Fig. 5. X-TSK induces the neural crest when in combination with Wnt. (A-E) Conjugate assay of ACs (stage 8-9) and the dorsolateral marginal zone (DLMZ) (stage10.5) analyzed by whole-mount in situ hybridization with a Sox9 probe. The numbers of phenotypic explants are described in the lower right-hand corners. (A) Sox9 expression in the conjugate of a non-injected animal cap and non-injected DLMZ. (B) The conjugate of X-TSK-si-injected AC and DLMZ; Sox9 expression is decreased. (C) The conjugate of AC and X-TSK-si-injected DLMZ. (D) The conjugate of X-TSK overexpressing AC and DLMZ; Sox9 is expressed in all explants. (E) The conjugate of AC and X-TSK expressing DLMZ. (F) The expression of Sox9 at the same stage as ACs. (G) RT-PCR analysis of the AC/DLMZ conjugate assay. X-TSK-si (0-4 pmol) was injected into the animal blastomeres evenly. (H-M) Whole-mount in situ hybridization analysis of the animal cap assay. ACs were prepared from stage 8-9 embryos injected with 0.5 ng X-TSK + 0.5 ng XWnt-8 (H,K), 0.5 ng X-TSK (I,L), or 0.5 ng XWnt-8 (J,M). ACs were harvested at the time equivalent to stage 21/22 and hybridized with XAG-1 (H-J) and Xslug (K-M) probes. (N) RT-PCR analysis of the animal cap assay. ODC was used as an the internal marker. The amount of injected X-TSK mRNA was 1 ng/embryo, which is enough to induce anterior markers. XWnt-8 mRNA (0.5 ng) was co-injected with X-TSK. X-TSK induced neural crest marker expression in combination with XWnt-8. (O-R) The embryos are transfected by the mRNA electroporation at stage 11.5 after gastrulation. The distribution of GFP fluorescence is indicated in the inset of each figure. (O) Sox9 expression in GFP mRNA transfected embryo. (P) A variation of X-TSK transfected embryo. Sox9 expression is extended to anterior side (arrowhead). (Q) A variation of X-TSK transfected embryo. Sox9 expression is ectopically observed (arrowhead), but endogenous expression was interfered by ectopic expression of X-TSK. (R) Xslug expression is also affected.