Structure, expression and biological activity of zebrafish TAp63, and molecular features of the mutant hu2525 allele.

(A) Schematics of structure of zebrafish p63 gene and of the encoded TAp63 (TA1 and TA4) and ΔNp63 isoforms. The different C-terminal isoforms (a,b,g) are not considered. The TA-specific transactivation domain is in dark (TA4) or light and dark (TA1) blue, the ΔN-specific N-terminal domain in red. Positions of primers used for RT-PCR analyses in (B–D) are indicated. Skipping of TA-specific exon 2 (indicated by dark blue lines) leads to the formation of the shorter TA4 isoform. The position of the TCA->TAA nonsense mutation (hu2525), and the resulting truncations of all TAp63 isoforms are indicated. (B) RT-PCR analyses of adult zebrafish skin, using indicated alternative TA-specific sense primers (for exact sequences and positions, see Figure S1), together with an antisense primer in exon 7 shared between TA and ΔNp63. TAs1 generates two bands, reflecting the presence or absence of exon2-encoded sequences and representing either TA1 (0.991 kb band; exon 2 present) or TA4 (0.862 kb band; exon 2 absent). TAs2 positioned in exon2 generates one band, representing TA1. TAs3 positioned in exon 3 shared by TA1 and TA4 generates one band. (C) RT-PCR analyses of TA1+TA4 and ΔNp63 expression at different stages of zebrafish development (10 hpf = end of grastrulation; 20 dpf = onset of metamorphosis) and in skin and ovaries of adult zebrafish (1 year). Identical results were obtained in three independent experiments. (D) Quantitative RT-PCR analyses of ΔNp63 and TAp63 transcript levels relative to standard rps23 transcript, and ratios of ΔNp63 and TAp63 isoforms in whole embryos/larvae at 10 hpf and 20 dpf and in adult skin and ovaries. (E–H) in situ hybridization with TA-specific TAp63 (E,F) and p53 (G,H) antisense (E,G) and sense control (F,H) RNA probes; transverse sections through breeding tubercles of lower jaw of wild-type fish, 1 year old. Breeding tubercles (bt), regular epidermis (ep), dermis (de) and melanocytes (mc) are indicated. (I,J) Tunel staining of apoptotic cells in uninjected embryo (I) and embryo injected with TA(1)p63γ mRNA (J); mid-gastrula stage (8 hpf). View on animal pole. (K) Quantification of TAp63-induced phenotypes (severe malformations and death) at 24 hpf, resulting from apoptosis during mid-gastrula stages, as shown in panels I,J (ratio between affected and total (n) embryos; in %; 3 independent injections). TA(1)p63γ(wt), n = 240; TA(4)p63γ(wt), n = 314; TA(1)p63γ(hu2525 mut), n = 234. (L,M) Representative examples of embryos at 32 hpf, injected with ΔNp63α1 mRNA (L) or co-injected with ΔNp63α1 and TA(4)p63γ mRNA (M); lateral views on head region. Over-expression of ΔNp63 leads to the loss of forebrain (fb) and eyes (ey), resulting from ventral shifts during early patterning of the embryonic ectoderm (L) [58], while these structures are rescued in the embryo over-expressing both ΔNp63 and TAp63 (M). (N) Quantification of headless phenotypes (in % of wild-type plus headless embryos (n); 3 independent injections; ΔNp63α1, n = 277; ΔNp63α1+TA(4)p63γ(wt), n = 354). For simplicity, dead and malformed embryos in the co-injection are not considered. However, frequencies were significantly lower (25%) compared to embryos only injected with TA(4)p63γ(wt) (52%; see panel K), indicating that ΔNp63 also alleviates the pro-apoptotic effect of TAp63. (O) MboI RFLP analysis of TAp63 cDNA fragment amplified from skin of adult wild-type or TAp63^{hu2525/hu2525} mutant fish. The mutant only contains transcripts harbouring the hu2525 mutation that destroys an MboI site (GATC; see Figure S3), resulting in a 208 bp instead of the two wild-type cleavage products of 101 bp and 107 bp. (P) Quantitative RT-PCR analyses of TAp63 and ΔNp63 mRNA levels in adult skin of wild-type and TAp63^{hu2525/hu2525} mutant fish (2 independent experiments), revealing that in the mutant, TAp63 levels are reduced to 13.3%, and ΔNp63 are elevated to 134% of the wild-type levels. (Q) Anti-Myc-tag Western blot of lysates from embryos injected with mRNA encoding wild-type (lane 1) or hu2525 mutant N-terminally Myc-tagged zebrafish TA(4)p63γ. Ponceau red staining of membrane is shown as loading control. Calculated protein masses of 6xMyc-TA(4)p63γ proteins are: wt, 65.3 kDa; mut, 14.4 kDa. Identical results were obtained in 3 independent experiments. Scale bars are: 20 µm (E–H), 200 µm (I,J).