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Figure 6. Prdm12 Is Induced by Retinoic Acid and Cooperates with Ngn1 and Ngn2 to Promote a Nociceptor Fate in Xenopus(A)Prdm12, Ntrk1, Ntrk2, and Ntrk3 are expressed in TG (arrowheads) and DRG (arrows) of stage 28Xenopustadpoles. Scale bar, 200mm.(B) qRT-PCR analysis ofPrdm12expression in stage 16AC explants derived from embryos injected with mRNA ofPax3-GR(250 pg), Zic1-GR(125 pg), orNgn2-GR(250 pg).The hatching gland markerXhe, the pre-placodal markerSix1, and the neuronal-specific geneTubb2bwere alsoexamined as controls of Pax3-, Zic1-, and Ngn2-inducingactivity, respectively. (C)Prdm12 expression in Xenopus neurula-stage embryos injected unilaterally at the 2-cell stage withPax3-GR(250 pg), Zic1-GR(125 pg), Ngn1(200 pg), orNgn2-GR(250 pg). Xhe that is upregulated by Pax3, Tubb2b that is induced by Ngn1/2, and pre-placodal markerFoxi1cthatis reduced by Zic1 were examined as controls. Arrow-heads indicate their altered expression in the trigeminalplacode on the injected side revealed by X-gal staining. Scale bar, 200mm.(D) Treatment ofZic1-GR(125 pg)-overexpressing ACexplants with citral (100mM) blocksPrdm12andSix1usedas controls. (E) Treatment of intact embryos at stage 11 with RA(0.01mM) expandsPrdm12.(F) qRT-PCR analysis of the expression of the indicated genes in stage 31 animal cap explants overexpressing Prdm12 (250 pg), Ngn1 (200 pg), andNgn2(200 pg), alone or in combination, as indicated, showing that Prdm12modulates the inducing activity of Ngn1/2.(G) qRT-PCR analysis ofNtrk1andEn1in stage 28Xen-opus laevisAC explants derived from embryos co-ex-pressing Ngn1 and Prdm12 WT or mutant constructs as indicated, showing that both the PR and the zinc-finger domain of Prdm12 are required for its activity. In (C) and (E), the total number of injected embryosanalyzed and among them those showing the observedphenotype is indicated. In all qRT-PCR experiments, mean ± SEM from at least 2 independent experiments is shown, and the individual values obtained are indicated (red dots). |
