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Fig. 4. Depletion of XPACE4 disrupts gastrulation, mesoderm induction and normal development. (A) XPACE4-depleted embryos [P(-)] compared with controls (Un) show delay in gastrulation and rescue by XPACE4 mRNA [P(-)+mRNA]. (B) Gastrulation delay of XPACE4 depletion is rescued by the reintroduction of XPACE4 mRNA. XPACE4 mRNA injected into control embryos (Un+mRNA) speeds up gastrulation and rescues the gastrulation delay of XPACE4-depleted embryos [P(-)+mRNA]. (C) XPACE4-depleted embryos develop patterning defects and anterior abnormalities. Depletion of XPACE4 [P(-)] results in a variety of late phenotypes ranging from ventralization to small heads. These phenotypes can be partially rescued by the reintroduction of XPACE4 mRNA [P(-)+mRNA]. (D) Equatorial explants (Eq explants) of XPACE4-depleted embryos fail to elongate. Explants dissected from control embryos (Un) show normal convergent extension movements and elongation. XPACE4-depleted embryos [P(-)] show severe reduction in elongation; XPACE4 mRNA rescues the elongation defect [P(-)+mRNA]. (E) Analysis of gene expression of equatorial explants from XPACE4-depleted embryos. Explants (Eq explants) dissected from control (AS-5MP-) and XPACE4-depleted (AS-5MP+) embryos at mid-blastula stage are cultured and analyzed together with stage 28 whole embryos (WE). The expression of general mesodermal marker MyoD, dorsal mesoderm marker cardiac actin and neural marker NCAM are all reduced in XPACE4-depleted whole embryos and explants. XPACE4 mRNA rescues the expression of markers. (F) Analysis of gene expression of XPACE4-depleted gastrulae. XPACE4-depleted whole embryos are harvested together with sibling control embryos. The expression of Xbra, chordin (Chd), Xnr1 and Xnr3 is analyzed using real-time RT-PCR. At stage 10.5, XPACE4-depleted embryos (AS-5MP+) show a reduction in the levels of these markers and the reduction is partially rescued by XPACE4 mRNA. The mRNA alone increases marker levels. (For rescue experiments 75-100 pg of XPACE4 mRNA is injected vegetally into both cells at the two-cell stage.)





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