligand.

3 Two other loci affect coat color. β-defensin (K locus) is a co-activator that binds to MC1R and prevents ASIP from binding. This locus is responsible for some pure black dogs. The micropthalmia transcription factor (MITF; the S locus) controls several developmental programs associated with neurogenesis and neural crest. After birth, MITF controls expression of 3 enzymes that are essential to melanin production. One of the enzymes, tyrosinase, is required for BOTH pheomelanin and eumelanin production. Mutations that block MITF prevent either color from being produced, and the animal's coat is white. The Loci Currently, 7 separate loci are thought to determine coat pattern. They control level and distribution of melanins, and thereby the vast majority of coat colors. There are a few breeds for which their coloring cannot be explained by the known genes and alleles. Work continues on uncovering these remaining regulators. Four loci (E, B, K, and A) control the relative levels and color of pigment, Three loci (D, S, and M) control distribution of pigment. There are additional minor loci that control less common coat features found in a few breeds. These have been omitted for simplicity. Coats and Noses Are Different! These 7 loci affect skin color differently than coat color. This point can confuse students. For example, a yellow Labrador retriever is that color because of the recessive e/e phenotype (explained under the E locus), but still may have black nose skin. Melanocytes in a dog's foot pads and nose skin still can produce eumelanin, even though the coat has none. Brown foot pads and nose skin are the result of a homozygous recessive genotype (b/b) for TYRP1, which also produces chocolate colored coats rather than black. If this were not sufficiently complex, cuts, scars, and normal aging can change the color of a dog's nose. The best strategy is to keep it simple, and have students focus on color of coat hair and whiskers, not nose or footpad skin, when evaluating phenotypes. (Instructors can use the black vs brown nose to double-check whether a particular dog is B/- (black nose) or b/b (brown nose) genotype.)

4 The Loci Controlling Melanin Production The Extension locus (E) This locus on CFA 5 codes for the MC1R protein, which controls whether cells can respond to melanocyte stimulating hormone (MSH), and how much eumelanin they make. There are 3 alleles at this locus: E codes for normal receptor activity. Animals will produce black eumelanin. This is the dominant allele. E M codes for enhanced eumelanin. Animals will have a black mask on their nose and face, but a lighter color on the rest of their body. e codes for a defective version of the MC1R. Dogs that are homozygous recessive e/e cannot respond to MSH, so cannot make eumelanin at all. Only colors and patterns with yellow-orange pheomelanin and white can occur. Colors associated with the E locus. 1. Karelian bear dog, showing fully black hairs, due to the E allele. 2. The black muzzle and eye-band on this German shepherd dog are due to the E M allele. 3. Homozygous recessive (e/e) dogs range from pale tan to red. Both are Labrador retrievers.

5 The Brown locus (B) This locus on CFA 11 codes for tyrosinase-related protein 1 (TRP1 or TYRP1). There are four alleles for this locus. B is wild type and dominant. It allows the normal production of black eumelanin. b is recessive. Lacking TRP1, eumelanin does not undergo final conversion, and is a milk chocolate brown rather than black. A dog that is homozygous recessive for any of these three variants will show the brown color phenotype. Chocolate Labrador retrievers show this color. o b d is the most common o b c o b s is the rarest of the recessive alleles Comparison of the black (genotype B/B or B/b; left) versus brown (b/b; right) coat color phenotypes. The Black locus (K) The K locus is responsible for the majority of all-black dog breeds. The recessive allele allows agouti coloration patterns to show. There are 3 alleles for this locus. They form an allele series; each is dominant to the allele below it in the series: K B or K Black is dominant. Dog is solid black or brown. k Br is recessive to K Br, but dominant to k y. Animals will have a mix of solid and agouti colored areas, known as brindle coloring. k y is recessive. Dogs that are homozygous for this allele will display whatever pattern the A (agouti) locus is coding for.

6 The CBD103 locus on CFA16 codes for beta-defensin 103. This immune modulator also controls coat color by binding to MC1R and increasing receptor activity. The dominant allele causes an animal to be a single solid color; it may be black or brown, depending on the alleles for the B locus. The K locus cannot produce a black dog unless there is at least one E allele for the E locus. A dog that is e/e does not produce eumelanin, so will be yellow or red regardless of the alleles at K or B. The coloration associated with alleles at the K locus. A. Black Labrador retrievers have K B /- genotype; the second allele may be any of the 3 alleles for the K locus. B. Greyhound showing the brindle pattern of bands of single and agouti colored hairs. This dog will be either k Br /k Br, or k Br /K y. C. Two yellow Labrador retrievers. They show the range in color that is possible for dogs that have only pheomelanin, from pale yellow (others can be nearly blonde) to orange-red. The Agouti locus (A) This locus on CFA 24 is unusual, in that no allele at this location is truly dominant. These alleles are dominant or recessive to each other, but all of them will be hidden by the K B or k Br allele. A dog must be k y /k y for any of these a alleles to be visible. The agouti signaling peptide (ASIP) competes with alpha-msh for MC1R binding. This results in alternating bands of dark black or brown eumelanin and yellow to red pheomelanin in individual hairs. It is important to note that agouti hair patterns still require both types of melanin; a dog that has an e/e genotype will not produce eumelanin, so it will not show agouti coloration. However, the dog can be a carrier or even homozygous for one of the a alleles. Four alleles are known for this locus:

7 a y codes for very high levels of ASIP. Animals are pale yellow to fawncolored, with a few scattered black hairs in their coat. a w is the ancestral allele. It is common in wolves but rare in all modern dog breeds. German shepherds are one of the few breeds where this allele is common. German shepherds carrying this allele are much darker on the legs, chest, and face. They lack the black and tan pattern we usually associate with German shepherds. a t codes for black and tan pattern. Typically dogs carrying this allele have black or very dark heads, backs and tails, with tan, yellow, red, or brown legs, bellies, and chests. a is the true recessive. Dogs that are a/a homozygous cannot produce pheomelanin, so are solid back. This genotype is identical to that of the dominant black locus, but has a very different inheritance pattern. Data Table 4 lists genotypes and phenotypes of dogs for the agouti locus. Different patterns produced by the A alleles. 1. Fawn color on a great Dane puppy. Like most fawn colored dogs, she has few black hairs overall. They are concentrated on her head and ears in a melanistic mask due to the E M allele. 2. Bred specifically to resemble wolves, tamaskans are a newly created breed developed from German shepherds. The distinctive mixed agouti patterns are especially clear on the dog in the back. Very likely these dogs are a w /a w genotype Large (3), mid-size (4) and small (5) dogs with the black and tan pattern associated with the a t /a t genotype for the agouti locus.

8 The Loci Controlling Pigment Distribution The Dilution locus (D) This locus on CFA 25 codes for MLPH (melanophilin). The recessive mutation at this locus does not affect total melanin production, but instead causes the pigment to remain in clumps inside hair cells rather than dispersing. This results in the color looking faded or diluted. This allele affects both types of melanin, but is most obvious when it affects black eumelanin; the result is a slate-blue or gray coat. D is the allele for normal melanin dispersion. d is the recessive allele that prevents dispersion. Left: this handsome slate blue pit bull is homozygous recessive (d/d). If it was D/D or D/d instead it would be entirely black. Right: brown dogs that are homozygous d/d can be more difficult to distinguish from normal color variation. This mixed breed dog appears to have the diluted phenotype combined with a b/b genotype, but its coat may simply be within the normal range of brown color. Other brown/dilute dogs are more obvious, because they are paler brown.

9 White & White Spotting (S) White hair in a dog's coat is due to a LACK of both melanins. Thus, the necessary alleles must prevent melanin production somehow. Evaluating white is the most challenging of all colors, because there is more than one possible pathway that can produce white dogs. A locus on CFA 20 codes for MITF (micropthalmia associated transcription factor). It is critical for development and migration of melanocytes AND controls MC1R expression. Inactivation of MITF should producea white coat. However the inheritance pattern is inconsistent, leading to different estimates of the number of alleles. There is evidence suggesting Samoyeds are pure white because they have a genotype of e/e and a/a. Pheomelanin synthesis is blocked by the "a" allele of ASIP, but the "e" alleles prevent eumelanin synthesis. This is an entirely separate path than MITF, and is not universally accepted yet. White ALSO can appear for non-genetic reasons. Dogs get white spots on their chest ( chest stars ), toes ("slippers" or socks ), and tail tip when melanocytes do not migrate completely into these locations during development. Many dogs have them, particularly mixed breeds, yet these patches of white are entirely the result of non-genetic developmental variation. Currently, we know at least 2 alleles exist for MITF: the wild type (S), and an alternate allele with a SINE insertion (s). In many pure breed dogs, S and s (inactivated by the SINE) behave as simple dominant/recessive. In these breeds a dog of genotype S/S or S/s will have little or no white in its coat. A dog that is homozygous recessive (s/s) will have a large patch of white on its belly. How far the white extends up its sides, shoulders, and chest varies a great deal. Some s/s dogs have white only on their legs chest, and belly (mantle pattern). Other s/s dogs have white extending up their shoulders and around their neck (Irish spotting pattern). Still others are randomly spotted with colors (piebald) or nearly entirely white. In a smaller number of breeds, S and s appear to be co-dominant, so that S/S dogs are fully colored, S/s dogs are mantled or Irish spotted, and s/s dogs are piebald to nearly white. Table 5 lists some of the dog breeds that have been mapped for the S locus, with genotypes and phenotypes.

10 Range of variation in white. All of these dogs are s/s (homozygous recessive) genotype. Top left: Nova Scotia tolling duck retrievers with varying degrees of white on their chests. The front two dogs have patches large enough to suggest they are not just white chest stars. As this group shows, white phenotype can be difficult to call accurately. Top right: A border collie with Irish spotting. The white forms a collar around his neck, then continues down his front legs and along his belly. Bottom left: a piebald Brittany spaniel. In piebald pattern the spots are unevenly shaped, on a mostly white background. Bottom right: Dogo Argentinos are nearly white, but will have occasional color patches. White boxers and bull terriers have similar color.

11 The Merle locus (M) This locus on CFA 10 is called SILV/PMEL17. The gene codes for two type I integral membrane proteins, PMEL17 and GP100. Both proteins are involved in pre-melanosome biogenesis in both skin and eyes. The merle pattern is a solid base color (usually red/brown or black) with lighter blue/gray or reddish patches, which gives a mottled or uneven speckled effect. Dogs often have eyes that are pale, unevenly colored or mismatched. This allele is an autosomal dominant mutation. Because it interferes with pigment cell development, dogs that are homozygous for this allele may be blind, deaf, or both. Reputable breeders will never knowingly mate two dogs that carry the merle mutation. However, merle coloring can be difficult to see in dogs that are also e/e, that is, lack eumelanin. M codes for spotty melanin dispersion. m is recessive, but is the major allele in most breeds. Left: an Australian shepherd. The mismatched, patchy coloring and mix of slate, black, orange-brown, and white indicate it is heterozygous M/m. The white collar, chest, throat, and forelegs are not controlled by the M locus, but by the S locus. This pattern of white indicates the dog is also heterozygous S/s. Right: a merle border collie. Dogs that are heterozygous M/m often have mismatched eye color. If homozygous M/M, they often are nearly white, and blind and/or deaf.