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Figure 2. Developmental expression of Tfap2e by in situ hybridization. (A,B) Tfap2e onset of expression at the NPB at Stage 13. (C and D) At Stage 16 Tfap2e expression extends to the entire length of the embryo, in region of the prospective trunk (arrowheads) and cranial NC. The most anterior segment of the cranial NC, the mandibular NC, shows stronger expression than the rest of the cranial NC (arrows). Panels (A and C), dorsal views, anterior to top. Panels (B and D), lateral views, anterior to right. (E) Transverse section of a Stage 16 embryo, dorsal to top, showing Tfap2e expression in the mandibular NC. (F–H) At Stage 22 as the neural tube closes, Tfap2e expression is restricted to the dorsal aspect of the spinal cord and in the most anterior and posterior segment of the migrating cranial NC (arrows). (I and J) On a transverse section of a Stage 22 embryo, Tfap2e and Sox8 have overlapping expression domains. (K–M) At Stage 25, Tfap2e expression is detected in the mandibular NC (arrows), the dorsal aspect of the spinal cord (arrowheads) and in the brain. (N–P) At Stage 30, Tfap2e expression is detected in the brain, dorsal neural tube and NC-derived melanocytes (arrows). (Q) Section of a Stage 30 embryo at the level of the hindbrain. Transverse (R) and longitudinal (S) sections of a Stage 35 embryo, showing expression in the brain and retina. Panels (A,K,N), dorsal views, anterior to right. Panels (G,L,O), frontal views, dorsal to top. Panels (H,M,P), lateral views, anterior to right. The stages are indicated in the lower left corner of each panel. ar, archenteron; hb, hindbrain; le, lens; no, notochord; ph, pharynx. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Figure 3. Comparative expression of Tfap2e with other NPB genes at the neurula stage. (A–F) Expression of Tfap2e, Snail2, Sox10, Xhe, Runx1, and Sox2 in stage-matched Stage 16 embryos. Dorsal views, anterior to top. (G–I′) In situ hybridization on adjacent transverse sections of a Stage 15 embryo (G), dorsal to top. Tfap2e (H) is coexpressed with Runx1 (I), as indicated by the red overlay in panels (H′) and (I′). ar, archenteron; no, notochord; np, neural plate. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Figure 4. Regulation of Tfap2e expression at the NPB. (A) Tfap2e expression at the NPB in a control embryo. (B and C) Unilateral injection of Pax3 (Pax3MO) or Zic1 (Zic1MO) morpholino antisense oligonucleotides at the two-cell stage resulted in a strong reduction of Tfap2e expression at the neurula stage. (D and E) Misexpression of Wnt8 or Fgf8 in one blastomere at the two-cell stage led to a lateral expansion of Tfap2e expression domain. (F and G) Conversely, morpholino-mediated knockdown of either Fgf8 (Fgf8MO) or Wnt8 (Wnt8MO) resulted in a loss of Tfap2e expression on the injected side. Embryos are viewed from the dorsal side, anterior to top. In all panels (B–G), the injected side is on the right, as indicated by the presence of the lineage tracer (Red-gal). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Figure 5. Tfap2e-depletion blocks NC formation and expands neural plate. (A) Target sequence (red) of Tfap2e morpholino antisense oligonucleotide (Tfap2eMO). The position of the start codon (ATG) is indicated. (B) In vitro coupled transcription/translation reactions with plasmid encoding Tfap2e. Increasing amounts of Tfap2eMO, 10 ng (+), 100 ng (++), and 1000 ng (+++) blocks translation directed by Tfap2e mRNA. (C–H) Embryos injected with 30 ng of Tfap2eMO in one blastomere at the two-cell stage displayed a reduction or loss of Snail2 (C), Sox10 (D), and Runx1 (E) expression, and a lateral expansion of Sox2 (F) and reduction of Keratin (G) expression domains. Tfap2a expression levels were largely unaffected in the morphant embryos, however, its domain of expression was shifted laterally (H). Dorsal views, anterior to top. Injected side (right side) is identified by the presence of the lineage tracer (Red-gal). (I) At tailbud stage, these embryos showed a severe reduction in the number of migratory cranial NC cells on the injected side as compared to the uninjected side (right panel). Embryo viewed from the lateral side, dorsal to top, anterior to right (left panel), or to left (right panel). (J) Snail2 expression in Tfap2e morphants can be efficiently rescued by coinjection of mRNA encoding a hormone inducible version of Tfap2e (Tfap2eGR). Dorsal views, anterior to top. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Figure 6. Tfap2e is sufficient to promote NC fate. (A–D) Injection of 0.5 ng of Tfap2e-GR mRNA at the two-cell stage expands Snail2 (A) and Sox10 (B) expression domains at the NPB while reducing Sox2 (C) and Runx1 (D) in the neuroectoderm. (E) In animal cap explants Tfap2eGR is sufficient to strongly activate Snail2 and Sox10 expression after 4 and 8 h in the presence of Dex, respectively. Comparatively, Sox2 and Runx1 were only weakly activated by Tfap2eGR while the epidermis marker, keratin, was reduced in these explants. Tfap2eGR was unable to activate the expression of Tfap2a and Msx1. The values were normalized to Ef1a and presented as mean ± SEM; (*), P < 0.05 versus uninjected (U), from four-independent experiments. (F) Proposed model for the sequence of induction of Tpaf2e and its putative downstream targets in NC progenitors, based on this study and published work (O'Donnell et al., 2006; Hong and Saint-Jeannet, 2007; de Croze et al., 2011). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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tfap2e (transcription factor AP-2 epsilon (activating enhancer binding protein 2 epsilon)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16. C. Dorsal view, anterior up. D. Lateral view, anterior right. E. Section.
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tfap2e (transcription factor AP-2 epsilon (activating enhancer binding protein 2 epsilon)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22. G. Anterior view, dorsal up. H. Lateral view, anterior right. I. Cross section.
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tfap2e (transcription factor AP-2 epsilon (activating enhancer binding protein 2 epsilon)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25. K. Dorsal view, anterior right. L. Anterior view, dorsal up. M. Lateral view, anterior right.
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tfap2e (transcription factor AP-2 epsilon (activating enhancer binding protein 2 epsilon)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30. O. Anterior view, dorsal up. P. Lateral view, anterior right. Q. Cross section through hindbrain.
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tfap2e (transcription factor AP-2 epsilon (activating enhancer binding protein 2 epsilon)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35. R. Cross-section through hindbrain/eye. S. Section.
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