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Polarized basolateral cell motility underlies invagination and convergent extension of the ascidian notochord

¹ Department of Zoology, University of Washington, Seattle, WA 98195, USA
² Friday Harbor Labs, Friday Harbor, WA, 98250, USA

View larger version (26K): [in a new window]   Fig. 1. Timeline showing major morphogenetic events at 10°C in Boltenia villosa between late gastrulation, approx. 14 hours after fertilization (AF), when notochord cells begin to divide for the final time, and tailbud stage when notochord cells have completed their rearrangements to form a stack of coins. We analyzed at least 6 and typically more than 10 embryos at each time point. Labeled segments associated with a given event span the period in which we observed that event in one or more embryos. The actual intervals in which that event occurs may be shorter.

View larger version (62K): [in a new window]   Fig. 2. Colorized confocal sections taken from whole-mounted phalloidin-stained Boltenia villosa embryos at selected stages. Dorsal is always up. In top and side views, anterior is to the left and posterior to the right. Red=primary notochord lineage; violet=secondary notochord lineage; dark blue=neural plate; orange=posterior muscle; light brown=mesenchyme/trunk lateral cells; yellow=endoderm/endodermal strand; light blue=epidermis. Scale bar, 10 µm in all panels. The above also refers to Fig. 3 and Fig. 5. A late gastrula stage embryo (approx. 15 hours AF). (A) Raw confocal data before false coloring to produced B; (B) Top view; (C) Lateral view. Several cells within the primary notochord lineage have just begun to cleave (asterisks). Secondary notochord lineage cells (violet) lie at the ends of the notochord plate just anterior to the posterior muscle. Note that apices of individual cells forming the anterior portion of the blastopore lip have not yet begun to constrict.

View larger version (97K): [in a new window]   Fig. 3. Confocal sections from embryos at late stage I (left column; approx. 16 hours AF), mid-stage II (middle column; approx. 17 hours AF) and early stage III (right column; approx. 18 hours AF). (A-C) Dorsal notochord plate. (D-F) Ventral notochord plate. Arrows indicate the lateral folds in D. In A, the secondary notochord cells (violet) are just rounding up to divide. Many primary cells have lost contact with the blastopore lip and joined the interior of the notochord plate, while others have greatly constricted apices (asterisks). (G-I) Lateral sections through the notochord plate. G and I show midline views. H shows a more lateral view to illustrate how cells at the lateral invaginating edges of the notochord plate are also elongated perpendicular to the AP axis. Note how the dorsal (basal) ends of many individual cells in I tend to extend posterior relative to the ventral (apical) ends of the same cells. The same phenomenon is apparent in F. The apices of these cells tend to become stretched along the AP axis by early stage III (not shown). Asterisks in I mark dividing cells within the neural tube. (J-L) Cross sections through the notochord plate taken near its anterior end where the lateral folds are least pronounced and the invagination progresses simultaneously across the width of the plate.

View larger version (53K): [in a new window]   Fig. 4. SEM views of Boltenia villosa embryos fractured to reveal the apical (ventral) surface of the notochord plate and the ventral folds at: (A) approx. 16 hours AF; (B) approx. 17 hours AF and (C) approx. 18 hours AF. Folds are present before cell rearrangement and notochord extension occurs (arrows in A), and then move towards the midline, meeting first at the posterior (asterisk in B) where they begin to fuse. Fusion then progresses posterior to anterior (fusion ‘front’ indicated by arrow in C). Posterior is top and left in all panels. Scale bar, 10 µm.

View larger version (25K): [in a new window]   Fig. 5. Progressive intercalation of cells within the cylinder during mid-late stage III (approx. 18-21 hours AF). Cross sections A-D are taken from different embryos at progressive stages to illustrate the expansion of wedge-shaped cells about the circumference of the notochord rod.

View larger version (200K): [in a new window]   Fig. 6. DIC sections taken from focus sweeps through a living Corella inflata embryo at three successive stages. (A-C) Sections through the neural plate/tube. (D-F) Sections through the dorsal notochord plate/cylinder/rod. (G-I) Sections through the dorsal notochord plate/cylinder/rod. (J-L) Sections through the endodermal strand. First column (A,D,G,J) just after completion of the final notochord cell division. Second column (B,E,H,K) mid-neurula stage, approximately 2 hours after the completion of the final division. Third column (C,F,I,L) stack of coins stage, approximately 6 hours after completion of the final division. By this stage, approximately half of the notochord has extended posterior beyond the field of view. Anterior is to the left in all panels. Asterisks in A,D,G mark the blastopore. Arrows in G and H indicate the boundaries of the notochord. Scale bar, 10 µm.

View larger version (57K): [in a new window]   Fig. 7. Reconstruction of cell movement and shape change during notochord formation in a living Corella inflata embryo (the same shown in Fig. 6). Cell outlines represent approximate cell boundaries as seen in cross section near the dorsal surface (left column) or ventral surface (right column) of the notochord plate. Anterior is to the left in all panels. Times shown are from completion of the final primary division. Blue cells remained within the dorsal plate; Grey cells started within or intercalated into the ventral folds. Cells 1-31 derive from the primary lineage. Cells B1-B8 derive from the secondary lineage. Cell 26 failed to divide for the last time but nevertheless changed shape and intercalated as other cells do. In the lowermost panels, part of the notochord has extended rightward out of the video camera’s field of view.

View larger version (23K): [in a new window]   Fig. 8. Morphometric analysis of cross-sectional cell shape changes during notochord formation in a living Corella inflata embryo. In each graph, the time represents minutes elapsed since final cleavage in the primary lineage.

View larger version (187K): [in a new window]   Fig. 9. F-actin organization within the notochord. Each image is a projection of 5 confocal sections taken at 0.2 µm intervals. (A) Top view section through a mid-stage II embryo (approx. 17 hours AF) illustrating general F-actin organization within the notochord plate and surrounding tissues. The interior cytoplasm of each notochord cell is filled with a dense meshwork of F-actin except where excluded by yolk granules (small dark holes) or nuclei (*). F-actin puncta similar to those described elsewhere (Foe et al., 2000; von Dassow and Schubiger, 1994) appear throughout the cytoplasm. Posterior is to the left. (B,C). Cross-sectional views of stage II embryos showing actin-rich protrusions (arrows) extending across the surfaces of adjacent neighbors. In C a protrusion lies just out of focus and to its right can be seen a dense meshwork of F-actin fibers in the cortex of the cell across which it is extending. Dorsal is up in B and C. Scale bar, 10 µm in all panels. B and C have same scale.

View larger version (198K): [in a new window]   Fig. 10. Stereo views of F-actin-rich protrusions within the notochord rudiment. (A) Cross-sectional view through the notochord of a mid-late stage 2 embryo. (B) Cross section through an early-mid stage III embryo. Arrows in each panel indicate individual protrusions.

View larger version (186K): [in a new window]   Fig. 11. SEM views of notochord cell protrusions. (A,B). Cross-sectional views of mid-stage II (A) and early stage III (B) embryos. Arrows indicate individual protrusions. Bright spots are yolk granules lying just under the membrane. (C) Outer (basal) surface of an early stage III embryo. In most (see arrows) but not all (arrowheads) cases, cells extend long continuous protrusions across the surfaces of adjacent neighbors. (D) Ventral surface of a late stage I embryo. Adjacent cells extend short interlocking protrusions across one another’s surfaces. Arrowheads outline a single cell, indicating in each case the direction of the protrusion. Scale bars, 10 µm in A and B and 5µm in C and D. Dorsal is up in A and B.

View larger version (31K): [in a new window]   Fig. 12. (A) Invagination and convergent extension lead to formation of a cylindrical intermediate. Arrows at left indicate convergent extension movements of cells within the notochord plate and its invagination, which occur simultaneously during late stage I and stage II. Dorsal is down and ventral is up. (B). Schematic view of an early-mid stage II notochord plate showing how individual cells extend their interior edges across the faces of adjacent notochord cell neighbors. (C). Textbook view of how an isolated cell crawls on a flat external substratum. (1) Localized actin-dependent protrusive forces (blue arrows) cause the leading edge to extend relative to adhesive contacts with the underlying substratum; (2) New adhesive contacts (green ovals) form at the leading edge with the underlying substratum, and subsequently stabilize through various mechanisms, including lateral clustering of adhesion proteins, and association with the underlying cortical cytoskeleton; (3) actin/myosin-dependent contractile forces within the cortical or interior cytoplasm (red arrows) set up a tug of war between different sites of attachment to the substratum. Directional movement occurs when this tug of war is biased to favor consolidation of leading edge attachments and release of adhesions at the rear (Chen, 1981; Jay et al., 1995; Palecek et al., 1996). (D) How the same machinery might operate within a monolayer epithelium. Each polygonal cell represents a cross section through an epithelial cell somewhere below the apical surface, each vertex represents an interior (basolateral) edge, analogous to the leading edge in (C), which attempts to extend (blue arrows) between adjacent neighbors. Homophilic associations between cadherin proteins replace the integrin-based adhesion used by most mesenchymal cells, but the underlying mechanics are entirely analogous. For simplicity, we consider contractile forces only within the cortex. (E) Mediolaterally biased protrusion (blue arrows) drives cells away from their preferred circular cross-sectional shapes. The cortical contractile forces that act to restore these shapes within each cell (red arrows) are joined by adhesive contacts to make contractile chains that span the width of the notochord plate and cause it to become longer and narrower.