Abnormal Nodal Flow Precedes Situs Inversus in iv and inv mice Yasushi Okada, Shigenori Nonaka, Yosuke Tanaka, Yukio Saijoh, Hiroshi Hamada, Nobutaka Hirokawa  Molecular Cell  Volume 4, Issue 4, Pages 459-468 (October 1999) DOI: 10.1016/S1097-2765(00)80197-5

Figure 1 Immotile Cilia and the Lack of Nodal Flow in iv/iv Embryos (A–C) Rapid movement of nodal cilia in an iv/+ embryo. (B) is a view of nodal cilia (red trace) 33 ms after (A). Arrowheads indicates the bases of each cilia. Exposure time, 1 ms. Note that the movement of the cilia is three-dimensional. Thus, the rotating movement of cilia is not very demonstrative in these snapshots. In a longer exposure view (2 s), rapidly moving cilia are blurred (C). (D) Frozen cilia in an iv/iv embryo. Almost all nodal cilia of iv/iv embryo were immotile (they do not even fluctuate by Brownian movement), so that they are clearly imaged in 2 s exposure view. (E) Laminar, leftward nodal flow in an iv/+ node. The positions of beads added to the extraembryonic fluid are shown. Different colors indicate different beads, and the interval of the trace is 33 ms. Arrows indicate the flow paths that most beads followed. Most beads linearly flowed toward the left side of the node (upper edge of the panel). Note that different beads (dots with different colors) flowed along similar paths. (F) Lack of the directional flow in an iv/iv node. Traces of the positions of three beads are shown. The beads were chased for 2 s with 100 ms interval. They did not show any directional movement. (A), (B), (C), and (E) were taken from one iv/+ embryo, and (D) and (F) were taken from its littermate. Bar, 50 μm. All panels are aligned with the anterior side of the node on left. Video clips are provided as supplementary material on the web at http://www.molecule.org/cgi/content/full/4/4/459. Molecular Cell 1999 4, 459-468DOI: (10.1016/S1097-2765(00)80197-5)

Figure 2 Slow, Turbulent Nodal Flow of inv Mutant Embryos (A and F) The movement of nodal cilia in inv/+ (A) and inv/inv (F) embryos. The gyrating movement of nodal cilia was visualized by attaching a fluorescent bead to the tip of cilium and imaged with 33 ms exposure and 16 ms interval (interlaced scan). Thus, the moving beads make arc-shaped images (traced by red arrows). Beads rotated clockwise when seen from above the node, reflecting the counterclockwise gyration of the cilia when seen from the nodal pit cell bodies. Beads rotated about 120° in 33 ms in both inv/+ and inv/inv embryos, or the speed of the rotation was about 600 rpm, a similar value to the wild-type embryos (a video clip of the movement of the wild-type nodal cilia is provided as supplementary material on the web at http://www.molecule.org/cgi/content/full/4/4/459). Bar, 5 μm. (B–D and G) Nodal flow in wild-type (B), heterozygous (inv/+) (C), transgene-rescued (inv/inv V15) (D), and homozygous (inv/inv) (G) mutants. Positions of the beads that entered the node from the right edge (left edge in the panel) were traced for 4 s with 1/3 s interval. Different marks indicate different beads. Most beads go straight to the left edge of the node (right edge in the panels) in wild-type (B) and heterozygous (C) embryos. In transgene-rescued embryos, the rapid leftward flow of beads was often impeded by the local whirl (arrows in [D]), but most of the beads reached the left side within 4 s. In homozygous (inv/inv) mutants, the local whirl severely impedes the leftward movement of the beads, so that most beads cannot reach the midline within 4 s (G). However, there exists a bias to the left. Most beads (>90%) entered the node only from the right side, and they eventually reached the left edge, though it took more than 20 s. (H) A 30 s trace of the same beads in (G). Two beads (red and green) were trapped in a local whirl, but others crossed the midline or reached the left edge within this time period. Note that the time interval of the plot is the same as (B–D). Thus, the shorter distances between adjacent marks in (H) reflect the slower movement of beads. That is, the leftward flow was slower in inv/inv embryos. (E and I) Trajectories of the beads in the node of inv/+ (E) and inv/inv (I) embryos. Four beads were selected from (C) and (H), and their trajectories are indicated, illustrating the laminar and the turbulent nature of the flow in the heterozygous (E) and the homozygous (I) embryos, respectively. Orientations of the nodes in (B–E) and (G–I) are the same and indicated in the panel; A, P, R, and L indicate anterior, posterior, right, and left, respectively. Bar, 20 μm. Video clips of (B)–(D), (G), and (H) are provided as supplementary material on the web at http://www.molecule.org/cgi/content/full/4/4/459. Molecular Cell 1999 4, 459-468DOI: (10.1016/S1097-2765(00)80197-5)

Figure 3 Slow Nodal Flow in inv Mutant Embryos (A) Distribution of the relative positions of the beads 4 s after entry from the right edge of the node. First forty beads that entered from the right edge of the node in the 2 min observation period were used. In inv/inv embryos, the frequency of the beads entrance to the node was lower, which will reflect the slow flow phenotype of these embryos. Therefore, all beads that entered from the right edge in the 2 min observation period were used for these embryos. Most beads reached the left side within 4 s in wild-type or heterozygotes, and more than half crossed the midline within 4 s in transgene-rescued mutants. However, more than half of the beads remained in the right side in homozygotes. (B) Distribution of the time to reach the midline for the beads that entered from the right edge of the node. The same beads that were analyzed in (A) were used. Note that most beads reached the midline within 4–5 s in normal situs embryos, but most beads could not reach the midline within 10 s in situs inversus, inv/inv mutant embryos. Molecular Cell 1999 4, 459-468DOI: (10.1016/S1097-2765(00)80197-5)

Figure 4 Deformation of the Nodes in inv Mutant Embryos Panels show the overall shape of the nodes of one- to two-somite stage embryos of inv mutants, with their anterior ends oriented to left and their left side up. Arrows indicate aberrant cell masses around the node, which was most prominent with inv/inv embryos but was less so with inv/+ or inv/inv V15 embryos. They were rarely found with inv/+ V15 or wild-type (+/+) embryos. These cell masses often deformed the shape of the nodes of inv/inv embryos. The arrowheads and the circles indicate the rising in the anterior edge of the node, which often makes steeper slope in the anterior part of the node than the posterior part. This rising was often found with inv/+ (18/30) and inv/inv (18/24) embryos and was sometimes found with inv/+ V15 (4/7) and inv/inv V15 (4/10) embryos, but was never found with wild-type embryos. Bar, 100 μm. Molecular Cell 1999 4, 459-468DOI: (10.1016/S1097-2765(00)80197-5)

Figure 5 Narrow Nodes in inv/inv Embryo Histograms of the left–right width to anterior–posterior length ratio of the nodes of 8.0 dpc embryos are shown. Arrows indicate the positions of the median values. The nodes of inv/inv embryos were significantly narrower than that of wild-type or heterozygous embryos (p < 0.001 by Kruskal-Wallis test and p < 0.01 by Scheffes test), though the distribution itself showed large overlap between the homozygous and the wild-type embryos. Molecular Cell 1999 4, 459-468DOI: (10.1016/S1097-2765(00)80197-5)

Figure 6 Delay in the Determination of Left Side in iv and inv Mutant Embryos Left side determination was monitored by the expression of the transgene lefty-2 ASE-hsp lacZ. The level of the expression is indicated by the shading. −, no detectable expression; +, the beginning of the expression in lateral plate mesoderm near the node; ++, the expression expanded to anterior and posterior in the lateral plate mesoderm, about two-thirds the length of the trunk; +++, the expression extended to the heart primordium in anterior and to the tail bud in posterior. Number of the embryos observed are shown on the right side of each bars. In wild-type and heterozygous embryos, the expression of the transgene started around the three-somite stage and reached the +++ level at the five-somite stage. However, both in iv/iv and inv/inv mutant embryos, the expression time course delayed by about one somite stage; the expression started around the four-somite stage and reached the +++ level at the six-somite stage. This indicates that these mutations delayed the determination of the left side by one somite stage. Molecular Cell 1999 4, 459-468DOI: (10.1016/S1097-2765(00)80197-5)

Figure 7 Model on the L–R Determination in Wild-Type and Mutant Embryos (A–C) Models to explain the delayed lefty expression by the loss of the nodal flow. See text for discussion. Keys: I, right side determinant or the inhibitor for the lefty expression; R, receptor for the morphogen; A, left side determinant or the activator for the lefty expression. (D) The flow-out model, postulating that the morphogen (A) is secreted in the node and that its receptor (R) exists outside the node. See text for discussion. (E and F) Two flow-in models, postulating that the morphogen (A) is secreted from outside the node and that its receptor (R) exists in the node. This model requires additional assumptions to generate the expected gradient of the morphogen in the wild-type embryo. (E) Delayed activation model, assuming that the morphogen is inactive (illustrated by white color) when secreted and that it is autonomously activated several seconds after secretion (illustrated by color change to red), conveys the left signal. Then, the position where left signal is activated depends on the speed of the nodal flow. With a rapid flow (+/+), the morphogen will reach the left side before autoactivation. With a slow flow (inv/inv), the morphogen will be activated before reaching the midline. With no flow mutant (iv/iv), the morphogen will diffusively enter the node bilaterally and will be activated there. (F) Slow binding model, assuming that the binding velocity of the morphogen is slower compared to the rapid nodal flow in the wild-type embryos. Then, the morphogen cannot bind to its putative receptor in the right side of the node, where the morphogen is transported by the rapid flow. On the left edge, the flow slows down and the morphogen can bind to its receptor there. In inv/inv embryos, the flow was slow and turbulent. Then, the morphogen binds to its receptor (R) on the right side of the node. Molecular Cell 1999 4, 459-468DOI: (10.1016/S1097-2765(00)80197-5)