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Review
. 2009 Jul;1(1):a000802.
doi: 10.1101/cshperspect.a000802.

Left-right determination: involvement of molecular motor KIF3, cilia, and nodal flow

Nobutaka Hirokawa  1 , Yosuke Tanaka, Yasushi Okada
Affiliations
Review

Left-right determination: involvement of molecular motor KIF3, cilia, and nodal flow

Nobutaka Hirokawa et al. Cold Spring Harb Perspect Biol. 2009 Jul.

Abstract

Mammalian left-right determination is a good example for how multiple cell biological processes coordinate in the formation of a basic body plan. The leftward movement of fluid at the ventral node, called nodal flow, is the central process in symmetry breaking on the left-right axis. Nodal flow is autonomously generated by the rotation of posteriorly tilted cilia that are built by transport via KIF3 motor on cells of the ventral node. How nodal flow is interpreted to create left-right asymmetry has been a matter of debate. Recent evidence suggests that the leftward movement of sheathed lipidic particles, called nodal vesicular parcels (NVPs), may result in the activation of the noncanonical hedgehog signaling pathway, an asymmetric elevation in intracellular Ca(2+) and changes in gene expression.

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Figures

Figure 1.
Figure 1.
(A) Left–right asymmetric arrangements of internal organs in the human body. (Left) Normal arrangement (situs solitus). Most humans (>99%) have the heart on the left side and the liver on the right side. (Right) Mirrored arrangement (situs inversus). Half of patients with Kartagener's syndrome have this arrangement, whereas the remaining patients are normal. Therefore, the left–right bilateral symmetry is randomly broken in this disease. (B–E) Scanning electron micrographs of wild-type (B, D) and Kif3b−/− (C, E) mouse embryos. (B, C) Full-length images. Wild-type embryos at this stage have already turned with a right-sided tail (B), whereas Kif3b−/− embryos remain unturned (C). In panel C, the dilated pericardial sac has been removed, and the heart loop is inverted (arrow). (D, E) Higher-magnification images and schematic representations of the heart loops showing a normal loop in the wild-type embryo (D) and an inverted loop in the mutant embryo (E). (F–I) Scanning electron micrographs of a mouse node. (F) Low-magnification view of a mouse embryo at 7.5 days postcoitum. Reichert's membrane is removed, and the embryo is observed from the ventral side. The node is indicated by a black rectangle. The orientation is indicated in the panel as anterior (A), posterior (P), left (L), and right (R). Scale bar = 100 µm. (G) Higher-magnification view of the mouse node. The orientation is the same as in panel A. Scale bar = 20 µm. (H) Higher-magnification view of the nodal cilia (arrows) and nodal pit cells. Scale bar = 5 µm. (I) Nodal pit cells of Kif3b−/− embryos. Nodal cilia are absent in these genetically manipulated embryos. (J) Intraflagellar transport. Protein components in the cilia and flagella are transported by KIF3A/B complexes (light and dark blue) along the doublet microtubules of the axoneme. (Panels A and J were reproduced with permission from JT Biohistory Research Hall/TokyoCinema. B–I were modified from Nonaka et al. 1998, Okada et al. 2005, and Hirokawa et al. 2006, with permission.)
Figure 2.
Figure 2.
(A) Ultrastructures of normal cilia and primary cilia. (Left) Normal cilia and flagella have nine pairs of doublet microtubules (yellow) and two central microtubules (yellow). Adjacent doublet microtubules are connected with dynein motors (blue and green). The orientation of the central pair of microtubules is considered to determine the beating plane (purple). (Right) The central pair of microtubules is missing in immotile primary cilia and nodal cilia. In nodal cilia, the dynein motors remain in a chiral arrangement and produce a rotation-like movement (purple). (B–E) Rotation of nodal cilia and leftward nodal flow. The images are views from the ventral side. The orientation is indicated in the panels as anterior (A), posterior (P), left (L), and right (R). (B) Trajectory of a fluorescent bead attached to the tip of a nodal cilium (Movies 1 and 2). Three consecutive video frames with 33-ms exposures at 16-ms intervals (interlaced scan) are shown. The moving bead produces arc-shaped images (traced by green arrows). The beads rotate clockwise when viewed above the node. (C) Trajectories of the tips of nodal cilia traced from a high-speed video sequence (500 frames/s) (Movie 3). The red circles show the positions of the ends of the cilia at 10-ms time intervals, and the yellow circles show the positions of the roots of the cilia. The white ellipses show the trajectories of the tips. Scale bar = 5 µm. (D) The positions of beads that entered the node from the right edge traced for 4 seconds at 0.33-second intervals. Different symbols indicate different beads. Most beads go straight to the left edge of the node. Scale bar = 20 µm. (E) The trajectories of four beads selected to illustrate the streamline of the nodal flow. The flow is mostly laminar and straight in the middle of the node, but often makes small vortices near the left edge (arrowheads). (Panel A was reproduced with permission from JT Biohistory Research Hall/TokyoCinema. B–E were modified from Okada et al. [1999, 2005] with permission.)
Movie 1
Movie 1
Leftward nodal flow. Nodal flow was visualized by adding fluorescent beads to the medium surrounding the ventral node of a mouse embryo at the early somite stage. 4× time lapse.
Movie 2
Movie 2
Rotatory movement of nodal cilia.
Movie 3
Movie 3
Posteriorly-tilted rotation of nodal cilia. Nodal cilia were observed by high-speed video microscopy (500 frames/s). The focus was adjusted to approximately 3 µm above the surface of the ventral node. The images of the cilia thus blur when they are near the floor and become clear when they come up to the focal plane. Note that the particle (highlighted by a red circle) does not go down to the surface but eventually goes toward the left side of the node due to the movement of the cilia. Reproduced from Okada et al. (2005) with permission.
Figure 3.
Figure 3.
(A, B) Posteriorly tilted rotation of nodal cilia. (A) Definition of the analysis parameters. The conic rotation of the nodal cilium was parameterized with the following parameters: tilt of axis, Θ; direction of the axis, Φ; slant height, ρ; and apex angle, Ψ̃. (B) Distributions of the parameters in mouse, rabbit, and medaka embryos. (C, D) Posterior bias in the positions of the roots of the nodal cilia. (C) Fluorescent micrograph of a rabbit node. Green dots show the roots of the nodal cilia, whereas red staining shows the cells' boundaries. The orientation is indicated in the panels as anterior (A), posterior (P), left (L), and right (R). Most of the green dots are located on the posterior side. Scale bar = 10 µm. (D) High-magnification scanning electron micrograph of rabbit nodal ciliated cells. Note the domelike curvature of the apical plasma membrane and posteriorly tilted projection of the monocilia nearly perpendicular to the plasma membrane. Scale bar = 5 µm. (E) Model of the generation of nodal flow. The orientation is indicated as anterior (A), posterior (P), left (L), and right (R). The conic rotation of the nodal cilia is posteriorly tilted. Therefore, the cilia move in a nearly perpendicular manner from right to left. The return movement from left to right occurs just above the cell surface. The viscous drag thus dumps the movement of the fluid to the right (purple). As a result, unidirectional leftward flow (blue) is generated. (Figure modified from Okada et al. 2005 and Hirokawa et al. 2006 with permission.)
Movie 4
Movie 4
Rapid leftward flow and slow counterflow in a mouse nodal pit. The focal plane is first adjusted to 4 µm above the floor of the nodal pit of the mouse (bottom), where the fluid flows rapidly to the left. The focal plane is then lifted by 6 µm (middle), where circulation of the fluid is evident. Beads flow upward near the right edge and go down near the left edge. Beads above the focal plane move toward the right, while beads below the focal plane move toward the left. Finally, the focal plane is lifted twice by 6 µm (top), where the slower rightward return flow is evident. Reproduced from Okada et al. (2005) with permission.
Figure 4.
Figure 4.
Fibroblast growth factor (FGF) signaling is essential for left-specific calcium elevation but not for the nodal flow. (A and B) Models for the mechanism of interpretation of the nodal flow to the downstream signaling. (A) Concentration gradient model. Left–right asymmetry of the concentration of morphogen(s) in the consequence of nodal flow (black arrow) stimulates the cells on the left edge of the node, which further propagate the signal to the downstream (red arrow). (B) Two-cilia model. Cilia on the edge are specifically differentiated to sensing the flow, and those on the left edge, but not on the right edge, are activated by the leftward nodal flow to activate the signaling cascade. (C) Leftward fluid flow is not affected by SU5402 treatment. Camera lucida of three traces of fluorescent beads for DMSO (carrier)- or SU5402-treated embryos at the 1–3 somite stage, plotted every 1 second for 10 seconds. Upper, anterior; lower, posterior. Scale bar, 10 µm. (D–F) FGF regulates left-specific Ca2+ elevation. Ventral view of Ca2+ imaging of wild type embryos, pharmacologically treated as indicated, and represented by pseudocolor (signal intensity: red > green > blue > black). Note that FGF inhibitor eliminates the left-specific Ca2+ elevation, and that addition of SHH-N peptide could replenish the Ca2+ elevation in cells on the left edge of the node (F, arrows). Upper, anterior; lower, posterior. Scale bars, 20 µm. (Figure modified from Tanaka et al. 2005 and Hirokawa et al. 2009 with permission.)
Figure 5.
Figure 5.
(A) Time-lapse optical microscope images of flowing nodal vesicular parcels (NVPs). The orientation is indicated in the panel as left (L) and right (R). NVPs (arrowheads) are transferred to the left side by the nodal flow (Movie 5). Scale bar = 10 µm. (B) Scanning electron micrograph of the ventral surface of nodal pit cells. Red arrowheads indicate NVP precursors. Scale bar = 2 µm. (C, D) Transmission electron micrographs of nodal pit cells. (C) Transition state of NVP release in an SU5402-treated embryo. Scale bar = 1 µm. (D) NVPs (red arrowheads) are associated with microvilli (yellow arrowheads). Scale bar = 1 µm. (E) Schematic representation of NVP flow. The orientation is indicated in the panel as left (L) and right (R). NVPs appear to be released from dynamic microvilli (Movie 6), transported to the left side by the nodal flow (Movie 5), and fragmented with the aid of cilia at the left periphery of the node (Movie 7). The green halos indicate calcium elevation as a sign of cell activation on the left side of the node. (Figure modified from Tanaka et al. 2005 with permission.)
Movie 5
Movie 5
Leftward flow of NVPs. A ventral view of a mouse embryo whose membrane lipids have been fluorescently labeled with a DiI dye is shown. The anterior side of the node (triangular blank structure in the center) is placed upward in the movie, so that the leftward flow of NVPs appears as rightward movement in the movie. Bar, 10 µm. 20x time lapse. Reproduced from Tanaka et al. (2005).
Movie 6
Movie 6
Release of NVPs from dynamically protruding microvilli. Detailed time-lapse observations of DiI-labeled NVPs reveal a unique ball-throwing mechanism for the release of NVPs from the tips of microvilli. Bar, 1 µm. 10x time lapse. Reproduced from Tanaka et al. (2005) with permission.
Movie 7
Movie 7
Fragmentation of an NVP on left nodal crown cells. An NVP of approximately 0.5 µm in diameter is approaching the left wall. When it comes within 1-2 µm of the wall, it suddenly expands to twice its size and bursts into several submicron pieces that spread out along the left wall. These pieces are most likely crushed by rotating cilia. Bar, 1 µm. 16x time lapse. Reproduced from Tanaka et al. (2005) with permission.
Movie 8
Movie 8
Animated model of NVP flow. An animated model summarizing the mechanisms of the release, transport and turnover of an NVP is shown. Copyright by Biohistory Research Hall/TokyoCinema Inc. (2005).
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