Xenbase Image ID: 144606

Figure 2. XGAP Is Required for the Intercalation of Dorsal Mesoderm Cells Undergoing Gastrulation (A) A morpholino-antisense oligonucleotide (Mo) for XGAP specifically inhibits the translation of XGAP mRNA. XGAP mRNA, constructed to generate a fusion protein with a variant of YFP (Venus, vn) (Nagai et al., 2002), was injected with or without each Mo into the animal pole of two-cell-stage embryos. XGAP-vn protein was detected by Western blotting with an anti-GFP polyclonal antibody. Flag-tagged β-globin was detected as the loading control. (B and C) XGAP-Mo causes gastrulation defects. The external appearance was examined at stage 35 ([B] and [C]; n = 120, gray and black indicate a short trunk and spina bifida, respectively). The gastrulation-defective phenotypes caused by the Mo were partially rescued by the coinjection of resXGAP (100 pg, 200 pg) in a dose-dependent fashion (C). In the absence of XGAP-Mo, resXGAP manifested the effects of XGAP overexpression, consistent with our initial observation (C). (D) Cell fates are unaffected by XGAP-Mo. Whole embryos or DMZ explants that had received injections of Ctrl Mo or XGAP-Mo were harvested at the midgastrula stage and then analyzed by RT-PCR with primer sets for Xbra, Xwnt11, myf5, gsc, chd, otx2, and ODC. (E) Mesodermal differentiation is unaffected by XGAP-Mo. Each Ctrl Mo- or XGAP-Mo-injected embryo was harvested at stage 30, and the notochord and somites were immunostained with the MZ15 and 12/101 antibodies, respectively. (F and G) XGAP-Mo attenuates the elongation of Keller explants. Keller explants from embryos given injections of XGAP mRNA (upper panels in [F], squares in [G]) or XGAP-Mo (lower panels in [F], triangles in [G]) or that did not receive the injections (uninj; the second row in [F], circles in [G]) were excised at stage 10.5, and the explants were cultured and their elongation monitored for 12 hr. Photographs were taken every 2 hr (F), and the length and width were then measured to determine the time course of elongation ([G], n = 20). The error bars represent standard error. (H) XGAP-Mo inhibits the intercalation in the dorsal mesoderm. XGAP-Mo or Ctrl Mo was coinjected with Venus with a membrane bound sequence (mb-Venus) into one of the two dorsal blastomeres at the four-cell stage. Ctrl Mo and RFP with a membrane bound sequence (mb-RFP) were coinjected into the other dorsal blastomere. We repeated this experiment more than three times. (I) XGAP-Mo disrupts the isodiametric cell morphology. Each cell in the intercalation experiment (H) was measured for length along the ML axis and width along the AP axis, and the length/width ratio was calculated (n = 25). The error bars represent standard error. Image published in: Hyodo-Miura J et al. (2006) Copyright © 2006. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

Return to previous page