TAp63 and p53 act via Notch signalling and activated caspase 3.
(A,B) Double anti-p63 (in red) and anti-GFP (in green) (A) or anti-aCasp3 (in red) and anti-GFP (in green) (B) immunofluorescences of Notch signalling reporter fish at 1 year of age; transverse sections through breeding tubercles in the disc region of the lower jaw; green channel in upper panels; merged images including DAPI staining in lower panels. Regular epidermis is indicated by arrows. (C,D) GFP and calcein-blue in vivo imaging of lower jaw of Notch signalling reporter fish at 21 dpf (C) and 28 dpf (D); ventral views; green channel to the left, blue channel to the right; the bilateral breeding tubercle disc regions are indicated by arrowheads. (E–P) GFP in vivo imaging of Notch signalling reporter (in green; E–J) and anti-aCasp3 immunofluorescence (in red; K–P) in untreated controls at 30 days (E,K), DMSO-treated controls (F,L), TAp63 mutants (G,M) p53 mutants (H,N), DAPT-treated wild types (I,O) or zDEVD-treated wild types (J,P), all at 50 days of age; transverse sections through breeding tubercles in disc region of lower jaw. For each condition, identical results were obtained in all of at least 10 investigated individuals (see Figure S5). (Q) Distribution of phenotypic strengths (normal; intermediate, C1; strong, C2) in lower jaw tubercles of DMSO-, DAPT- or zDEVD-treated wild-type fish; all fish (numbers indicated; n) were evaluated at 50 days of age after calcein staining (not shown). (R,S) anti-BrdU immunofluorescence, combined with nuclear DAPI staining; transverse sections through breeding tubercles on the lower jaw of DAPT- (M), and DMSO-treated control fish (N) at an age of 60 days and directly after incubation with BrdU for 24 hours. Scale is as in panels (E–P). (T) Ratios between keratinocyte proliferation rates in lower layers of breeding tubercles and regular epidermis (epidermal rates were identical; BrdU+ nucleibt/total nucleibt//BrdU+ nucleiep/total nucleiep; in %). Standard deviations are indicated. Numbers of evaluated samples were: DMSO, 30 sections from 5 fish; DAPT, 19 sections from 4 fish. Difference is statistically significant (Student′s t-test; p = 0.0008). (U) Distribution of phenotypic strengths (normal; intermediate, C1; strong, C2) in lower jaw tubercles of TAp63 mutant and wild-type siblings versus Tg(5xUAS-E1b:6xMYC-notch1a); Tg(-1.5hsp70l:Gal4) double transgenic TAp63 mutants or wild-type siblings after heatshock-treatments from 20–50 days of development; all fish (numbers indicated; n) were evaluated at 50 days of age after calcein staining (see V–X for examples). (V–X) 50 day old non-transgenic control (WT; V), non-transgenic TAp63 mutants (W) and double transgenic TAp63 mutant after transgenic NICD expression from 20–50 days of development (X); calcein in vivo staining, lateral views on head. Arrows in (V,X) point to stained breeding tubercles in disc region of lower jaw. Abbreviations: ep, regular epidermis; bt, breeding tubercle.