Limits...
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.

Show MeSH

Related in: MedlinePlus

Depletion of TYR protein in skin in Tyr-shRNA; rtTA3 mice.(A) Four-mm skin punch biopsies were taken at P100 from three different coat color backgrounds. Total protein was extracted and used to immunoblot for TYR and GFP. The numerical values present below the TYR protein bands in the western blot represent the relative intensity of the TYR band normalized to the beta-actin band (loading control) for each lane divided by the relative expression of the TYR band in the rtTA3 control sample. (B) Four-mm skin punch biopsies taken from the indicated mice at P100 were formalin fixed, dehydrated, and paraffin embedded. Next, seven-μm thick sections of the skin were cut and immunostained for GFP.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664286&req=5

pone.0143702.g002: Depletion of TYR protein in skin in Tyr-shRNA; rtTA3 mice.(A) Four-mm skin punch biopsies were taken at P100 from three different coat color backgrounds. Total protein was extracted and used to immunoblot for TYR and GFP. The numerical values present below the TYR protein bands in the western blot represent the relative intensity of the TYR band normalized to the beta-actin band (loading control) for each lane divided by the relative expression of the TYR band in the rtTA3 control sample. (B) Four-mm skin punch biopsies taken from the indicated mice at P100 were formalin fixed, dehydrated, and paraffin embedded. Next, seven-μm thick sections of the skin were cut and immunostained for GFP.

Mentions: To better assess how effectively our Tyr-shRNAs could inhibit TYR expression in vivo, we performed a more detailed analysis of the expression of the Tyr-shRNA and TYR in Tyr-shRNA/rtTA2 and Tyr-shRNA/rtTA3 knockdown mice. To do so, we performed western blot analysis on skin harvested at P50 from Tyr-shRNA/rtTA3 knockdown mice and Tyr-shRNA control mice. Western blot analysis indicated knockdown of TYR with simultaneous substantial expression of GFP, providing evidence of Tyr knockdown in the skin tissue (Fig 2A). Densitometry analysis showed a 65%-73% reduction in TYR protein (Fig 2A, numerical values under TYR protein bands). We also utilized harvested skin from mice at P50 and performed immunohistochemistry using an anti-GFP antibody. When compared to rtTA2, rtTA3, or Tyr-shRNA only littermate controls, Tyr-shRNA/rtTA2 and Tyr-shRNA/rtTA3 mice showed robust staining of GFP in the epidermis and the hair follicle in all coat color backgrounds, confirming co-expression of Tyr-shRNA when mice are fed a DOX diet (Fig 2B). Further IHC experiments demonstrated that GFP expression co-localized with Melan-A expression, indicating that GFP is expressed in melanocytes (S4 Fig).


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)

Depletion of TYR protein in skin in Tyr-shRNA; rtTA3 mice.(A) Four-mm skin punch biopsies were taken at P100 from three different coat color backgrounds. Total protein was extracted and used to immunoblot for TYR and GFP. The numerical values present below the TYR protein bands in the western blot represent the relative intensity of the TYR band normalized to the beta-actin band (loading control) for each lane divided by the relative expression of the TYR band in the rtTA3 control sample. (B) Four-mm skin punch biopsies taken from the indicated mice at P100 were formalin fixed, dehydrated, and paraffin embedded. Next, seven-μm thick sections of the skin were cut and immunostained for GFP.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143702.g002: Depletion of TYR protein in skin in Tyr-shRNA; rtTA3 mice.(A) Four-mm skin punch biopsies were taken at P100 from three different coat color backgrounds. Total protein was extracted and used to immunoblot for TYR and GFP. The numerical values present below the TYR protein bands in the western blot represent the relative intensity of the TYR band normalized to the beta-actin band (loading control) for each lane divided by the relative expression of the TYR band in the rtTA3 control sample. (B) Four-mm skin punch biopsies taken from the indicated mice at P100 were formalin fixed, dehydrated, and paraffin embedded. Next, seven-μm thick sections of the skin were cut and immunostained for GFP.
Mentions: To better assess how effectively our Tyr-shRNAs could inhibit TYR expression in vivo, we performed a more detailed analysis of the expression of the Tyr-shRNA and TYR in Tyr-shRNA/rtTA2 and Tyr-shRNA/rtTA3 knockdown mice. To do so, we performed western blot analysis on skin harvested at P50 from Tyr-shRNA/rtTA3 knockdown mice and Tyr-shRNA control mice. Western blot analysis indicated knockdown of TYR with simultaneous substantial expression of GFP, providing evidence of Tyr knockdown in the skin tissue (Fig 2A). Densitometry analysis showed a 65%-73% reduction in TYR protein (Fig 2A, numerical values under TYR protein bands). We also utilized harvested skin from mice at P50 and performed immunohistochemistry using an anti-GFP antibody. When compared to rtTA2, rtTA3, or Tyr-shRNA only littermate controls, Tyr-shRNA/rtTA2 and Tyr-shRNA/rtTA3 mice showed robust staining of GFP in the epidermis and the hair follicle in all coat color backgrounds, confirming co-expression of Tyr-shRNA when mice are fed a DOX diet (Fig 2B). Further IHC experiments demonstrated that GFP expression co-localized with Melan-A expression, indicating that GFP is expressed in melanocytes (S4 Fig).

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
Related in: MedlinePlus