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Figure 1. XArx is expressed in a dynamic pattern in the developing diencephalon and telencephalon. A-L: Double whole-mount in situ hybridization (WISH) using antisense riboprobes specific for xArx (blue color) and other forebrain markers (in red): Rx2A (A,B), otx1 (C), en-2 (D), FoxG1 (E-H), dlx5 (I), emx1 [note typological error- 'mex1' sic] (J), Nkx2.1 (K), and eomesodermin (L). Regions of overlap appear purple. At neural plate and neural tube stages, xArx expression is caudal/posterior to telencephalic markers such as Rx2A and FoxG1. XArx expression is rostral to otx1 and en-2. E-H: xArx is expressed caudal to FoxG1 at neural plate and neural tube stages. During tailbud stages, xArx expression spreads rostrally to overlap a portion of the FoxG1 expression domain (arrow in H). XArx expression also overlaps expression of dlx5, mex1, Nkx2.1, and eomesodermin. Anterior views are shown in A-I. Lateral views are shown in J-L.
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arx (aristaless related homeobox ) gene expression in Xenopus laevis, as assayed via in situ hybridization ( light blue), NF stage 20 embryo, anterior view, dorsal up.
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Fig. 2. Overexpression of xArx results in shortened forebrain, loss of anterior midline structures, and dorsal displacement of the nasal organ. A,B: Lateral and dorsal views of a normal tadpole, respectively. C,E: Lateral view of xArx-injected tadpole shows apparent anterior truncation. D,F: Dorsal views of the same embryos show displacement of the eyes toward the midline. G: Visualization of neural structures in control and xArx-injected embryos by whole-mount immunostaining with the pan-neural antibody Xen-1. G,H: Lateral and dorsal views of uninjected tadpoles. I,J: lateral and dorsal views of two different xArx-injected tadpoles. Tadpole in I exhibits incomplete retinal development and dorsal displacement of nasal organ (arrowhead). Tadpole in J exhibits single nasal organ (arrowhead) and fused rostral brain. K,L: Whole-mount in situ hybridization (WISH) of control and xArx-injected embryos with sonic hedgehog (shh; blue staining). XArx-injected embryos lack anterior/rostral shh expression. LacZ RNA was used as a lineage tracer in the control and xArx-injected embryos (turquoise and red staining, respectively). M: Histological staining of transverse sections of control (M) and xArx-injected (N,O) embryos. Sections were taken at the level of the eye. N: Thickening of ventral brain in embryos injected with xArx RNA on both sides. O: Section through embryo with fused eyes. P: WISH of xArx-injected embryos using antisense probes for Xic1 (purple/blue staining). LacZ RNA was used as a lineage tracer in xArx-injected blastomeres (red staining). P: Anterior view of unilaterally injected neural tube stage embryo. Q,R: Lateral views of uninjected (Q) and injected (R) sides of unilaterally injected tail bud embryo. S: Antisl-1 immunofluorescence of control (S) and xArx-injected (V) embryos to label ventral forebrain cells. Progressively caudal transverse sections from the telencephalon (Q) to the diencephalon (S) show gradual thickening and medial concentration of ganglion cells in the forebrain. T: Isl-1 ositive cells are arranged in a broad thick ventral layer and in an ectopic lateral dorsal patch (arrowhead) in xArx-injected embryo.
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Fig. 3. Molecular analysis of xArx-injected em- bryos. Expression of forebrain markers was an- alyzed in control or xArx-injected embryos by whole-mount in situ hybridization (WISH). Em- bryos were injected with xArx and lacZ RNA (A22, A44) or lacZ RNA alone (A11, A3H3). A12: Embryos were fixed at neural tube stages (A11, A22) or tailbud stages (A3H3, A4 4, I, J), processed for -galactosidase activity using X-gal (control embryos, turquoise color) or Red-gal (xArx-injected embryos, red color) and WISH (blue-purple color) using anti- sense riboprobes for otx2 (A), pax6 (B), Rx2A (C), FoxG1 (D), emx1 (E), lhx2 (F), Nkx2.1 (G), vax2 (H), lhx1 (I), or lhx9 (J). Anterior views are shown in A11, A22, D3, and D4. Lateral views are shown in A3, B3, F33, J1, A4, B4, F44, and J2. Dorsal views are shown in C3, E3, I1, C4, E4, and I2.
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Fig. 4. Expression of some forebrain markers enhanced by xArx expression. A: Whole-mount in situ hybridization (WISH) of control (A,C,E,G,K,M) or xArx-injected (B,D,F,L,N) embryos using antisense probes for wnt8b (A), dlx5 (E), pax2 (K,L), or otx1 (M,N). LacZ was used as a lineage tracer (blue in control embryos, red in xArx-injected embryos). I,J: Transverse sections of embryo shown in H. Endogenous and ectopic otic structures are indicated (arrows in IJ). NT, neural tube; OV, otic vesicle; 2OV, secondary otic vesicle. Filled arrowheads in E,K,M indicate normal otic vesicles. Open arrowheads in F,H,L,N indicate secondary or duplicated otic structures.
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Fig. 5. Morphological and molecular analysis of embryos injected with xArx antisense mor- pholino oligonucleotides (ASMO). A: The xArx ASMO specifically inhibits translation of an xArxreen fluorescent protein fusion (Arx- GFP) but not GFP lacking the xArx ASMO tar- get (myc-GFP) fusion. Embryos were injected with control (CO, A,C) or xARX ASMO (ARX, B,D), CMV::myc-GFP (A,B) or CMV::xArx-GFP (C,D) plasmid DNA and dsRedExpress RNA. Embryos were cultured to midneurula stage and analyzed for GFP fluorescence (A) or dsRedExpress fluorescence (A ). E: Phe- notype of embryos injected with control (CON) or xArx (ARX) ASMO. H: Whole-mount in situ hybridization of embryos injected with control (H,K,N) or xArx (I,J,L,M,O,P) ASMO using anti- sense probes for FoxG1 (E), Nkx2.1 (H), or pax6 (K).
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Fig. 6. injected embryos. E: Expression of xArx and xArx-enR but not xArx-VP16 results in expansion of wnt8b expression. I: Injection of xArx or xArx-VP16 but not xArx-enR results in ectopic expression of dlx5 and ectopic otic structures (arrowheads). Control embryos (A,E,I) embryos injected with 100 pg/blastomere of xArx (B,F,J), xArx-enR (C,G,K), or xArx-VP16 (D,H,L). LacZ was used as a lineage tracer in E. A,I show lateral views, and E show dorsal views of control and injected tadpoles.
Morphological and molecular analysis of obligate transcriptional repressor and activator forms of xArx. A: Phenotypes of control (CON) and
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arx (aristaless related homeobox ) gene expression in Xenopus laevis, as assayed via double in situ hybridization with nxk2.1 (arx in light blue), NF stage 31 embryo, lateral view, anterior left, dorsal up.
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arx (aristaless related homeobox ) gene expression in Xenopus laevis, as assayed via double in situ hybridization with dlx5.1 (arx in light blue), NF stage 26 embryo, anterior view, dorsal up.
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