Fig 1.
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.
Table 1.
Knockdown of Tyr in vivo is not sufficient to induce significant melanin loss in the black mouse coat.
Table 2.
Knockdown of Tyr in vivo is not sufficient to induce significant melanin loss in the agouti mouse coat.
Fig 2.
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.
Fig 3.
Tyr depletion inhibits the normal deposition of melanin within the melanosome.
(A) Fresh whole mouse skin was excised from the indicated 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. DOPA histochemistry was performed to assess the relative activity of tyrosinase within the melanosome. The top row contains the relevant control images for the images listed in the middle and bottom rows, respectively. Images are representative of 15 melanocytes from 2 mice per group. (B) The relative accumulation of stage I-IV melanosomes in each coat color background was quantified as described in the methods. For black and yellow agouti coat colors, the rtTA3 driver was used. For white-bellied agouti coat color, the rtTA2 driver was used. N = nucleus; G = Golgi area. Bar = 0.5 microns (Bar on inset = 1.5 microns). Graph: * = p ≤0.05, or ** = p ≤0.005, Bars = standard deviation (n = 15 melanocytes from 2 mice per group).
Fig 4.
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).
Fig 5.
The partial depletion of Tyr impacts key genes involved in melanogenesis.
Four-mm skin punch biopsies were acquired from anesthetized (A) control rtTA3 mice and experimental Tyr-shRNA/rtTA3 mice, (B) control Luc-shRNA mice and experimental Luc-shRNA/rtTA2 mice, and (C) control C57BL/6J wild-type and Tyrc/Tyrc albino mice and immediately stabilized in RNAlater. Extracted RNA was subjected to Nanostring analysis on the genes listed below the bar graphs. Data shown for all three Nanostring bar graphs (A-C) is mean normalized counts of mRNA for each gene (n = 3 mice as indicated by the error bars). * = p ≤0.05 or ** = p ≤ 0.01. (D) Three littermates (genotypes from left to right as shown in graphs above each figure) on continuous DOX feed were photographed at P50 (left images). After P110, DOX was removed from the diet for 60 days and the mice were photographed once more (middle images). Finally, the mice were fed a DOX-free diet for an additional 30 days and a final set of photographs were taken (right images). Pigment accumulation was measured spectrophotometrically as described in the methods. 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%.