Tube formation by complex cellular processes in Ciona intestinalis notochord - PubMed (original) (raw)

Tube formation by complex cellular processes in Ciona intestinalis notochord

Bo Dong et al. Dev Biol. 2009.

Abstract

In the course of embryogenesis multicellular structures and organs are assembled from constituent cells. One structural component common to many organs is the tube, which consists most simply of a luminal space surrounded by a single layer of epithelial cells. The notochord of ascidian Ciona forms a tube consisting of only 40 cells, and serves as a hydrostatic "skeleton" essential for swimming. While the early processes of convergent extension in ascidian notochord development have been extensively studied, the later phases of development, which include lumen formation, have not been well characterized. Here we used molecular markers and confocal imaging to describe tubulogenesis in the developing Ciona notochord. We found that during tubulogenesis each notochord cell established de novo apical domains, and underwent a mesenchymal-epithelial transition to become an unusual epithelial cell with two opposing apical domains. Concomitantly, extracellular luminal matrix was produced and deposited between notochord cells. Subsequently, each notochord cell simultaneously executed two types of crawling movements bi-directionally along the anterior/posterior axis on the inner surface of notochordal sheath. Lamellipodia-like protrusions resulted in cell lengthening along the anterior/posterior axis, while the retraction of trailing edges of the same cell led to the merging of the two apical domains. As a result, the notochord cells acquired endothelial-like shape and formed the wall of the central lumen. Inhibition of actin polymerization prevented the cell movement and tube formation. Ciona notochord tube formation utilized an assortment of common and fundamental cellular processes including cell shape change, apical membrane biogenesis, cell/cell adhesion remodeling, dynamic cell crawling, and lumen matrix secretion.

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Figures

Fig. 1

Fig. 1

Timeline of notochord morphogenesis. Shown are the major morphogenesis events occurring at 16°C in Ciona intestinalis notochord following cell intercalation (from 14 hours past fertilization, hpf). Other major concurrent events of Ciona embryogenesis are listed.

Fig. 2

Fig. 2

Cell shape changes during notochord tube formation. (A–H) Confocal images of notochord cells expressing GFP. (A′-H′) 3-D reconstruction of the same cell(s) in (A to H). (A″-H‴′) Cross-section of notochord cell at the plane(s) shown in (A–H) respectively. At the beginning of stage IV, notochord cells have a “coin” shape (A and A′). In the next four hours cells elongate along the A/P axis (B and B′) and narrow in cross section (compare A″ and B″). The nucleus in this elongated cell is localized at the posterior end (“N” in B), while a constriction can be observed midway (white arrow in B and B′). At stage V, lumens emerge (“L” in C and C″) and enlarge (“L” in D and D‴). Cells remain radially symmetrical with respect to the cylindrical axis of the notochord (C′ and D′). The cell’s main body in cross section resembles a round disc (C‴ and D″), whereas the portion encompassing the lumen resembles a ring (C″ and D‴). During stage VI, a certain point on the anterior edge and its opposing point on the posterior edge (designated as anterior trailing edge, or ATE, and posterior trailing edge, or PTE, red arrows in E) on each cell recede along the A/P axis, while the opposite points around the circumference (designated as anterior leading edge, or ALE, and posterior leading edge, or PLE, white arrows in E) advance along the A/P axis. The cell thus loses its radial symmetry (E′ and E″). Some portion of the cell remains in basal contact with the notochordal sheath at the entire circumference (E‴). At the end of stage VI, two trailing edges of the same cell meet and merge into one (designated as joint trailing edge, or JTE, red arrow in F and F′). The only portion of the cell that retains basal contact around the entire circumference is at the level of the JTE (F″, compared with F‴). This 360° basal contact is lost at stage VII, as the JTE detaches from the basal lamina and splits into two trailing edges (designated as trailing edge 1, or TE1, and trailing edge 2, or TE2, red arrows in G, G′ and G″, also in H″) which continue to crawl across the basal lamina. The vacant basal area is taken over by either the anterior or the posterior neighbor cell. Concurrently, two lumens are allowed to connect (white arrow in G′). Notochord cells further flatten and eventually assume an endothelial-like morphology (H and H′). In cross sections, the notochord now has two cells surrounding a central lumen (“L” in H″-H‴′, white arrows indicate cell-cell boundaries). Anterior is to the left. Scale bars: 10 μm.

Fig. 3

Fig. 3

Biometric analysis of Ciona notochord during tubulogenesis. The calculations were done from measurements of five notochords at each of three time points. (A) Length of notochord. (B) Average length of individual notochord cells. (C) Average diameter of notochord. (D) Average diameter of notochord cells at 14 and 18 hpf, and average thickness of endothelial-like notochord cells at 36 hpf. (E) Total volume of entire notochord (black) and its two components, cells (purple) and lumen (white, at 36 hpf). For calculations see Materials and Methods and supplementary materials Fig. 1.

Fig. 4

Fig. 4

Notochord cells crawl and shape the adjacent extracellular lumens. (A–C) Median sections of notochord mosaically expressing GFP at 23 hpf. The confocal images are merged with bright field images to show the position of cells, cell extensions, and extracellular lumens in the notochord. (A′–C′) 3-D reconstructions of cells shown in AC. Notochord cells (* in A–C′) at this stage send out long anterior, posterior, or bidirectional extensions (white arrows in A–C), respectively, forcing the adjacent lumens (outlined by dashed lines) to tilt. The space between dashed lines in C′ indicates the space occupied by the cell before protrusions are made. The edge of lamellipodia has a wavy outline (arrows in C′). Anterior is to the left. (D) 3-D reconstruction of a section of notochord mosaically expressing turboGFP-actin at stage VI (see supplementary movie). Actin is concentrated at the leading edges of crawling notochord cells (arrows). (E and F) Control and Latrunculin B-treated embryos at 26 hpf. Inhibition of actin polymerization prevents the cell shape changes and tubulogenesis. L, lumen; CB, main cell body; NE, new cell extension; LA, lamellipodia; ALE, anterior leading edge; PLE, posterior leading edge; TE, trailing edge. Scale bar: 10 μm.

Fig. 5

Fig. 5

Emergence and expansion of the luminal domain before and during lumen formation. Confocal images from the midline of notochord cells stained with antibody against Ciona homolog of SLC26-2 (red) for luminal domain, and BODIPY phalloidin for filamental actin (F-actin, green) to highlight cell membrane. (A) SLC26-2 positive domains (arrowheads) first appear in the center of anterior and posterior cell surfaces. (B) The SLC26-2 positive domains enlarge and occupy the portions of notochord cell contacting extracellular lumens. (C) The luminal domains expand as the lateral domains abutting two notochord cells retract. (D) The anterior and posterior luminal domains of the same cell meet and fuse (arrow). (E) After the lumens have fused, the SLC26-2 positive domain occupies the luminal side of the endothelial-like notochord cell. Unknown structures that express SLC26-2 appear inside the luminal spaces (* in C, D, and E). (F) The emergence of luminal domain lags behind in the posterior-most notochord and predicts the temporal order of the lumen formation (arrowheads indicate lumen formation proceeds at different paces; arrows indicate that lumen has not formed among last three cells). Anterior is to the left. Scale bar: 10 μm.

Fig. 6

Fig. 6

Biometric analysis of notochord cell membrane domains. The surface areas were calculated from measurements of 4 to 5 GFP-labeled cells located in the central region of notochord (see Materials and Methods and supplementary materials Fig. 2). (A) In the middle of stage IV (16 hpf) the lateral domain of notochord cells is larger than the basal domain. (B) After elongation (18 hpf), the total surface of notochord cells is reduced, and the basal domain is now larger than the lateral domain. (C) The lateral domain further reduces as the luminal domain emerges (21 hpf). (D) The luminal domain continues to enlarge while the lateral domain shrinks, as the lumens increase (22 hpf). The sum of both domains is larger than in the previous time point.

Fig. 7

Fig. 7

Adhesion remodeling as notochord cells undergo mesenchymal-epithelial transition. (A–C′) Subcellular localization of E-cadherin and GFP (to outline the cells and extracellular lumens) in notochord at three stages viewed in median section by confocal imaging. (A″–C″) 3-D reconstruction of E-cadherin localization in the same cells shown A–C′. Before lumen formation the E-cadherin-positive domain occupies the entire anterior and posterior surface of notochord cells (A-A″). As the lumen forms, the E-cadherin domain recedes from the center to the periphery (B-B″). When lumens fuse, lateral junctions between cells a and b and between cells b and c merge, and new junction (arrow in C′ and C″) is established between cells a and c, which were not in contact previously. The cell body of cell b is off the section plane in C-C′. Anterior is to the left. Scale bar: 10 μm.

Fig. 8

Fig. 8

Mature notochord cells assume an endothelial-like shape and arrangement. (A-A″) Median confocal section of a fragment of notochord after tubulogenesis is complete (36 hpf). Notochord cells are mosaically labeled with E-cadherin (A) to reveal the cell/cell junctions (arrows), and mGFP (A′) to outline the cell shape (A″, merged). (B-B″) 3-D reconstruction of notochord fragment in A-A″. (C) A diagram of the cell/cell boundaries. Anterior is to the left. Scale bar: 10 μm.

Fig. 9

Fig. 9

Schematic views of Ciona notochord during tube formation. (A) Cartoon depicting the median section of three consecutive notochord cells (a, b, and c) at different stages of tubulogenesis. (B) Cartoon depicting the median section of cell a in (A). At the beginning of stage V, novel membrane domains (red) appear that encompasses the emerging extracellular lumens. From this point, each notochord cell possesses three membrane domains: apical/luminal domain (red), lateral domain (green), and basal domain (light blue). The cell can be viewed as a highly modified epithelial cell, with two separate apical domains and two separate lateral domains at the opposing ends, and curved A/B axes (brown arrows, only two are drawn). Concurrent with the enlargement of extracellular lumens, apical domains expand and lateral domains retract. At the end of stage V notochord cells in median section resemble steep biconcave lenses. The radial symmetry along the cylindrical axis of the notochord is still maintained. During stage VI, as the lateral domains of a notochord cell are reduced to a minimum, a certain point on the anterior rim (anterior trailing edge, ATE) and its opposing point on the posterior rim (posterior trailing edge, PTE) of an individual cell begin to retract (orange arrows). While at the opposite points in the circumferences, anterior and posterior edges (anterior leading edge, ALE, and posterior leading edge, PLE) of the same cell begin to protrude and extend (magenta arrows). The direction of these movements is parallel with the cylindrical axis of the notochord. The extent by which these edges retract and extend is not equal. As the result of these movements the radial symmetry of the cell with respect to the cylindrical axis is lost. At the end of stage VI, the ATE and PTE meet and merge (designated as joint trailing edge, JTE). At the same time, the basal domain previously located between ATE and PTE are eliminated, and the anterior and posterior apical domains join. At the beginning of stage VII, the JTE further retracts away from the inner surface of notochordal sheath in a direction perpendicular to this surface. The space vacated by the JTE at the basal side is filled by either the ALE of the posterior cell or the PLE of the anterior cell. For the first time the single notochord cell does not line the entire circumference of the emerging notochord tube. The anterior and posterior lumens now connect. At the end of tubulogenesis, the notochord cell has assumed an endothelial-like morphology, with a single and large apical domain, a shallow lateral domain, and an extended basal domain. The apical/basal axis runs orthogonally to the apical and basal surfaces (brown arrow). (C) Cross section of notochord cell a. Up to the end of stage VI, the basal domain of the notochord cell contacts the entire circumference of the notochord. At the beginning of stage VII, after the JTE has detached from the inner surface of the sheath and the vacated surface has been occupied by the leading edge of neighbor cell (b), lumens fuse. The existence of JTE is transient as it quickly splits into two trailing edges (designated as trailing edge 1 and 2, or TE1 and TE2, respectively). Both TE1 and TE2 retracts across the inner surface of the sheath in opposite directions (orange arrows), effectively bringing themselves closer to each other. The endothelial-like notochord cells after tube formation occupy only a portion of the notochord circumference. (D) Schematic diagrams of the basal domains as viewed from the basal surface of the notochord flatten onto a two dimensional space. Each notochord cell simultaneously extends bi-directionally (to different extents) along the longitudinal axis at one point of the circumference (for example, at 180°), and retracts at the opposite point (at 0°). This movement transforms the shape of basal domain from a rectangle to an uneven hexagon. When both ATE and PTE merge and form a transient JTE, the shape of the basal domain is roughly a diamond at the end of stage VI. JTE quickly splits into TE1 and TE2 as it loses its contact with the notochordal sheath. The direction of retraction then turns from previously parallel, to the direction of extension of leading edges, to orthogonal. These movements eventually transform the shape of the basal domain back to a rough hexagon that is elongated along the longitudinal axis of the notochord. At stage VII, the cross-section at any point along the tube is composed of two cells instead of one, as is the case at stages IV to VI. This configuration effectively reverses the result of intercalation that occurs prior to the stage IV, which arranges the notochord cells into a single file. (E) Schematic illustration of notochord cell/cell junctions viewed from the basal surface of the notochord flatten out onto a two dimensional space. New cell junction forms during stage VII between two notochord cells (a and c) which are previously separated (by cell b).

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