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Figure 1. Analysis of δNp63 expression in developing embryos. A: Analysis of δNp63 temporal expression pattern. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on total RNA extracted from embryos at different embryonic stages. EF1α amplification was used as control. B–P: Whole-mount in situ hybridization analysis of spatiotemporal δNp63 expression. B: Lateral view. C,D,J–L: Lateral view, anterior on the right, dorsal at the top. E,M–P: Anterior view, dorsal at the top. F,H,I: Dorsal view, anterior on the right. G,M′–P′: Transversal sections. B,F,G,H: In situ hybridization for δNp63. C,D: Double in situ hybridization for δNp63 (purple) and XK81a (turquoise). G: Small arrow, δNp63 expression in the internal layer of the ectoderm. Inset, δNp63 (purple) and XK81a (turquoise). (H) Small arrow, two medial stripes of δNp63 expression. E,N,N′: δNp63 (purple) and Sox2 (turquoise). I,J,M,M′: δNp63 (purple) and FoxD3 (turquoise). L,P,P′: δNp63 (purple) and Dlx5 (turquoise). K, O, O′: In situ hybridization for Six1. Asterisk, δNp63 expression in the prospective epidermis. Red star, ventral anterior prospective epidermis with no δNp63 expression. Arrowhead, δNp63 expression in the most external limit of the neural crest overlapping with Six1 and Dlx5 expression. Large arrow, anterior δNp63 expression overlaps with Six1 and Dlx5. i.l., internal layer; e.l., external layer. An, animal; Vg, vegetal; D, dorsal; V, ventral; np, neural plate.Download figure to PowerPoint
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Figure 2. Np63 expression is decreased by inhibiting bone morphogenetic protein (BMP) signaling. A,A′,B,B′: One dorsal blastomere of 8- to 16-cell stage embryos was injected with 500 pg of CM-BMP4 (A,A′) or dnBMPR mRNA (B,B′) and the expression of Np63 was analyzed at stage 16. A,B: Dorsal view, anterior on the right. A′,B′: Anterior view, dorsal at the top. The injected side was recognized by FLDx staining (A,A′) or fluorescence (B,B′) and is indicated by an arrowhead. C–F: One-cell stage embryos were injected with CM-BMP4 (D,F) or dnBMPR mRNA (E). Control embryos were not injected. At stage 9, animal caps were dissected and processed for reverse transcriptase-polymerase chain reaction (RT-PCR) or in situ hybridization when sibling embryos reached neurula stage 16. C–E: Note the reduced Np63 expression analyzed by in situ hybridization in injected animal caps (D,E) compared with control caps (C). F: Total RNA was isolated from stage 14 embryos and treated and control caps and the expression of Np63, epidermal markers XK81a and Dlx3, neural marker Sox2 and the mesodermal marker XBra was analyzed by RT-PCR. EF1α amplification was used as control.Download figure to PowerPoint
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Figure 3. Overexpression of δNp63 induces epidermal fate and suppresses neural induction. A–F: δNp63 mRNA (700 pg) was injected into one dorsal blastomere of 16-cell stage embryos. Embryos were fixed at stage 16 and whole-mount in situ hybridization was performed for (A) XK81a, (B) Dlx3, (C) Rexp52, (D) XAG, (E) FoxD3, and (F) Sox2. A–C,E,F: Dorsal view, anterior on the right. D: Anterior view, dorsal at the top. Dashed lines indicate the midline, small arrowheads indicate increased expression, and brackets indicate the width of the neural plate. Arrowheads indicate the injected side, recognized by FLDx staining. G–L: One-cell stage embryos were injected with 500 pg of Chd (H,K) or 500 pg of Chd + 500 pg δNp63 mRNA (I,L). G,J: Control embryos were not injected. Embryos were cultured until the blastula stage when animal caps were dissected out and processed when sibling embryos reached the equivalent of stage 16. The expression of XK81a and Sox2 was assessed by in situ hybridization. M: Total RNA was isolated from treated and control caps and the expression of δNp63, epidermal markers XK81a and Dlx3, neural marker Sox2 and the mesodermal marker XBra was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR).Download figure to PowerPoint
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Figure 4. delta-Np63 is required for epidermal initial development. A: Both blastomeres of two-cell stage embryos were injected with delta-Np63(Ct)-GFP mRNA (3.5 ng/embryo). δNp63(Ct)-GFP was coinjected with CoMO (40 ng/embryo) or with δNp63MO (10, 30 or 40 ng/embryo). Injected and control embryos were cultured until stage 16 and processed for total protein extraction. delta-Np63(Ct)-GFP protein was detected by western blotting using an anti-green fluorescent protein (GFP) antibody. Each lane was loaded with 100 μg of total protein. delta-Np63MO inhibits translation of δNp63 in a dose dependent manner. B–G: delta-Np63MO (40 ng/embryo; B–F) or CoMO (40 ng/embryo; G) was injected into one blastomere of 8- to 16-cells stage embryos. Embryos were fixed at stage 16, and the expression of different genes was analyzed by in situ hybridization. B–D,G: Dorsal view, anterior at the top. F: Anterior view, dorsal at the top. B′,C′,E: Transverse sections. Arrowhead indicates the injected side evidenced by the linage tracer FLDx. Dashed lines indicate the midline. Brackets indicate the width of the neural plate. B,E,F: δNp63-depleted embryos fail to correctly express epidermal markers XK81a (B), Rexp52 (E), and cement gland marker XAG (F). C,D: Neural plate marker Sox2 and neural crest marker FoxD3 are expanded in the delta-Np63MO-injected side of embryos. F: Injection of control morpholino (CoMO) showed no effect on FoxD3 expression. np, neural plate.Download figure to PowerPoint
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Figure 5. delta-Np63(Ct)-GFP acts as a dominant negative of delta-Np63. A–F: One dorsal blastomere of eight-cell stage embryos was injected with 0.5 ng (F), 2.0 ng (F), or 3.5 ng (A–C,F) of delta-Np63(Ct)-GFP mRNA or with different combinations of delta-Np63(Ct)-GFP and delta-Np63 (D–F). Embryos were fixed at stage 16 and the expression of several genes was analyzed. The injected side is evidenced by green fluorescent protein (GFP) fluorescence. A–C: Dorsal view, anterior at the top. A′–C′: Transverse sections. Dashed lines indicate the midline of embryos. Brackets indicate the width of the neural plate. The injected side of the embryos treated with delta-Np63(Ct)-GFP shows a reduction in XK81a expression (A,A′) and an increase in Sox2 (B,B′) and FoxD3 (C,C′) expression. D,E: The coinjection of delta-Np63(Ct)-GFP and δNp63 (1:1 ratio) rescued XK81a (D) and Sox2 (E) expressions. F: Quantification of delta-Np63(Ct)-GFP dose effects and rescue experiments. np, neural plate.Download figure to PowerPoint
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Figure 7. δNp63 inhibits apoptosis in embryos and animal caps. A–I: One dorsal blastomere of 8-cell stage embryos (A) or one-cell stage embryos (B–I) were injected with 1 ng of δNp63 (A,C,G), 1 ng of δNp63 + 1 ng of Bax (C,H), or 1 ng of δNp63 + 500 pg of Bax (I). A: Embryos were fixed at stage 16 or cultured. B–D: A set of embryos was grown until stage 13 and epidermis explants were dissected and cultured until the equivalent of stage 16. F–I: Another set was cultured until the blastula stage when animal caps were dissected and cultured until the equivalent of stage 16. Apoptosis was analyzed by terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) staining. A: Dorsal view, anterior on the right. The arrowhead indicates the injected side containing FLDx as lineage tracer. B,C: Inset: FLDx evidenced by fluorescence. E: Quantification of the epidermis explants experiments. J: Quantification of the animal cap experiments. a.n., apoptotic nuclei. K: One-cell stage embryos were injected with 1ng of δNp63 mRNA; control embryos were not injected. Embryos were cultured until the blastula stage when animal caps were dissected out and processed for reverse transcriptase-polymerase chain reaction (RT-PCR). Caspases 2, 3, 9, Bax and Bcl2 expression was analyzed. EF1α was used as a loading control. L: Quantification of the gel that is shown in (K). Results are expressed as Relative Intensity (Sample/EF1αX10). Student's t-test was used to analyze the differences between each group and the corresponding control group. Differences were considered statistically significant at P < 0.001(*).Download figure to PowerPoint
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