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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. 6. (A-D) Embryos injected with Xrx1 were treated with HUA at stage 10.5 and the expression of Zic2 (A), Xhairy2 (B), X-ngnr-1 (C) and p27Xic1 (D) was analyzed at stage 13. (A,B) Black brackets indicate the anterior expression domains in the uninjected side; white brackets indicate the corresponding enlarged domains in the injected side. (C,D) Black arrowheads indicate anterior expression domains in the uninjected side, white arrowheads indicate the absence of this expression domain in the injected side. (E-I) HUA treatment dramatically reduced anti-phosphoH3 staining (E,F; stage 16) as well as Xoptx2 ability of expanding Xrx1 (Zuber et al., 1999) (G,H; stage 18). This treatment also results in a reduction of the optic vesicle size (I; stage 26; Co, control untreated embryo). (A-D,G,H) Frontal views, dorsal towards the top; (E,F,I) lateral views, anterior towards the left. Red staining in A-D and turquoise staining in G,H represent expression of co-injected lacZ lineage tracer. The injected side of the embryos (to the right of vertical bars representing the midline) is indicated (inj). |
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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. |
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Fig. 7. Xrx1 induces Xhairy2 but does not affect several other markers in animal caps. Xrx1 injected animal caps were dissected at stage 9, cultured to stage 17 and analyzed for the expression of the indicated genes. The column on the right (Embryo) shows the expression of the indicated genes in control embryos. |
