Hong CS , Devotta A , Lee YH , Park BY , Saint-Jeannet JP .

R01 DE014212 NIDCR NIH HHS, R01-DE014212 NIDCR NIH HHS

	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.]
	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.]
	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.]
	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.]
	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.]
	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.
	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.
	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.
	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.
	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.

Aoki, Sox10 regulates the development of neural crest-derived melanocytes in Xenopus. 2003, Pubmed, Xenbase

Aoki, Sox10 regulates the development of neural crest-derived melanocytes in Xenopus. 2003, Pubmed , Xenbase
Arduini, Genetic ablation of neural crest cell diversification. 2009, Pubmed
Bae, Identification of Pax3 and Zic1 targets in the developing neural crest. 2014, Pubmed , Xenbase
Betancur, Assembling neural crest regulatory circuits into a gene regulatory network. 2010, Pubmed
Christen, FGF-8 is associated with anteroposterior patterning and limb regeneration in Xenopus. 1997, Pubmed , Xenbase
de Crozé, Reiterative AP2a activity controls sequential steps in the neural crest gene regulatory network. 2011, Pubmed , Xenbase
Ebert, TFAP2E-DKK4 and chemoresistance in colorectal cancer. 2012, Pubmed
Feng, Disorganized olfactory bulb lamination in mice deficient for transcription factor AP-2epsilon. 2009, Pubmed
Feng, Cloning and characterization of the mouse AP-2 epsilon gene: a novel family member expressed in the developing olfactory bulb. 2003, Pubmed
Fletcher, FGF8 spliceforms mediate early mesoderm and posterior neural tissue formation in Xenopus. 2006, Pubmed , Xenbase
Garnett, BMP, Wnt and FGF signals are integrated through evolutionarily conserved enhancers to achieve robust expression of Pax3 and Zic genes at the zebrafish neural plate border. 2012, Pubmed
Harland, In situ hybridization: an improved whole-mount method for Xenopus embryos. 1991, Pubmed , Xenbase
Henry, TGF-beta signals and a pattern in Xenopus laevis endodermal development. 1996, Pubmed , Xenbase
Hilger-Eversheim, Regulatory roles of AP-2 transcription factors in vertebrate development, apoptosis and cell-cycle control. 2000, Pubmed
Hoffman, Tfap2 transcription factors in zebrafish neural crest development and ectodermal evolution. 2007, Pubmed , Xenbase
Hong, The activity of Pax3 and Zic1 regulates three distinct cell fates at the neural plate border. 2007, Pubmed , Xenbase
Hong, Fgf8a induces neural crest indirectly through the activation of Wnt8 in the paraxial mesoderm. 2008, Pubmed , Xenbase
Jonas, Epidermal keratin gene expressed in embryos of Xenopus laevis. 1985, Pubmed , Xenbase
Katagiri, Molecular cloning of Xenopus hatching enzyme and its specific expression in hatching gland cells. 1997, Pubmed , Xenbase
Knight, Skeletal and pigment cell defects in the lockjaw mutant reveal multiple roles for zebrafish tfap2a in neural crest development. 2004, Pubmed
Knight, lockjaw encodes a zebrafish tfap2a required for early neural crest development. 2003, Pubmed
Kolm, Efficient hormone-inducible protein function in Xenopus laevis. 1995, Pubmed , Xenbase
Lamborghini, Rohon-beard cells and other large neurons in Xenopus embryos originate during gastrulation. 1980, Pubmed , Xenbase
Lamborghini, Disappearance of Rohon-Beard neurons from the spinal cord of larval Xenopus laevis. 1987, Pubmed , Xenbase
Luo, Induction of neural crest in Xenopus by transcription factor AP2alpha. 2003, Pubmed , Xenbase
Luo, Transcription factor AP-2 is an essential and direct regulator of epidermal development in Xenopus. 2002, Pubmed , Xenbase
Mayor, Induction of the prospective neural crest of Xenopus. 1995, Pubmed , Xenbase
Meulemans, Gene-regulatory interactions in neural crest evolution and development. 2004, Pubmed
Milet, Pax3 and Zic1 drive induction and differentiation of multipotent, migratory, and functional neural crest in Xenopus embryos. 2013, Pubmed , Xenbase
Mizuseki, Xenopus Zic-related-1 and Sox-2, two factors induced by chordin, have distinct activities in the initiation of neural induction. 1998, Pubmed , Xenbase
Monsoro-Burq, Msx1 and Pax3 cooperate to mediate FGF8 and WNT signals during Xenopus neural crest induction. 2005, Pubmed , Xenbase
O'Donnell, Functional analysis of Sox8 during neural crest development in Xenopus. 2006, Pubmed , Xenbase
Park, Xaml1/Runx1 is required for the specification of Rohon-Beard sensory neurons in Xenopus. 2012, Pubmed , Xenbase
Park, Hindbrain-derived Wnt and Fgf signals cooperate to specify the otic placode in Xenopus. 2008, Pubmed , Xenbase
Prasad, Induction of the neural crest state: control of stem cell attributes by gene regulatory, post-transcriptional and epigenetic interactions. 2012, Pubmed , Xenbase
Rada-Iglesias, Epigenomic annotation of enhancers predicts transcriptional regulators of human neural crest. 2012, Pubmed , Xenbase
Sato, Neural crest determination by co-activation of Pax3 and Zic1 genes in Xenopus ectoderm. 2005, Pubmed , Xenbase
Sauka-Spengler, A gene regulatory network orchestrates neural crest formation. 2008, Pubmed
Schorle, Transcription factor AP-2 essential for cranial closure and craniofacial development. 1996, Pubmed
Slack, An interaction between dorsal and ventral regions of the marginal zone in early amphibian embryos. 1980, Pubmed , Xenbase
Stuhlmiller, Current perspectives of the signaling pathways directing neural crest induction. 2012, Pubmed , Xenbase
Tummala, Molecular cloning and characterization of AP-2 epsilon, a fifth member of the AP-2 family. 2003, Pubmed
Van Otterloo, Novel Tfap2-mediated control of soxE expression facilitated the evolutionary emergence of the neural crest. 2012, Pubmed
Van Otterloo, Differentiation of zebrafish melanophores depends on transcription factors AP2 alpha and AP2 epsilon. 2010, Pubmed
Winning, Developmental regulation of transcription factor AP-2 during Xenopus laevis embryogenesis. 1991, Pubmed , Xenbase
Wolda, Overlapping expression of Xwnt-3A and Xwnt-1 in neural tissue of Xenopus laevis embryos. 1993, Pubmed , Xenbase
Zhang, Expression of TFAP2beta and TFAP2gamma genes in Xenopus laevis. 2006, Pubmed , Xenbase
Zhang, Neural tube, skeletal and body wall defects in mice lacking transcription factor AP-2. 1996, Pubmed