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Fig. 4. Antisense morpholino oligonucleotides directed against Xcyr61 inhibit gastrulation movements but have little effect on mesodermal specification. (A,B) Morpholino oligonucleotide MO1 causes a severe retardation in blastopore closure (B) compared with control stage 12 embryos (A). (C,D) Morpholino oligonucleotide MO1 causes a decrease in Xbra expression and shifts the Xbra expression domain towards the animal pole. Embryos are at stage 11.5. (E,F) Morpholino oligonucleotide MO1 causes expansion of the goosecoid expression domain. Embryos are at stage 11.5. (G,H) Morpholino oligonucleotide MO1 (30 ng) causes shortening of the anteroposterior axis. Embryos are at stage 35. (I-K) Bisection of embryos injected with morpholino oligonucleotide MO1 reveals changes in the structure of the blastocoel roof and of the marginal zone. (I) Embryo previously injected with a control morpholino oligonucleotide at stage 11. The blastocoel roof and marginal zone are thin and compact, as indicated by the two white lines. (J,K) Morpholino oligonucleotide MO1 (30 ng) causes a thickening of the blastocoel roof and marginal zone (lines), and a separation of cell layers (arrows). (L,M) Scanning electron microscope images of a control embryo at stage 11 (L) and an embryo at the same stage previously injected with 30 ng antisense morpholino oligonucleotide MO1 (M). Note the tightly packed epithelial appearance of the cells in the blastocoel roof of the control embryo (L), and the more loosely packed appearance of cells in the MO1-injected embryo, with some cells apparently about to detach (arrows; M). Note also that the migration of the large flat mesendodermal cells visible at the bottom of (L) is impaired in MO1-injected embryos (M). (N,O) Morpholino oligonucleotide MO2 causes a decrease in fibronectin assembly in the blastocoel roof. (N) Fibronectin forms an elaborate fibrillar network in the blastocoel roof of control embryos. (O) Fibronectin assembly is reduced in the blastocoel roof of morpholino-injected embryos. (P) Western blot analysis indicates that levels of fibronectin are similar in control and morpholino-injected embryos. HSP-70 was used as a loading control. Scale bars: in L, 100 μm for L,M; in O, 100 μm for O,N. |
