Huang X , Hong CS , O'Donnell M , Saint-Jeannet JP .

DE14212 NIDCR NIH HHS, R01 DE014212 NIDCR NIH HHS

	Fig. 1. Developmental expression of XDmrt4 by whole-mount in situ hybridization. (A) XDmrt4 expression at the early neurula stage is detected at the anterior edge of the neural plate. (B–D) Six1 (B), Xdlx5 (C), and XBF1 (D) expression at a similar stage are shown for comparison. (E) Early neurula stage embryo stained with both XDmrt4 and Six1 probes show that XDmrt4 is expressed in a subdomain of the preplacodal ectoderm. The arrowhead indicates the posterior boundary of XDmrt4, and the arrow marks the posterior domain of Six1. (F and G) Comparison of XDmrt4 (F) and Pax6 (G) expression in sibling embryos illustrates that both genes are expressed in the presumptive olfactory placode (arrowheads). (H and I) Double in situ hybridization showing the relationship of the expression domain of the neural crest-specific gene Slug and XDmrt4 at stage 17. The arrowheads indicate the posterior and anterior boundary of XDmrt4 and Slug, respectively. Views are dorsal-anterior (A–D and F–H) and lateral (E and I, anterior to right). (J) At stage 23, XDmrt4 is detected bilaterally in the prospective nasal placode (arrowheads), anterior view. (K–M) By stages 29/30 XDmrt4 is strongly detected in the developing olfactory organ and the telencephalon (arrowheads). At this stage XDmrt4 is also detected in the foregut (arrows, K and L). Lateral (K) and anterior (M) views, dorsal to top. (L) Transverse section. (N and O) In stage-35 embryos XDmrt4 expression domains in the telencephalon (arrows) and the olfactory organ (arrowheads) are segregated. (N) Dorsal view, anterior to right. (O) Longitudinal section through the olfactory epithelium and the telencephalon. (P–S) At stage 45, XDmrt4 expression persists in the olfactory epithelium (arrowheads, P), the olfactory bulb (arrowheads, Q), and forebrain (arrows, R and S). Whole-brain lateral (Q) and dorsal (R) views. (S) Transverse section through the forebrain. (T) XDmrt4 is also strongly expressed in the gall bladder (arrow) and more diffusely throughout the intestine. (Magnifications: ×10 in A–D, H, and I; ×12 in E, F, and J–N; ×20 in O and S; and ×15 in P, Q, and T.)
	Fig. 3. Caudalizing factors restrict XDmrt4 expression domain. Activation of Wnt signaling pathway by injection of Wnt-3a blocks XDmrt4, XBF1, and Six1 expression at the neurula stage. Conversely, inhibition of Wnt signaling pathway by injection of β-catenin morpholino (β-cat-mo) or Xdkk1 mRNA expands posteriorly XDmrt4, XBF1, and Six1 placodal expression domains. At the tail bud stage, the same manipulations of the Wnt signaling pathway resulted in a reduction or an expansion of Xebf2 and XEmx2 expression in the nasal placode. In most cases, Xdkk1 injection leads to an expansion of these markers on both sides of the injected embryos presumably because of Xdkk1 protein diffusion. Overexpression of Fgf8 completely blocks XDmrt4, XBF1, and Six1 placodal expression at the neurula stage, as well as the nasal placode expression of Xebf2 and XEmx2 at the tail bud stage. Anterior views are shown, dorsal to the top. RNA encoding the lineage tracer β-gal was coinjected in all cases to identify the injected side (red staining, left, arrows). (Magnifications: ×8.)
	dmrt4 (DMRT like family A1) gene expression gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up. Key: arrow: anterior-dorsal foregut endoderm, arrowhead: olfactory bulb.
	Fig. 2. Signaling pathways involved in inducing XDmrt4. (A) Two-cell stage embryos are injected in both blastomeres with a combination of Noggin, Wnt3a, and Fgf8 mRNAs. Animal explants are dissected at the blastula stage, cultured until neurula stage, and analyzed by RT-PCR for expression of XDmrt4 and XBF1. (B) Noggin induces strong expression of XDmrt4 and XBF1. Coinjection of Fgf8 or Wnt3a severely reduces Noggin-mediated XDmrt4 and XBF1 induction. Controls include uninjected animal explant (–), whole embryo (WE) at stage 17, and reaction minus reverse transcriptase (–RT). Elongation factor 1α is used as a loading control.
	Fig. 4. XDmrt4-mo and XDmrt4ΔC specifically blocks Xebf2 expression. (A) In vitro-coupled transcription/translation reactions with plasmid encoding WT XDmrt4 and a mutated version of XDmrt4 (muXDmrt4) in the presence of increasing amounts of XDmrt4-mo. (B) Embryo injected in with a control morpholino shows normal expression of Xebf2 on the injected side. (C and D) Injection of 20 ng of XDmrt4-mo in one blastomere at the eight-cell stage results in a reduction or loss of Xebf2 on the injected side. (E) Another Olf/Ebf family member, Xebf3, is also reduced upon XDmrt4-mo injection. (F) However, the olfactory placode marker, XEmx2, remains unaffected under these conditions. (G–J) XBF1 (G and H) and Dlx5 (I and J) expression at stage 17 (G and I) or stage 25 (H and J) are unperturbed in XDmrt4-mo-injected embryos. (K) Schematic representation of WT and XDmrt4 deletion construct (XDmrt4ΔC). The blue box indicates the position of the DM domain. (L) Injection of 1 ng of XDmrt4ΔC mRNA results in a loss of the Xebf2 expression domain. (B–J and L) RNA encoding the lineage tracer β-gal was coinjected in all cases to identify the injected side (red staining, arrows). Anterior views are shown, dorsal to the top. (Magnifications: ×10.)
	Fig. 5. Impaired neurogenesis in the olfactory epithelium of embryos lacking XDmrt4 function. (A) XDmrt4-mo or XDmrt4ΔC mRNA-injected embryos show a marked reduction in NCAM expression in the olfactory epithelium (arrows). Injection of a control morpholino (Co-mo) has no effect on NCAM expression levels. Anterior views are shown, dorsal to the top. (B) Transverse section through the head of an XDmrt4-mo-injected embryo stained for NCAM shows a reduced number of neurons in the olfactory epithelium on the injected side (arrow). cg, cement gland; f, forebrain. (Magnifications: ×15.)
	Fig. 6. Regulation of neurogenin, Xebf2, and NCAM expression by XDmrt4. (A–C) Real-time RT-PCR of animal explants isolated from embryos injected with various combinations of mRNA and morpholinos as indicated and collected when sibling embryos reached stages 14 or 22. (A) Injection of XDmrt4 induces Xeb2 and NCAM expression. This activity is inhibited by coinjection of XDmrt4ΔC. (B) XDmrt4 induces robust expression of neurogenin at stage 14 and to a lesser extend at stage 22. (C) Noggin-mediated activation of neurogenin, Xebf2, and NCAM expression is blocked in the context of XDmrt4-depeleted embryos (+XDmrt4-mo). (D) Based on these findings and other studies that have positioned Xebf2 downstream of neurogenin (29) we propose that XDmrt4 is an upstream regulator of neurogenin and Xebf2 in the molecular cascade, leading to neuronal differentiation in the olfactory system.
	dmrta1 (DMRT like family A1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, anterior view, dorsal up.
	dmrta1 (DMRT like family A1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up.
	dmrta1 (DMRT like family A1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 45, dorsal view, anterior up.
	dmrta1 (DMRT like family A1) gene expression in dissected brain of Xenopus laevis embryo, assayed via in situ hybridization, NF stage 45, dorsolateral view (Q) and dorsal view (R), anterior up. Arrow heads= olfactory bulb; arrows =forebrain.
	dmrt4 () gene expression in the dissected alimentary system of Xenopus laevis embryo, assayed via in situ hybridization, NF stage 45, dorsal view, anterior up.

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