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Fig. 2. Xrx1 inhibits neurogenesis. The effects of Xrx1 overexpression on (A) X-ngnr-1 (stage 12), (B,D,E) X-Delta-1 (stage 13), (C) N-tubulin (stage 16), (F) Sox2 (stage 13) and (G) XBF-1 (stage 13) are shown. (D,E) Transverse sections at the level of the anterior (D) and posterior (E) neural plate. The black arrowhead indicates the anterior expression domain of X-Delta-1, while the white arrowhead indicates the repression of the corresponding domain in the injected side. (H) Stage 16 embryo injected with 150 pg of XBF-1 RNA showing suppression of endogenous N-tubulin (white arrowheads) as well as ectopic N-tubulin activation at the border of the injected area (arrow). A-C,F,G,H Frontodorsal views. (I,K) Stage 16 embryos co-injected with either X-ngnr-1 and lacZ (I) or X-ngnr-1, Xrx1 and lacZ (K); lateral views, anterior towards the left. (J,L) Animal caps co-injected with either X-ngnr-1 and lacZ (J) or X-ngnr-1 and Xrx1 (L) analyzed at stage 16. Both in whole embryos and in animal caps Xrx1 inhibits N-tubulin expression induced by X-ngnr-1. Red staining represents expression of co-injected lacZ lineage tracer. |
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Fig. 1. Xrx1 expression in the proliferative region of the anterior neural plate is controlled by Hedgehog and neurogenin signaling. (A,B) Expression of Xrx1 (light blue) in relation to the expression of (A) X-Delta-1 (purple) and (B) X-ngnr-1 (purple) in stage 13 embryos; frontodorsal views. (C) Sagittal section of a stage 13 embryo showing Xrx1 expression in the deep sensorial layer of the neuroectoderm. (D,E) Stage 14 embryos injected with X-chh (D) and X-shh (E) showing ectopic expression of Xrx1 (blue); frontal views, dorsal towards the top. (F,G) Embryos injected with X-ngnr-1 displaying repression of Xrx1 (blue, F, stage 13) and ectopic expression of N-tubulin (blue, G, stage 16); frontodorsal views. The injected side of the embryos (to the right of vertical bars representing the midline) is indicated (inj). Red staining represents expression of co-injected lacZ lineage tracer. d, deep neuroectodermal layer; s, superficial neuroectodermal layer. |
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Fig. 3. Xrx1 counteracts retinoic acid-mediated neuronal differentiation. (A) Comparison of the anterior expression of XRALDH2 (purple) with that of Xrx1 (light blue) in a stage 13 embryo. (B) Stage 13 embryo injected with XRALDH2 showing reduction of Xrx1 expression. (C) Stage 13 embryo injected with Xrx1 showing repression of the anterior XRALDH2 expression domain. Black arrowheads indicate the expression domains of Xrx1 (B) and XRALDH2 (C) in the uninjected side of the embryos. White arrowheads indicate the repression of the corresponding domains in the injected side. (A-C) Frontal views, dorsal towards the top. Red staining represents expression of co-injected lacZ lineage tracer. (D) Animal caps injected with chordin and analyzed at stage 16 express Xrx1 but not N-tubulin. If chordin-injected caps are treated with RA at stage 9 and analyzed at stage 16, Xrx1 expression is suppressed and N-tubulin expression is induced. (E) Stage 16 animal caps co-injected with either chordin and lacZ (control) or chordin and Xrx1, and treated with RA at stage 9. Xrx1 strongly inhibits the induction of N-tubulin expression. |
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Fig. 5. Xrx1 regulates the expression of genes that control cell proliferation and differentiation, and does not work through the Notch-Delta pathway at early neurula. (A-C,G,H,M) Comparison of the expression of Xrx1 to that of Zic2 (A,B), Xhairy2 (C), Xhairy1 (G), p27Xic1 (H) and X-Notch-1 (M) in stage 13 embryos. (D-F,I-L,N) Xrx1-injected embryos analyzed at stage 14 (D-F,J,K,N) or stage 18 (I,L). The probes used and the respective staining are indicated, color-coded, on the bottom of each panel. (A-D,F-O) Frontal views, dorsal towards the top; (E,P,Q) dorsoanterior views. The injected side of the embryos (to the right of vertical bars representing the midline) is indicated (inj). Red staining in F,I,J,K,L,N-Q and turquoise staining in D,E represent expression of co-injected lacZ lineage tracer. (F,J,K) Black arrowheads indicate the lateroventral expression domain of Xhairy2 (F), Xhairy1 (J) and p27Xic1 (K) in the uninjected control side of the embryos. White arrowheads indicate the corresponding expression domain in the injected side, which is expanded in the case of Xhairy2 (F) and repressed in the case of Xhairy1 (J) and p27Xic1 (K). Black brackets indicate the anterior expression domains of Zic2 (D,E), cyclinD1 (I) and Xoptx2 (L) in the control uninjected side; white brackets indicate the corresponding enlarged domains in the injected side. (O) Stage 14 embryo injected with Notch-ICD showing no significant change in Xrx1 expression. (P,Q) Stage 16 embryos injected with X-Delta-1stu and lacZ (P) or co-injected with Xrx1, X-Delta-1stu and lacZ (Q). Xrx1 represses N-tubulin expression in the trigeminal ganglion but does not affect N-tubulin posterior expansion. Arrows indicate the increase in density of N-tubulin-positive cells within the posterior neurogenic stripes caused by the block of lateral inhibition. The black arrowhead indicates N-tubulin expression in the trigeminal ganglion of the uninjected side; the white arrowhead indicates the absence of this expression domain in the injected side. |
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Fig. 8. Effects of Xrx1 loss of function on genes regulating anterior neurogenesis. (A-C) Phenotypes of stage 41 embryos injected with MoXrx1 (A), MoXrx1 and Xrx1 (B), and control morpholino oligo (C). (F,J,K,M,N,Q) Embryos injected with MoXrx1 in both dorsoanimal blastomeres at the eight-cell stage and analyzed at stage 14 (F,K,M,N,Q) and stage 18 (J). (D,G,H,L,P) Control uninjected embryos analyzed at stage 14 (D,G,L,P) and stage 18 (H). (E,I) Embryos bilaterally injected with Xrx1-EnR analyzed at stage 14 (E) and stage 18 (I). (O) Stage 14 embryo treated with HUA. (R,S,V) Stage 14 embryos bilaterally injected with Xhairy2 (R), MoXrx1 and Xhairy2 (S), and MoXrx1 and XBF-1 (V). (T,U) Dorsal (T) and frontal (U) views of a stage 14 embryo injected with XBF-1. Red staining represents co-injected lacZ. Black brackets indicate the size of the anterior expression domain in the uninjected embryo (P); white brackets indicate the size of the corresponding domain in the injected embryo (Q). Arrows in D,E,F,S,V indicate the anterior boundaries of X-ngnr-1 expression; the arrow in T indicates X-ngnr-1 ectopic expression. Black arrowheads in I,J indicate the continuous anterior extension of X-ngnr-1 expression. White arrowheads in U and R indicate the repressed anterior expression domain of X-ngnr-1. |
