Ribosomal mutations cause p53-mediated dark skin and pleiotropic effects

Abstract

Mutations in genes encoding ribosomal proteins cause the Minute phenotype in Drosophila and mice, and Diamond-Blackfan syndrome in humans. Here we report two mouse dark skin (Dsk) loci caused by mutations in Rps19 (ribosomal protein S19) and Rps20 (ribosomal protein S20). We identify a common pathophysiologic program in which p53 stabilization stimulates Kit ligand expression, and, consequently, epidermal melanocytosis via a paracrine mechanism. Accumulation of p53 also causes reduced body size and erythrocyte count. These results provide a mechanistic explanation for the diverse collection of phenotypes that accompany reduced dosage of genes encoding ribosomal proteins, and have implications for understanding normal human variation and human disease.

Figure 1

Dsk3 and Dsk4 pigmentary phenotype. (a,b) Whole footpads and tails (a) or separated tail epidermis and dermis (b) from adult animals. (c) Skin darkness (mean ± s.e.m.) of epidermis and dermis for each genotype. (d) Histological sections of footpad epidermis show pigment accumulation in the epidermis (arrow). (e–h) Xgal-stained adult footpad epidermis (e, inset), whole footpad (f) and E15.5 embryos (h) from +/+ and Dsk4/+ animals that also carry the Dct-lacZ transgene. A dashed circle denotes the area without Xgal-positive cells. (g) Number of Xgal-positive cells (mean ± s.e.m.) from the location shown in e (inset). For c and g, P values for mutant versus control (based on a two-tail t-test) are 1.53 × 10^–5 and 0.0036 (c), and 0.0017 and 0.0036 (g), for Dsk3/+ and Dsk4/+, respectively. Scale bars: b, 500 μm; d,f, 50 μm; e,h, 300 μm.

Figure 2

Positional cloning of Dsk mutations. (a) Genetic and physical maps of the Dsk4 critical interval on mouse chromosome 4. Recombination frequencies (stated as the number of recombinant chromosomes between the marker and Dsk3, over the number of informative chromosomes evaluated) are given immediately below each marker. Approximate physical coordinates in megabases (Mb) are given below. (b) The position and sequence of the Dsk4 point mutation is shown relative to the exon–intron structure of Rps20 where untranslated and protein-coding regions are represented by blue and yellow, respectively. (c,d) Predicted protein sequences for Rps20^Dsk4 (c) and Rps19^Dsk3 (d), aligned with homologous sequence in other species.

Figure 3

Tissue-specific modulation of Rps6 gene dosage. (a) One copy of Rps6 was removed either from keratinocytes (pink, using Tg.K5Cre) or from melanocytes (yellow, using Tg.MitfCre). (b–d) Whole footpads and tails (b), ears (c) and separated tail epidermis and dermis (d) from animals of the indicated genotype. (e) Skin darkness (mean ± s.e.m.); P values for mutant versus control (based on a two-tail t-test) are 1.7 × 10^–5 and 6.7 × 10^–5 for keratinocyte (Tg.K5Cre)- and melanocyte (Tg.MitfCre)-specific Rps6, respectively. Scale bar in d, 500 μm.

Figure 4

Gene expression and Kit signaling in Rps mutants. (a–c) Expression of Kitl mRNA in footpad epidermis relative to Gapdh mRNA (mean ± s.e.m.) in animals of the indicated genotype at different times. Mutant versus control P values (two-tail) are Rps6 P3 P = 0.002, P30 P = 0.003; Rps19^Dsk3/+ P0 P = 0.039, P3 P = 0.007, P10 P = 0.005, P30 P = 0.0004; Rps20^Dsk4/+ P3 P = 0.017, P10 P = 0.036, P30 P = 0.038 (n = 3–4 for each assay). (d) Appearance of P8 whole footpads from animals of the indicated genotype 6 days after intraperitoneal injection with PBS or Kit blocking antisera. Representative results are shown for each genotype and treatment condition (n = 4–6 for each class). (e) Microarray results for keratinocyte-specific Rps6 versus control footpad epidermis for each of 24,611 transcripts represented on the array. Each dot represents the mean for three mutant (Rps6^lox/+;Tg.K5Cre/+) and three control (Rps6^lox/+) samples; transcripts associated with P values ≤0.05 (after multiple testing correction) are indicated in red and identified in Supplementary Table 1.

Figure 5

p53 is sufficient and necessary to induce dark skin. (a,b) Immunofluorescence for p53 in footpad sections; white lines mark the dermal-epidermal junction. (c,d) Whole footpads from adult animals; all results shown are representative of at least three animals for each genotype. (e,f) Expression of Kitl mRNA in P30 footpad epidermis relative to Gapdh mRNA (mean ± s.e.m.) in animals of the indicated genotypes (two-tail P values are Trp53^QS/+ versus Trp53^QS/+; Tg.K5Cre/+ P = 0.00016; Rps6^+/+ vs. Rps6^lox/+; Tg.K5Cre P = 5 × 10^–7; Rps6^lox/+; Tg.K5Cre vs. Rps6^lox/+; Tg.K5Cre; Trp53^KO/KO P = 3.2 × 10^–7). Scale bars: a,b, 25 μm.

Figure 6

Effect of Rps19^Dsk3 on bone marrow. (a) Representative photomicrographs of bone marrow aspirates (stained with Wright-Giemsa) from animals of the indicated genotypes. Scale bar, 40 μm. (b) The number of annexin V–positive bone marrow progenitor cells (lineage-cKit+) in nonmutant and Rps19^Dsk3/+ animals (± s.e.m.) at 8 weeks of age; n = 3 for each genotype. Rps19^Dsk3/+ differed significantly from Rps19^+/+ (P = 0.013 based on a two-tail t-test).

Figure 7

Pathophysiology of mutations affecting ribosomal proteins (Rp). As described in the text, reduced dosage of Rps6, Rps19 or Rps20 triggers stabilization and/or activation of p53, which gives rise to a pleiotropic phenotype whose components depend on the sensitivity and response of individual cell types and on specific downstream targets of p53. All of the phenotypes can be rescued by deficiency for Trp53.