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Fig. 1. Misexpression of XSRF suppresses mesodermal cell fates. (A-C) Spatial expression of XSRF at the four-cell, blastula and early gastrula stages. Lateral views are shown. (D) XSRF expression in dissected gastrula embryos. Animal cap (AC), vegetal pole (VP), dorsal marginal zone (DMZ) and ventral marginal zone (VMZ) explants were dissected from stage 10.5 gastrula embryos and subjected to RT-PCR analysis. -RT, control RT-PCR in the absence of reverse transcriptase. The following controls were included as markers for the dissected tissues: Chordin for dorsal mesoderm, Xbra for pan-mesoderm, XMsx1 for animal cap and ventral mesoderm and XSox17β for endoderm. ODC, ornithine decarboxylase loading control. (E-G) Phenotypic effects of overexpression of wild-type (wt) XSRF RNA. Four-cell stage embryos were injected in the dorsal (F) or ventral (G) marginal regions with wt XSRF mRNA (2 ng), or not injected as a control (E), and cultured until sibling embryos reached stage 30. Lateral views are shown and anterior is to the left. (H-P) Ectopic wt XSRF interferes with the expression of mesodermal genes. Four-cell stage embryos were injected in the dorsal or ventral marginal regions with wt XSRF mRNA (2 ng) and, along with uninjected controls, cultured to stage 10.25-10.5 (H-M,P) and 28 (N,O) and then analyzed by whole-mount in situ hybridization using antisense Xenopus Wnt8 (H,I), brachyury (J,K), chordin (L,M) and myoD (N,O) probes or RT-PCR (P). Arrowheads in I,K,M,O indicate the absence or reduction of expression of genes. (H,I) Ventro-vegetal views are shown. (J-M) Vegetal views are shown with dorsal side at top. (N,O) Lateral views are shown with anterior side to the left. (P) Ventral marginal zone explants were dissected at stage 10.25 and subjected to RT-PCR. VMZ Co, uninjected ventral marginal zone tissue. |
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Fig. 4. Depletion of XSRF leads to expansion of mesoderm. (A) XSRF morpholino oligonucleotides (MOs) specifically knockdown the translation of C-terminally Myc-tagged XSRF protein in animal cap cells. Four-cell stage embryos were injected in the animal pole region with C-terminally 6Myc-tagged XSRF RNA (2 ng) with or without CO MO (60 ng), XSRF MO1 (60 ng) or XSRF MO2 (40 ng), and then animal cap explants dissected at late blastula stages were subjected to western blotting. Uninjected, animal caps without injection; Control, animal caps injected with XSRF-6Myc only. (B-H) Phenotypes of XSRF-depleted embryos. Embryos were injected at the four-cell stage with the indicated reagents (60 ng CO MO; 60 ng XSRF MO1; 40 ng XSRF MO2; 100 pg wt XSRF) dorsally (B,D,E,G,H) or animally (C,F) and cultured to stage 31. (I-W) Knockdown of XSRF expands the expression of mesodermal markers. Four-cell stage embryos were injected in the dorsal or ventral marginal region with CO MO (60 ng), XSRF MO1 (60 ng), XSRF MO2 (40 ng) or DN XSRF (2 ng) and then analyzed at the mid-gastrula stages by in situ hybridization against Goosecoid (I,L,O,R,U), Xbra (J,M,P,S,V) or Wnt8 (K,N,Q,T,W). |
