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Fig. 1. Expression pattern of Xenopus laevis Irx genes. (A1–5) Vegetal views at early gastrula show that Irx1 (A1) and Irx3 (A3) are expressed in two dorso-lateral mesodermal bands (arrowhead). In addition, Irx1 and Irx2 are expressed in the presumptive neural ectoderm (A1 and A2, insets). (B1–5) Dorsal views of early neurula embryos illustrating the similar expression domains of all Irx genes (n, notochord). Arrowheads in (B1) and (B2) point at the placodal expression of Irx1 and Irx2. (C1) Early neurula embryo double-stained for Six3 (purple) and Irx1 (cyan) genes. The expression patterns of these genes are separated a few cell diameters (arrowheads). (C2–C5) Early neurula embryo double-stained for Irx genes (purple) and Fezf2 (cyan). Inset in (C2) show a similar embryo double-stained for Irx1 (purple) and Fezf2 (cyan). Insets in (C3–5) show double in situs developed in a single colour (purple) for Fezf2 and Irx3 (C3), Irx4 (C4) and Irx4 (C5). All Irx genes show similar anterior border abutting Fezf expression (arrowheads). (D1, D2) Double staining for Irx1 and Irx2 (D1) or Irx3 (D2) also show that these genes share their anterior expression limit (arrowheads). (D3, D4). This border is rostral to Pax2, as shown in double-stained embryos for this gene and Irx1 (D3) or Irx3 (D4). Inset in (D3) show a sagittal section of a double-labelled embryo for Pax2 (purple) and Irx1 (cyan). Note that Irx1 is anterior to that of Pax2. (D5) The Irx anterior limit overlaps with the posterior expression of Pax6 in the forebrain. (E1–G5) At tailbud stage, in lateral views (E1–5) or in dorsal views (F1–5, G1–5) all Irx genes show similar expression patterns in the brain although with different intensities in different regions. This is more clearly seen in double-stained embryos for Irx genes (purple) and Krox20 (cyan) (G1–G5). Red, black and blue arrowheads point at the M/H boundary, rhombomere 3 (r3) and rhombomere 5, respectively. Green arrowheads and red arrows point at the otic vesicle and pronephros, respectively. Stg, stage.
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Fig. 2. Irx MOs causes antero-posterior neural defects. Dorsal views of Xenopus tropicalis embryos at early neurula (stg 14–15; A–R) or tadpoles (stg 42; S–X) injected with 10 ng of MOIrx1 (A, G, M, S), MOIrx2 (B, H, N, T), MOIrx3 (C, I, O, U), MOIrx4 (D, J, P, V), MOIrx5 (E, K, Q, W) or a mix of 2 ng of each MO (F, L, R, X). The MOs were co-injected with LacZ mRNA as a tracer. In all embryos, red or black arrowheads point at the injected or control side, respectively. (A–F) In Irx morphant embryos, Gbx2 is reduced and shifted caudally. (G–L). Impairment of Irx genes also caused Otx2 posterior displacement and Krox20 downregulation but does not affect Cad3 expression. (M–R) In contrast, Wnt4 expression in the spinal cord, as well as in the midbrain, is reduced. A caudal shift of the midbrain is also observed in some cases. (S–X) Later, all injected embryos show brain malformations and some of them eye defects.
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Fig. 3. Irx genes are required for neural patterning. All panels, except (M) that show a stage 18 embryo, show dorsal views of stage 14–16 embryos co-injected with a mix of all five Irx MOs and LacZ mRNA. Black and red arrowheads point at the control or injected side, respectively. (A–C) Forebrain markers Rx1, Six2 and Fezf2 are expanded posteriorly (compare red with black arrowheads). (D) In addition, the anterior Pax6 domain is also shifted caudally at the expenses of the midbrain territory that lack the expression of this gene (compare red with black arrowhead). (E–F) The expression of Wnt1 and Irx3 in the posterior diencephalon is strongly downregulated (compare red with black arrowheads). (G–I) The midbrain is also reduced in the Irx morphant, as determined by the expression of En2, Pax2 and Wnt4. In addition, Wnt4 expression in the spinal cord in strongly impaired (I). (J–L) The rhomboencephalon markers Gbx2, Krox20 and Nhf1β are also downregulated in the injected embryos. (M) At stage 18, Sox2 expression shows and altered morphology of brain structures in the Irx impaired side. (N, O) The proneural gene Ngnr1 (N) and the primary neurons markers Ntubulin (O) are also downregulated in the injected side.
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Fig. 4. Rescue of Irx MOs defects. All embryos are at stage 14–16. (A, C, E and G) are anterior and (B, D, F, H) are dorsal views, respectively. Black and red arrowheads point at control and injected sides, respectively. Embryos injected with MT-Irx1-GR (A, C, E, G) or MT-Irx3-GR (B, D, F, H) mRNAs alone (A–D) or with a mix of Irx MOs (E–H). (A–D) In the presence of Dexamethasone, the injected mRNAs caused expansion of Ngnr1 (C) and Gbx2 (D). This is not observed in the absence of the hormone (A, B). (E–H) In embryos co-injected with MT-Irx-GR mRNA and Irx MOs, in the absence of Dex, Ngnr1 (E) and Gbx2 (F) are downregulated. (G, H) This phenotype is rescued in the presence of Dex.
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Fig. 5. Fezf represses Irx genes. All embryos are dorsal views at stage 14–15. Black and red arrowheads point at the control or injected side, respectively. (A, B) In embryos injected with Fezf1 mRNA Irx1 (A) and Irx3 (B) are downregulated. (C, D) Injection of EnR-Znf-GR mRNA caused similar Irx1 downregulation in the presence of Dex (C) but not in its absence (D). (E, F) Injection of VP16-Znf-GR mRNA caused anterior Irx1 expansion in the presence of Dex (E) but not in the absence of the hormone (F). (G–J) Irx3 is also activated by VP16-Znf-GR mRNA even in the presence of cycloheximide.
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Fig. 6. Irx repress Fezf genes. All embryos are dorsal views at stage 14–15. Black and red arrowheads point at the control or injected side, respectively. (A, B) In embryos injected with Irx3-MT-GR mRNA Fezf1 is downregulated in the presence (A) but not in the absence (B) of Dex. (C, D) Injection of HD-GR-EnR mRNA caused similar Fezf1 downregulation. (E, F) In contrast, injection of HG-GR-E1A mRNA caused posterior Fezf1 expansion in the presence (E) but not in the absence of the hormone (F). (G–J) Fzf2 is also activated by HG-GR-E1A mRNA even in the presence of cycloheximide.
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Fig. 7. Arx represses Irx genes. All embryos are dorsal views at stage 14–15. Black and red arrowheads point at the control or injected side, respectively. (A) Double staining of Fezf2 (cyan) and Arx (purple). Both genes share their posterior limits (arrowheads). (B) Double staining of Irx1 (cyan) and Arx (purple) showing the complementary expression domains of these genes. (C) Injection of Fezf1 mRNA expanded Arx posteriorly. (D) In embryos injected with VP16-Znf-GR mRNA Arx expression is shifted anteriorly. (E–F) Overexpression of Arx (E) or Arx-EnR (F) mRNAs downregulated Irx1 (E) and Irx3 (F). (G) Impairment of Arx function with a specific MO expands Irx1 anteriorly. (H) Injection of Arx-VP16 mRNA caused a similar rostral expansion of Irx3. (I) Injection of VP16-Znf-GR mRNA expands anteriorly Irx3 expression. (J) This effect is reverted by overexpressing Arx.
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Fig. 8. Irx repress Arx. All embryos are dorsal views at stage 14–15. Black and red arrowheads point at the control or injected side, respectively. (A) In embryos injected with Irx3-MT-GR mRNA Arx is downregulated in the presence of Dex. This effect was not observed in the absence of the hormone (not shown). (B) Impairment of Irx activity caused caudal expansion of Arx. (C, D) Injection of HG-GR-E1A mRNA caused posterior Arx expansion in the presence (C) but not in the absence of the hormone (D). (E–F) This expansion also occurred in the presence of cycloheximide.
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