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First published online 11 June 2008
doi: 10.1242/dev.014704

Development 135, 2435-2444 (2008)
Published by The Company of Biologists 2008
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Convergence and extension at gastrulation require a myosin IIB-dependent cortical actin network

Paul Skoglund1,*, Ana Rolo1,{dagger}, Xuejun Chen2, Barry M. Gumbiner2 and Ray Keller1

1 Department of Biology, University of Virginia, Charlottesville, VA 22903, USA.
2 Department of Cell Biology, University of Virginia, Charlottesville, VA 22903, USA.


Figure 1
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  		Fig. 1. Myosin IIB protein localization. (A-C) In tailbud-stage Xenopus embryos, MHC-B protein is localized to the dorsal axis (A), both at the surfaces of the somites (B) and in a polarized distribution in mature notochord (C). (D) Posterior notochordal cells actively undergoing convergent extension (CE) exhibit MHC-B localized to triple cell junctions inside the notochord (*), where invasive protrusions extend between adjacent cells during intercalation, and at cell-cell-matrix junctions at the notochord-somite boundary (NSB) (arrowheads). (E) Basal (mesodermal side) view of MHC-B staining in a deep neural-over-mesoderm explant reveals that MHC-B exhibits a fibrillar distribution over notochord and somites. BA, brachial arches; N, notochord; S, somitic mesoderm. Scale bars: 500 µm, except 50 µm in D.

 

Figure 2
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  		Fig. 2. Myosin IIB is required for morphogenesis at grastrulation. (A) Percentage of Xenopus embryos failing to close the blastopore plotted for 10 µM MHC-B (IIB) MO (n=74), 10 µM control (n=72) and uninjected (n=84) embryos, with s.e.m. (B) Western blots showing MHC-B (IIB), MHC-A (IIA) and actin (Act) levels in uninjected, 5 µM and 10 µM MO, and 10 µM control MO embryos. The normalized ratio of MHC-B or MHC-A to actin (R) is also shown. (C-E) A representative morphant embryo (C), a unilaterally (right-side) morphant embryo (D), and a control embryo (E) at stage 19. (F,G) Time-lapse movie frames showing vegetal (blastopore) view of development of a 10 µM MHC-B MO morphant (F) and a 10 µM control MO embryo (G) with time in minutes shown. Insets in C and G show bottle cells (brackets indicate regions magnified in insets). Scale bars: 500 µm, except 1 mm in D.

 

Figure 3
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  		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 5 µM morphant embryos exhibit some variability in notochordal extension (G), but 10 µM morphant embryos essentially lack notochord extension (H).

 

Figure 4
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  		Fig. 4. Partially myosin IIB-depleted notochordal cells exhibit polarization and adhesion phenotypes. (A) Explanting the dorsal tissues of a Xenopus embryo neurula above the blastopore (Bp) and removing the endodermal archenteron roof (ar) exposes the notochord (N) and somitic mesoderm (S) for imaging during CE. (B) In such explants, confocal time-lapse imaging of a notochord mosaic for cells injected with 5 µM MHC-B MO reveals that morphant cells (labeled), in the background of a notochord expressing a membrane-bound GFP (revealing cell outlines), exhibit loss of regulation of polarized cell motility. Asterisks, NSB. (C) An epifluorescent time-lapse sequence of one partially morphant labeled cell shows that it exhibits loss of regulation of polarized cell motility and eventually is excluded from the notochord (cell outlines are normal notochordal cells in this mosaic explant). (D) Control notochordal cells at or near the NSB exhibit monopolar motile behaviors, with few protrusions toward the NSB, whereas 5 µM MO morphant cells express increased motility and improperly direct this motility towards the boundary. One unit represents 6% of the total protrusive activity directed towards that sector, and each sector is oriented relative to the NSB. Scale bars: 50 µm.

 

Figure 5
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  		Fig. 5. Partially myosin IIB-depleted notochordal cells exhibit a cortical actin phenotype. (A) Confocal microscopy of Xenopus notochordal control cells injected with moesin-GFP reveals a dynamic cortical actin network in a basket-like distribution, with 3-5 foci of actin visible per cell. (B) These actin-rich foci (asterisk) visibly move within the cell on a sub-minute timescale. (C) Plotting the relative displacement of foci along the embryonic axis in the mediolateral (M) and anterior-posterior (A) directions, reveals that the rate of displacement of these foci is greater in the mediolateral dimension than in the anterior-posterior dimension for notochordal cells (No) (P<0.02 by Student's t-test), and this polarity is also detectable in pre-notochordal cells (PN) but not in animal cap cells (AC). (D) In contrast to control cells, MHC-B MO-injected cells exhibit dramatic disruption of the cortical actin network. Asterisks (also in E) indicate the NSB. (E) Confocal time-lapse sequence of a morphant cell exhibiting aberrant motility concomitant with cortical actin breakdown. Elapsed time is shown in minutes. (F,G) Mildly morphant notochordal cells (1 µM MO) remain in the notochord with intact cortical actin networks, but exhibit profuse filopodia (F) as compared with control notochordal cells (G), revealing a threshold cell polarity phenotype in response to MHC-B depletion. Scale bars: 50 µm.

 

Figure 6
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  		Fig. 6. Cell-cell and cell-matrix adhesion are affected in morphant cells. (A) Morphant dorsal axial cells at stage 13 exhibit a dramatic dose-dependent reduction in adhesion to recombinant extracellular domain of C-cadherin (C-Cad). (B) However, cell-surface levels of C-Cad are unchanged in morphant cells, as similar levels of control and morphant cell C-Cad is available on the cell surface for trypsin digestion. (C) Morphant cells also show a dramatic reduction in adhesion to fibronectin (FN). (D) Cell-surface levels of integrin {alpha}5 are not affected by myosin IIB depletion. (E) Failure of blastopore closure due to MHC-B depletion is not rescued by exogenous expression of C-Cad, consistent with the hypothesis that C-Cad levels are not limiting in morphant embryos. (F) Adhesion to FN in morphant cells is rescued by co-expression of exogenous human MHC-B.

 

Figure 7
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  		Fig. 7. A myosin IIB-dependent cortical actin network functions to integrate cell adhesion and polarization to generate directed forces driving morphogenesis. (A) Cell intercalation in the Xenopus notochord requires two distinct cell activities: cell contraction in the cell body (gray arrows) and polarized protrusive activity (black arrows). (B) Contraction events are driven by the myosin IIB-dependent cortical actin network (green lines), which is organized into dynamic foci (black) and interacts with myosin IIB at adhesion sites (red). (C) Integrating this episodic cell shortening with polarized protrusive activity and dynamically regulated myosin IIB-dependent adhesion leads to cell intercalation (white arrows) and tissue-level convergence and extension (large black arrows). The deeply interdigitated notochordal cells at this stage have adopted a monopolar protrusive activity (small black arrows). The extracellular matrix of the NSB is lateral in each case.

 

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