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Tyrosinase Depletion Prevents the Maturation of Melanosomes in the Mouse Hair Follicle.

Paterson EK, Fielder TJ, MacGregor GR, Ito S, Wakamatsu K, Gillen DL, Eby V, Boissy RE, Ganesan AK - PLoS ONE (2015)

Bottom Line: Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair.Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis.These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California Irvine, Irvine, CA, United States of America.

ABSTRACT
The mechanisms that lead to variation in human skin and hair color are not fully understood. To better understand the molecular control of skin and hair color variation, we modulated the expression of Tyrosinase (Tyr), which controls the rate-limiting step of melanogenesis, by expressing a single-copy, tetracycline-inducible shRNA against Tyr in mice. Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair. Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis. These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.

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Expression of a control shRNA does not affect pigment accumulation or melanosome maturation.(A) Coat color in Luc-knockdown mice and appropriate control mice was compared both visually (genotypes of each mouse are listed above each photo) and spectrophotometrically (the percentage value below each mouse corresponds to the absorbance at 492 nm for that particular mouse divided by the absorbance at 492 nm for its control littermate, which is set to 100%). (B) Fresh whole mouse skin was excised from Luc-knockdown mice and corresponding control mice using a four-mm round punch biopsy and fixed in Karnovsky’s fixative before electron microscopy analysis. Melanosomes within the Golgi area of the cell body were evaluated for maturation stage and ultra-structural morphology. Images are representative of 15 melanocytes from 2 mice per group. (C) The melanosomes were also quantified as described in Fig 3B. N = nucleus; G = Golgi area. Bar = 0.5 microns (Bar on inset = 1.5 microns). Graph: * = p ≤0.05. Bars = standard deviation (n = 2 mice per group, 15 melanocytes per mouse analyzed).
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pone.0143702.g004: Expression of a control shRNA does not affect pigment accumulation or melanosome maturation.(A) Coat color in Luc-knockdown mice and appropriate control mice was compared both visually (genotypes of each mouse are listed above each photo) and spectrophotometrically (the percentage value below each mouse corresponds to the absorbance at 492 nm for that particular mouse divided by the absorbance at 492 nm for its control littermate, which is set to 100%). (B) Fresh whole mouse skin was excised from Luc-knockdown mice and corresponding control mice using a four-mm round punch biopsy and fixed in Karnovsky’s fixative before electron microscopy analysis. Melanosomes within the Golgi area of the cell body were evaluated for maturation stage and ultra-structural morphology. Images are representative of 15 melanocytes from 2 mice per group. (C) The melanosomes were also quantified as described in Fig 3B. N = nucleus; G = Golgi area. Bar = 0.5 microns (Bar on inset = 1.5 microns). Graph: * = p ≤0.05. Bars = standard deviation (n = 2 mice per group, 15 melanocytes per mouse analyzed).

Mentions: To verify that the phenotypes observed in our Tyr-knockdown mice were not due to possible side effects caused by GFP expression, we generated mice on the black background that expressed an shRNA directed towards Luciferase (Luc), a gene that is not expressed in the mouse. First, we examined Luc-knockdown mice and appropriate control mice and determined that there were no visual differences in coat color between the two groups (Fig 4A). Next, we analyzed shaved hairs from Luc-knockdown mice and control mice using the previously described melanin assay and found no differences in absorbance at 492 nm, indicating that expression of GFP does not affect the production of melanin within the mouse hair follicle (Fig 4A, percentage values below image). To determine whether the expression of a control shRNA or GFP affected the deposition of melanin within the melanosome, fresh skin harvested from Luc shRNA/rtTA2 mice and appropriate Luc-shRNA controls on the black background and was analyzed by TEM. Melanosomes from Luc-shRNA/rtTA2 mice demonstrated no differences in morphology when compared to melanosomes obtained from Luc-shRNA only mice (Fig 4B). Both the Luc-shRNA and the Luc-shRNA/rtTA2 mice contained appropriate numbers of both early and late stage melanosomes, with stage IV melanosomes comprising the majority of the total melanosome number (Fig 4C).


Tyrosinase Depletion Prevents the Maturation of Melanosomes in the Mouse Hair Follicle.

Paterson EK, Fielder TJ, MacGregor GR, Ito S, Wakamatsu K, Gillen DL, Eby V, Boissy RE, Ganesan AK - PLoS ONE (2015)

Expression of a control shRNA does not affect pigment accumulation or melanosome maturation.(A) Coat color in Luc-knockdown mice and appropriate control mice was compared both visually (genotypes of each mouse are listed above each photo) and spectrophotometrically (the percentage value below each mouse corresponds to the absorbance at 492 nm for that particular mouse divided by the absorbance at 492 nm for its control littermate, which is set to 100%). (B) Fresh whole mouse skin was excised from Luc-knockdown mice and corresponding control mice using a four-mm round punch biopsy and fixed in Karnovsky’s fixative before electron microscopy analysis. Melanosomes within the Golgi area of the cell body were evaluated for maturation stage and ultra-structural morphology. Images are representative of 15 melanocytes from 2 mice per group. (C) The melanosomes were also quantified as described in Fig 3B. N = nucleus; G = Golgi area. Bar = 0.5 microns (Bar on inset = 1.5 microns). Graph: * = p ≤0.05. Bars = standard deviation (n = 2 mice per group, 15 melanocytes per mouse analyzed).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4664286&req=5

pone.0143702.g004: Expression of a control shRNA does not affect pigment accumulation or melanosome maturation.(A) Coat color in Luc-knockdown mice and appropriate control mice was compared both visually (genotypes of each mouse are listed above each photo) and spectrophotometrically (the percentage value below each mouse corresponds to the absorbance at 492 nm for that particular mouse divided by the absorbance at 492 nm for its control littermate, which is set to 100%). (B) Fresh whole mouse skin was excised from Luc-knockdown mice and corresponding control mice using a four-mm round punch biopsy and fixed in Karnovsky’s fixative before electron microscopy analysis. Melanosomes within the Golgi area of the cell body were evaluated for maturation stage and ultra-structural morphology. Images are representative of 15 melanocytes from 2 mice per group. (C) The melanosomes were also quantified as described in Fig 3B. N = nucleus; G = Golgi area. Bar = 0.5 microns (Bar on inset = 1.5 microns). Graph: * = p ≤0.05. Bars = standard deviation (n = 2 mice per group, 15 melanocytes per mouse analyzed).
Mentions: To verify that the phenotypes observed in our Tyr-knockdown mice were not due to possible side effects caused by GFP expression, we generated mice on the black background that expressed an shRNA directed towards Luciferase (Luc), a gene that is not expressed in the mouse. First, we examined Luc-knockdown mice and appropriate control mice and determined that there were no visual differences in coat color between the two groups (Fig 4A). Next, we analyzed shaved hairs from Luc-knockdown mice and control mice using the previously described melanin assay and found no differences in absorbance at 492 nm, indicating that expression of GFP does not affect the production of melanin within the mouse hair follicle (Fig 4A, percentage values below image). To determine whether the expression of a control shRNA or GFP affected the deposition of melanin within the melanosome, fresh skin harvested from Luc shRNA/rtTA2 mice and appropriate Luc-shRNA controls on the black background and was analyzed by TEM. Melanosomes from Luc-shRNA/rtTA2 mice demonstrated no differences in morphology when compared to melanosomes obtained from Luc-shRNA only mice (Fig 4B). Both the Luc-shRNA and the Luc-shRNA/rtTA2 mice contained appropriate numbers of both early and late stage melanosomes, with stage IV melanosomes comprising the majority of the total melanosome number (Fig 4C).

Bottom Line: Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair.Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis.These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California Irvine, Irvine, CA, United States of America.

ABSTRACT
The mechanisms that lead to variation in human skin and hair color are not fully understood. To better understand the molecular control of skin and hair color variation, we modulated the expression of Tyrosinase (Tyr), which controls the rate-limiting step of melanogenesis, by expressing a single-copy, tetracycline-inducible shRNA against Tyr in mice. Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair. Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis. These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.

Show MeSH