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Force-producing convergence (narrowing) and extension (lengthening) of tissues by active intercalation of cells along the axis of convergence play a major role in axial morphogenesis during embryo development in both vertebrates and invertebrates, and failure of these processes in human embryos leads to defects including spina bifida and anencephaly. Here we use Xenopus laevis, a system in which the polarized cell motility that drives this active cell intercalation has been related to the development of forces that close the blastopore and elongate the body axis, to examine the role of myosin IIB in convergence and extension. We find that myosin IIB is localized in the cortex of intercalating cells, and show by morpholino knockdown that this myosin isoform is essential for the maintenance of a stereotypical, cortical actin cytoskeleton as visualized with time-lapse fluorescent confocal microscopy. We show that this actin network consists of foci or nodes connected by cables and is polarized relative to the embryonic axis, preferentially cyclically shortening and lengthening parallel to the axis of cell polarization, elongation and intercalation, and also parallel to the axis of convergence forces during gastrulation. Depletion of MHC-B results in disruption of this polarized cytoskeleton, loss of the polarized protrusive activity characteristic of intercalating cells, eventual loss of cell-cell and cell-matrix adhesion, and dose-dependent failure of blastopore closure, arguably because of failure to develop convergence forces parallel to the myosin IIB-dependent dynamics of the actin cytoskeleton. These findings bridge the gap between a molecular-scale motor protein and tissue-scale embryonic morphogenesis.
Fig. 3. Myosin IIB MO-mediated failure of gastrulation is dose dependent. (A-D) Still images from simultaneous time-lapse videorecordings showing vegetal views of control (A) and morphant Xenopus embryos injected with 2.5 μM (B), 5 μM (C) and 10 μM (D) MHC-B MO. Embryos are oriented with their dorsal side up. At stage 10 (t=0), bottle cells form in the dorsal lip of the blastopore of all control and morphant embryos. By control stage 12 (t=2.5), ventral bottle cells have formed in all control and morphant embryos, but blastopore closure is delayed in a MHC-B MO dose-dependent manner. The site of blastopore closure in 2.5 μM (B, t=7.5) morphant embryos is not located as ventrally as in control embryos (A, t=7.5). Asterisks indicate the center of the yolk plug at t=0 and the point of blastopore closure at t=7.5. (E-H) RNA in situ hybridizations of stage-13 embryos for brachyury expression reveals the extent of notochordal morphogenesis in a control embryo (E), and reduced notochordal morphogenesis in a 5 μM morphant (F). The 5μ M morphant embryos exhibit some variability in notochordal extension (G), but 10 μM morphant embryos essentially lack notochord extension (H).
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