Luo T et al. (2003), Induction of neural crest in Xenopus by transcr...

XB-ART-5946

Proc Natl Acad Sci U S A 2003 Jan 21;1002:532-7. doi: 10.1073/pnas.0237226100.

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Induction of neural crest in Xenopus by transcription factor AP2alpha.

Luo T , Lee YH , Saint-Jeannet JP , Sargent TD .

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We report experiments with Xenopus laevis, using both intact embryos and ectodermal explants, showing that the transcription factor AP2alpha is positively regulated by bone morphogenetic protein (BMP) and Wnt signaling, and that this activation is an essential step in the induction of neural crest (NC). Ectopic expression of AP2alpha is sufficient to activate high-level expression of NC-specific genes such as Slug and Sox9, which can occur as isolated domains within the neural plate as well as by expansion of endogenous NC territories. AP2alpha also has the property of inducing NC in isolated ectoderm in which Wnt signaling is provided but BMP signaling is minimized by overexpression of chordin. Like other NC regulatory factors, activation of AP2alpha requires some attenuation of endogenous BMP signaling; however, this process occurs at a lower threshold for AP2alpha. Furthermore, AP2alpha expression domains are larger than for other NC factors. Loss-of-function experiments with antisense AP2alpha morpholino oligonucleotides result in severe reduction in the NC territory. These results support a central role for AP2alpha in NC induction. We propose a model in which AP2alpha expression, along with inactivation of NC inhibitory factors such as Dlx3, establish a feedback loop comprising AP2alpha, Sox9, and Slug, leading to and maintaining NC specification.

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Species referenced: Xenopus laevis
Genes referenced: chrd dlx3 snai2 sox2 sox9 tfap2a
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	Figure 1 Comparison of neurula stage expression of AP2α and other NC marker genes. (A) Whole-mount in situ hybridization with probes for AP2α, Slug, and Sox9 are shown in dorsal and lateral views. AP2α transcripts appear to extend more laterally than for the other two genes. This is shown more clearly in cross section in B, where the bracket indicates the Slug domain and the arrows point to the more extensive lateral AP2α. Note that under the conditions used here the epidermal expression of AP2α, which is considerably lower than for NC, is not apparent. The notochord is indicated by n. (Magnifications: A, ×7; B, ×42.)
	Figure 2 NC expression of AP2α depends on Wnt signaling. Whole-mount in situ hybridizations with NC markers Sox9, Slug, and AP2α to embryos injected into one cell at the two-cell stage with 100 pg of plasmid DNA encoding Xenopus Xwnt-1 (Left) and Xwnt-3A (Center), or 1 ng RNA encoding GSK3β (Right), along with a β-galactosidase lineage tracer (red staining). In all cases the injected side is oriented to the right (red staining). Both Wnt treatments resulted in lateral and posterior expansion of NC territory, including expression of AP2α as well as the other NC markers. The reciprocal treatment with GSK3β, which interferes with downstream Wnt signaling (50), had the opposite effect: AP2α, Sox9 and Slug all were repressed. (Magnification: ×15.)
	Figure 3 Loss of function by MO antisense injections. (A) In vitro-coupled transcription/translation reactions with plasmid encoding the AP2α ORF in the presence of either control (Co-mo) or perfect-match AP2α MO (AP2-mo; 50 ng; see Materials and Methods for sequences). (B) Neurula stage embryos injected into one cell at the two-cell stage with 30 ng of AP2α MO or the control MO along with β-galactosidase mRNA as a lineage tracer and probed for expression of NC markers Slug and Sox9 and the neural plate marker Sox2. The injected sides are on the right in these dorsal views (reddish β-galactosidase staining). The AP2α MO reduces NC and slightly expands neural plate laterally. (C) AP2α-MO inhibits Slug expression in a dose-dependent manner (●). Values in parentheses indicate the number of embryos analyzed for each concentration of injected MO. Injection of 30 ng of a control MO had a minor effect on Slug expression (■). (D) Cross section of AP2α MO-injected embryo showing reduction in Slug. The injected side is to the right and the uninjected side is to the left (arrow). This also shows the reduced elevation of neural fold after AP2α MO injection. (Magnifications: B, ×7; D, ×42.)
	Figure 4 Ectopic expression of AP2α converts neural plate into NC. Whole-mount in situ hybridization of probes for Slug, Sox9 (NC), and Sox2 (neural plate) to embryos injected at the 16-cell stage with 100 pg of RNA encoding AP2α, along with β-galactosidase lineage tracer RNA (5-bromo-4-chloro-3-indolyl-β-d-galactoside, turquoise staining). Discrete domains of intense ectopic expression of both NC marker genes was observed (arrows) within the neural plate. Endogenous Slug and Sox9 expression is indicated by the arrowheads. Concomitant repression of neural plate identity also occurred, as indicated by the reduction in Sox2 expression (arrow). (Magnification: ×22.)

References [+] :

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