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

<p>(A) Schematics of structure of zebrafish <i>p63</i> 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 <a href="" target="_blank">Figure S1</a>), 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 <i>ΔNp63</i> and <i>TAp63</i> transcript levels relative to standard <i>rps23</i> transcript, and ratios of <i>ΔNp63</i> and <i>TAp63</i> 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 <i>TA(1)p63γ</i> 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) <a href="" target="_blank">[58]</a>, 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) <i>Mbo</i>I RFLP analysis of TAp63 cDNA fragment amplified from skin of adult wild-type or <i>TAp63<sup>hu2525/hu2525</sup></i> mutant fish. The mutant only contains transcripts harbouring the hu2525 mutation that destroys an <i>Mbo</i>I site (GATC; see <a href="" target="_blank">Figure S3</a>), 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 <i>TAp63<sup>hu2525/hu2525</sup></i> 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).</p>