Ring C , Ogata S , Meek L , Song J , Ohta T , Miyazono K , Cho KW .

HD29507 NICHD NIH HHS , R01 HD029507 NICHD NIH HHS

Figure 2. Expression analysis of XWBSCR11 duringXenopus development. (A) RT–PCR analysis with RNA isolated from whole embryos demonstrates the expression of XWBSCR11 throughout Xenopus embryogenesis. (B) RT–PCR of tissue explants from blastula (stage 8/9) and early gastrula (stage 10) embryos. The schematic diagram shows the location of the dissection for the various tissue explants. XWBSCR11 does not appear to be differentially expressed between animal cap (AC), marginal zone (MZ), or vegetal pole (VG) tissues in blastula and gastrula embryos, nor between dorsal (DMZ) and ventral marginal zone (VMZ) in gastrula embryos. RT–PCR of histone H3 is shown as a loading control in bothA and B. (C) Whole mount in situ analysis of XWBSCR11 expression. (a) Gastrula stage; (b–g) neurula stages; (h–i) tailbud stages. In a–c, e, and g–i, anterior is left.

Figure 5. Effects of XWBSCR11 morpholinos on reporter gene activity and endogenous gene expression. (A) XWBSCR11 morpholinos were coinjected with activin (4 pg/embryo) and the DE(6×)gsc/Luc reporter gene into animal caps at the two- to four-cell stage, then assayed for luciferase activity at stage 10.5. The XWBSCR11 morpholino specifically abolished activin responsiveness of the reporter gene, as a control morpholino similarly injected had no effect. (B) This effect was rescued by the injection of a wild-type form of XWBSCR11 lacking a portion of the 5′ sequence corresponding to the region bound by the morpholino. (C) XWBSCR11 morpholino (0.5, 1, and 2 ng/embryo) was coinjected with activin mRNA (4 pg/embryo) in animal caps and assayed for gene expression. gsc expression decreased with increasing XWBSCR11 morpholino concentration, whereas levels of histone H3appeared constant and Mix2, Mixer and Otx2expression was unaffected. However, injection of XWBSCR11 morpholino oligonucleotides at concentrations >8 ng per embryo inhibitedMix2 expression (data not shown). A control morpholino had no effect. (D) WXBSCR11 morpholino-injected (0.5 ng/embryo) embryos had disrupted anterior structures whereas they appeared to go through normal gastrulation movements. At higher concentrations (1.5 ng), head truncation was more prominent and gastrulation movements were affected. Control morpholino-injected embryos were normal.

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