Mizuseki K et al. (1998), Xenopus Zic-related-1 and Sox-2, two factors in...

XB-ART-15238

Development 1998 Feb 01;1254:579-87. doi: 10.1242/dev.125.4.579.

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Xenopus Zic-related-1 and Sox-2, two factors induced by chordin, have distinct activities in the initiation of neural induction.

Mizuseki K , Kishi M , Matsui M , Nakanishi S , Sasai Y .

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In a differential screen for downstream genes of the neural inducers, we identified two extremely early neural genes induced by Chordin and suppressed by BMP-4: Zic-related-1 (Zic-r1), a zinc finger factor related to the Drosophila pair-rule gene odd-paired, and Sox-2, a Sry-related HMG factor. Expression of the two genes is first detected widely in the prospective neuroectoderm at the beginning of gastrulation, following the onset of Chordin expression and preceding that of Neurogenin (Xngnr-1). Zic-r1 mRNA injection activates the proneural gene Xngnr-1, and initiates neural and neuronal differentiation in isolated animal caps and in vivo. In contrast, Sox-2 alone is not sufficient to cause neural differentiation, but can work synergistically with FGF signaling to initiate neural induction. Thus, Zic-r1 acts in the pathway bridging the neural inducer with the downstream proneural genes, while Sox-2 makes the ectoderm responsive to extracellular signals, demonstrating that the early phase of neural induction involves simultaneous activation of multiple functions.

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Species referenced: Xenopus
Genes referenced: chrd fgf2 fst h4c4 hoxa9 hoxb9 hoxc9 ncam1 neurog1 neurog2 nrp1 otx2 prph snai2 sox13 sox2 tbxt tubb2b zic1

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	Fig. 1. Structural comparison of Xenopus Zic-r1, mouse Zic1 and odd-paired related factors (A) and of Xenopus and chick Sox-2, 3 (B). The black boxes show the zinc finger (A) and HMG (B) domains. The striped boxes (A) show well-conserved regions. Percentages of identical amino acid residues are shown.
	Fig. 2. Spatio-temporal expression of Zic-r1 and Sox-2. Whole-mount in situ hybridization analyses of Zic-r1 (labeled as ZCR; A, C, E, G, I, K, M) and Sox-2 (labeled as SOX; B, D, F, H, J, L, N) at the early gastrula stage (stage 10.5; A-D), the early neurula stage (stage 14; E, F), the midneurula (stage 16; G, H) and the larva stage (stage 32; I, J). (C and D) show 10 mm paraffin sections. The signals are detected in superficial dorsal ectoderm but not in the deep mesoderm. The arrow and arrowhead in J show the branchial arch neural crest and the olfactory placode, respectively. (K-N) Radial injection of Chd mRNA (150 pg/blastomere; K, L) and of BMP-4 mRNA (75 pg/blastomere; M, N) into the 4-cell embryo. Chd injection induced Zic-r1 (P) and Sox-2 (Q) expression in the animal caps (excised at stage 9 and harvested at stage 11.5) but not the mesodermal marker Xbra (O). Activin-treated animal caps (excised at stage 9 and treated with 10 ng/ml activin until stage 11.5) for positive control for Xbra (O, inset). (P,Q, insets) Uninjected animal caps. The arrowheads show the dorsal lip in A-D and the closing blastopore in E-H.
	Fig. 3. Onset of Zic-r1 and Sox-2 expression is between those of Chd and Xngnr-1. A whole-mount in situ hybridization study comparing the spatio-temporal expression of Chd (A,E,I,M), Zic-r1 (ZCR; B,F,J,N), Sox-2 (SOX; C,G,K,O) and Xngnr-1 (Xngnr; D,H,L,P) at -75 minutes (A,B,C,D), -30 minutes (E,F,G,H), +15 minutes (I,J,K,L) and +75 minutes (M,N,O,P) relative to the onset of gastrulation, which was judged by both external and internal appearances of albino embryos and by the pigmentation pattern of pigmented embryos fertilized at the same time (Nieuwkoop and Faber, 1967). (A-O) Dorsal views (looking down on the neuroectoderm). (P) Dorso-lateral view (Tg, trigeminal ganglion; m, i and l stand for medial, intermediate and lateral primary neurons). Arrowheads show the dorsal lip.
	Fig. 4. Zic-r1 injection promotes neural and neuronal differentiation in animal caps and in vivo. After 2 days culture, when siblings reached stages 38-39, animal cap explants injected with Zic-r1 mRNA (A) contained a large neural mass. V, ventricle-like cavities; arrow, roof plate-like thin layer. The control animal cap contained atypical epidermis (B). When siblings reached stage 17, Zic-r1-injected animal caps expressed the neural marker nrp-1 (C; detected by in situ hybridization). Inset (C) shows control animal caps. Injection of 200 pg Zic-r1 mRNA into one blastomere of the 2-cell embryo expanded expression of Xngnr-1 (D; primary neurons shown by triangles; arrow, midline), N-tubulin (E; note the width of lateral neuron bands, l) and slug (F; arrow, ectopic expression) and suppressed that of keratin (G, H) in vivo at neural plate stages. (I) Expression of hairy 2, which marks the midline and borders of neural plate (np) (Turner and Weintraub, 1994). The width of neural plate (arrows) is significantly increased on the injected side compared to the control side. epi, epidermis. The injected side (inj; cont, control side) was determined by coinjecting a GFP (D-F, I; not shown) or b-gal (arrows in G) mRNA.
	Fig. 5. RT-PCR analysis of Zic-r1 (ZCR)-injected animal caps (AC) stage 12 (A) and stage 17 (B). Ngnr-1, Xngnr-1; XFS, Follistatin; H4, histone H4. Zic-r1 mRNA injection induced neural, neuronal and neural crest markers without accumulation of the mesodermal markers or neural inducers.
	Fig. 6. Sox-2 alone is not sufficient for neural induction but enhances the responsiveness of ectoderm to the FGF neuralizing signal. Histological analysis of animal caps (corresponding stages were 38-39) injected with Sox-2 mRNA (A; atypical epidermis), treated with bFGF (B; atypical epidermis), injected with Sox-2 and treated with bFGF (C; neural mass with cavity) (D; neural mass in an elongated explant like a tail). In situ hybridization analyses with animal caps (E-H; when siblings reached stage 17). (E-G) N-CAM signals in animal caps treated with Sox-2, bFGF and both, respectively. (H) N-tubulin expression in the Sox-injected and FGFtreated caps (arrowhead; the inset shows the caps injected with Sox mRNA alone). Animal caps (I-K) and a control embryo (L; harvested at the same time as the caps at stage 39) from an albino mother and a pigmented father, fixed with Bouin’s and cleared with Murray’s solution. (I) Sox-2 injected, (J) bFGF-treated, (K) both Sox-2 injected and bFGF-treated.
	Fig. 7. RT-PCR analysis of the explants at the stage 17. Lane 1, control embryo; lane 2, control animal caps; lane 3, Sox-2-injected caps; lane 4, FGF-treated caps; lane 5, Sox-2-injected and FGFtreated caps. Otx-2 (forebrain and also weakly in cement gland), Xif3 (anterior neural plate), Krox 20 (hindbrain), XlHbox6 (spinal cord), CG13 (cement gland); other markers as in Fig. 5.