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
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.