TAp63 and p53 mutants display reduced proliferation in basal layers and reduced differentiation in upper layers of tubercle remnants.

<p>(A,B) anti-p63 immunofluorescence; transverse sections through lower jaw of wild-type (A) and TAp63 mutant (B), 1 year of age. Breeding tubercles (bt) and adjacent regular epidermis (ep) are indicated. For larger field images and p53 mutant, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004048#pgen.1004048.s004" target="_blank">Figure S4E</a>–G. (C,D,E) SEM images of lower jaw, ventral view. The TAp63 mutant (D; intermediate phenotype; C1) has a smaller disc (arrowheads in C,D) with smaller and fewer tubercles, while the posterior row of tubercles (arrows in C) is missing. The p53 mutant (E; strong phenotype; C2) lacks all breeding tubercles. Asterisks mark smaller, more randomly distributed appendages, which most likely are unculi <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004048#pgen.1004048-Roberts1" target="_blank">[85]</a> that are unaffected in the mutants. (F) Distribution of phenotypic strengths (n, normal; C1, intermediate; C2, strong) in lower jaw tubercles of wild-type, TAp63 mutant, p53 mutant and TAp63/p53 double mutant fish at an age of 50 days (upper panel) and 1 year (lower panel), determined via calcein staining (for example, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004048#pgen-1004048-g008" target="_blank">Figure 8V,X</a>). Numbers of evaluated specimens are indicated. Similar results were obtained for the pectoral fins of males (not shown). (G–I) Hematoxylin & eosin staining; transverse sections through breeding tubercle disc region of lower jaw of wild-type (G), TAp63 mutant (H; strong phenotype; C2), and p53 mutant (I; intermediate phenotype; C1) fish at 1 year. For larger field images, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004048#pgen.1004048.s004" target="_blank">Figure S4A</a>–D. (J–L) <i>tgm1 in situ</i> hybridization; transverse sections through breeding tubercle disc region of lower jaw at an age 1 year. In TAp63 mutant (K), <i>tgm1</i> levels in upper layers of breeding tubercles are strongly reduced compared to wild type (J), whereas in the p53 mutant (L), the effect is weaker. Identical results were obtained in three independent experiments. (M–O) anti-BrdU immunofluorescence, combined with nuclear DAPI staining; transverse sections through breeding tubercles on the lower jaw of wild-type (M), TAp63 mutant (N), and p53 mutant (O) fish at an age of 60 days and directly after incubation with BrdU for 24 hours. (P) Ratios between keratinocyte proliferation rates in lower layers of breeding tubercles and regular epidermis (epidermal rates were unaltered in all cases; BrdU+ nuclei<sub>bt</sub>/total nuclei<sub>bt</sub>//BrdU+ nuclei<sub>ep</sub>/total nuclei<sub>ep</sub>; in %). Standard deviations are indicated. Numbers of evaluated samples were: WT, 36 sections from 4 fish; TAp63 mut, 28 sections from 4 fish; p53 mutant, 31 sections from 4 fish; TAp63/p53 double mutant, 21 sections from 3 fish. Observed decreases in keratinocyte proliferation in tubercles of mutants are statistically significant (Student's t-test; WT - TAp63 mut, p = 0.0041; WT - p53 mut, p = 0.008; WT – TAp63/p53 double mut, p = 0.0033).</p>