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

Tyr depletion leads to coat color changes in three unique coat color backgrounds.(A) B16 mouse melanoma cells infected with the lentiviral constructs containing individual shRNAs were harvested for quantification of Tyr mRNA (left panel) and protein (right panel). The values below the TYR protein bands in the western blot represent the relative intensity of the TYR band normalized to the tubulin band (loading control) for each lane divided by the relative expression of the TYR band in the mismatch-control (mm control) sample. (B) Mouse KH2 ES cells containing a FRT-hygro-pA cassette on chromosome 11 and a reverse tet-transactivator (rtTA) on chromosome 6 were co-electroporated with pCAGGS-FLPe and the targeting vector, pCol-TGM-Tyr. Resulting FLPe-mediated recombination between the FRT site at the Col1a1 locus and the FRT sites present within pCol-TGM-Tyr results in colonies that survive hygromycin selection. (C-F) Effect of Dox-mediated Tyr shRNA on coat color was analyzed in (C,F) yellow agouti, (D) white-bellied agouti, or (E) non-agouti (black) mice by comparison of littermates. The genotype of each mouse is listed above each photo. 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%. (C) Yellow-agouti littermates with the rtTA2 driver; (D) agouti littermates with the rtTA2 driver; (E) black (non-agouti) littermates with the rtTA3 driver and (F) yellow agouti littermates with the rtTA3 driver were shaved on their dorsal side and then photographed. rtTA3 and Tyr-shRNA/rtTA3 littermates each maintained on a doxycycline diet were photographed in daylight at P100 to demonstrate the presence of GFP in the eye.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143702.g001: Tyr depletion leads to coat color changes in three unique coat color backgrounds.(A) B16 mouse melanoma cells infected with the lentiviral constructs containing individual shRNAs were harvested for quantification of Tyr mRNA (left panel) and protein (right panel). The values below the TYR protein bands in the western blot represent the relative intensity of the TYR band normalized to the tubulin band (loading control) for each lane divided by the relative expression of the TYR band in the mismatch-control (mm control) sample. (B) Mouse KH2 ES cells containing a FRT-hygro-pA cassette on chromosome 11 and a reverse tet-transactivator (rtTA) on chromosome 6 were co-electroporated with pCAGGS-FLPe and the targeting vector, pCol-TGM-Tyr. Resulting FLPe-mediated recombination between the FRT site at the Col1a1 locus and the FRT sites present within pCol-TGM-Tyr results in colonies that survive hygromycin selection. (C-F) Effect of Dox-mediated Tyr shRNA on coat color was analyzed in (C,F) yellow agouti, (D) white-bellied agouti, or (E) non-agouti (black) mice by comparison of littermates. The genotype of each mouse is listed above each photo. 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%. (C) Yellow-agouti littermates with the rtTA2 driver; (D) agouti littermates with the rtTA2 driver; (E) black (non-agouti) littermates with the rtTA3 driver and (F) yellow agouti littermates with the rtTA3 driver were shaved on their dorsal side and then photographed. rtTA3 and Tyr-shRNA/rtTA3 littermates each maintained on a doxycycline diet were photographed in daylight at P100 to demonstrate the presence of GFP in the eye.

Mentions: To develop a model to study pigment diversity in vivo, we generated a murine model where we could selectively modulate the expression of any gene using a tet-regulatable single-copy shRNA linked to a GFP reporter [38]. First, we utilized an algorithm [35] to identify two shRNA sequences that could effectively silence Tyr. Lentiviral constructs that expressed Tyr shRNA or non-targeting scrambled shRNA (negative control) were used to infect B16 mouse melanoma cells at a MOI of 0.1 to ensure that each cell was only infected with one lentivirus (single copy shRNA) [38]. Quantitative RT-PCR, western blot, and densitometry confirmed that one of the two tested shRNAs (Tyr-shRNA #1) efficiently suppressed Tyr mRNA (Fig 1A, left panel) and protein expression (Fig 1A, right panel). Tyr-shRNA #1 was cloned into the pCol-TGM targeting vector [37] (hereafter referred to as pCol-TGM-Tyr shRNA; Fig 1B). To introduce the Tyr-shRNA into the mouse genome immediately downstream of the Col1a1 locus, pCAGGS-Flpe and pCol-TGM-Tyr shRNA were co-electroporated into KH2 mouse embryonic stem cells to generate Flpe-mediated recombination between the Frt site at the Col1a1 locus and the Frt site on the targeting vector (Fig 1B). After recombination, the tetracycline response element (TRE), GFP, and Tyr-shRNA are all inserted downstream of the Col1a1 locus in chromosome 11 in the KH2 cells (Fig 1B), a C57BL/6J x 129S4 F1 (agouti) ES cell line that is pre-engineered to express a reverse tet-trans-activator from the ROSA26 locus on chromosome 6 [37]. Southern blot analysis confirmed the correct integration of the Tyr-shRNA into the Col1a1 locus in 12/12 clones tested (S1A Fig). Modal chromosome number was verified in a sub-set of clones that were then injected into C57BL/6NTac blastocysts, and implanted into CD-1 pseudo-pregnant female foster mice. Chimeric male agouti mice confirmed to contain both the R26-M2rtTA (hereafter referred to as rtTA2) and the Tyr-shRNA via genotyping were then bred to eight-week old C57BL/6J females. To verify that the Tyr-shRNA was expressed in the hair in a doxycycline-dependent manner, breeding pairs were placed on a 600-mg/kg doxycycline chow prior to conception of litters. Resultant pups were shaved at post-natal day (P) 50 and GFP expression within the hair was monitored by fluorescence microscopy. Tyr-shRNA/rtTA2 (Tyr-knockdown mice) mice but not Tyr-shRNA (control mice) mice exhibited robust expression of GFP in the hair, indicating that Tyr-shRNA is expressed within the hair in a tet-regulatable manner (S1B Fig, left two columns).


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)

Tyr depletion leads to coat color changes in three unique coat color backgrounds.(A) B16 mouse melanoma cells infected with the lentiviral constructs containing individual shRNAs were harvested for quantification of Tyr mRNA (left panel) and protein (right panel). The values below the TYR protein bands in the western blot represent the relative intensity of the TYR band normalized to the tubulin band (loading control) for each lane divided by the relative expression of the TYR band in the mismatch-control (mm control) sample. (B) Mouse KH2 ES cells containing a FRT-hygro-pA cassette on chromosome 11 and a reverse tet-transactivator (rtTA) on chromosome 6 were co-electroporated with pCAGGS-FLPe and the targeting vector, pCol-TGM-Tyr. Resulting FLPe-mediated recombination between the FRT site at the Col1a1 locus and the FRT sites present within pCol-TGM-Tyr results in colonies that survive hygromycin selection. (C-F) Effect of Dox-mediated Tyr shRNA on coat color was analyzed in (C,F) yellow agouti, (D) white-bellied agouti, or (E) non-agouti (black) mice by comparison of littermates. The genotype of each mouse is listed above each photo. 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%. (C) Yellow-agouti littermates with the rtTA2 driver; (D) agouti littermates with the rtTA2 driver; (E) black (non-agouti) littermates with the rtTA3 driver and (F) yellow agouti littermates with the rtTA3 driver were shaved on their dorsal side and then photographed. rtTA3 and Tyr-shRNA/rtTA3 littermates each maintained on a doxycycline diet were photographed in daylight at P100 to demonstrate the presence of GFP in the eye.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143702.g001: Tyr depletion leads to coat color changes in three unique coat color backgrounds.(A) B16 mouse melanoma cells infected with the lentiviral constructs containing individual shRNAs were harvested for quantification of Tyr mRNA (left panel) and protein (right panel). The values below the TYR protein bands in the western blot represent the relative intensity of the TYR band normalized to the tubulin band (loading control) for each lane divided by the relative expression of the TYR band in the mismatch-control (mm control) sample. (B) Mouse KH2 ES cells containing a FRT-hygro-pA cassette on chromosome 11 and a reverse tet-transactivator (rtTA) on chromosome 6 were co-electroporated with pCAGGS-FLPe and the targeting vector, pCol-TGM-Tyr. Resulting FLPe-mediated recombination between the FRT site at the Col1a1 locus and the FRT sites present within pCol-TGM-Tyr results in colonies that survive hygromycin selection. (C-F) Effect of Dox-mediated Tyr shRNA on coat color was analyzed in (C,F) yellow agouti, (D) white-bellied agouti, or (E) non-agouti (black) mice by comparison of littermates. The genotype of each mouse is listed above each photo. 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%. (C) Yellow-agouti littermates with the rtTA2 driver; (D) agouti littermates with the rtTA2 driver; (E) black (non-agouti) littermates with the rtTA3 driver and (F) yellow agouti littermates with the rtTA3 driver were shaved on their dorsal side and then photographed. rtTA3 and Tyr-shRNA/rtTA3 littermates each maintained on a doxycycline diet were photographed in daylight at P100 to demonstrate the presence of GFP in the eye.
Mentions: To develop a model to study pigment diversity in vivo, we generated a murine model where we could selectively modulate the expression of any gene using a tet-regulatable single-copy shRNA linked to a GFP reporter [38]. First, we utilized an algorithm [35] to identify two shRNA sequences that could effectively silence Tyr. Lentiviral constructs that expressed Tyr shRNA or non-targeting scrambled shRNA (negative control) were used to infect B16 mouse melanoma cells at a MOI of 0.1 to ensure that each cell was only infected with one lentivirus (single copy shRNA) [38]. Quantitative RT-PCR, western blot, and densitometry confirmed that one of the two tested shRNAs (Tyr-shRNA #1) efficiently suppressed Tyr mRNA (Fig 1A, left panel) and protein expression (Fig 1A, right panel). Tyr-shRNA #1 was cloned into the pCol-TGM targeting vector [37] (hereafter referred to as pCol-TGM-Tyr shRNA; Fig 1B). To introduce the Tyr-shRNA into the mouse genome immediately downstream of the Col1a1 locus, pCAGGS-Flpe and pCol-TGM-Tyr shRNA were co-electroporated into KH2 mouse embryonic stem cells to generate Flpe-mediated recombination between the Frt site at the Col1a1 locus and the Frt site on the targeting vector (Fig 1B). After recombination, the tetracycline response element (TRE), GFP, and Tyr-shRNA are all inserted downstream of the Col1a1 locus in chromosome 11 in the KH2 cells (Fig 1B), a C57BL/6J x 129S4 F1 (agouti) ES cell line that is pre-engineered to express a reverse tet-trans-activator from the ROSA26 locus on chromosome 6 [37]. Southern blot analysis confirmed the correct integration of the Tyr-shRNA into the Col1a1 locus in 12/12 clones tested (S1A Fig). Modal chromosome number was verified in a sub-set of clones that were then injected into C57BL/6NTac blastocysts, and implanted into CD-1 pseudo-pregnant female foster mice. Chimeric male agouti mice confirmed to contain both the R26-M2rtTA (hereafter referred to as rtTA2) and the Tyr-shRNA via genotyping were then bred to eight-week old C57BL/6J females. To verify that the Tyr-shRNA was expressed in the hair in a doxycycline-dependent manner, breeding pairs were placed on a 600-mg/kg doxycycline chow prior to conception of litters. Resultant pups were shaved at post-natal day (P) 50 and GFP expression within the hair was monitored by fluorescence microscopy. Tyr-shRNA/rtTA2 (Tyr-knockdown mice) mice but not Tyr-shRNA (control mice) mice exhibited robust expression of GFP in the hair, indicating that Tyr-shRNA is expressed within the hair in a tet-regulatable manner (S1B Fig, left two columns).

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