1. Volume 29, Issue 6, Pages 716-728 (June 2014)
Left-Right Organizer Flow Dynamics: How Much Cilia Activity Reliably Yields Laterality? 
Pedro Sampaio, Rita R. Ferreira, Adán Guerrero, Petra Pintado, Bárbara Tavares, Joana Amaro, Andrew A. Smith, Thomas Montenegro-Johnson, David J. Smith, Susana S. Lopes 
Developmental Cell 
Volume 29, Issue 6, Pages (June 2014)
DOI: /j.devcel
Copyright © 2014 Elsevier Inc. Terms and Conditions

2. Figure 1 Genetically Generating a Range of Fluid Flow Speeds
(A) Cartoon representing a Kupffer’s vesicle in 3D; light blue denotes central region, dark blue illustrates the peripheral region, and the arrow shows how we scanned the KV for native particles (represented in red) according to the dorsal-ventral axis.
(B–E) Representative KV flow map (B) for a WT embryo, (C and D) for two different dld−/− mutant embryos, and (E) for a MO-dnah7-injected embryo displaying what seems to be Brownian motion (arrows). Left is to the left and anterior to the top throughout.
(F) Zebrafish embryo with 14 hpf showing expression of dnah7 mRNA in the KV.
(G) Expression of dnah7 mRNA in the KV amplified region.
(H and I) Immunofluorescence with anti-acetylated α-tubulin to visualize the presence of KV cilia in a dnah7-MO-injected embryo that showed (H) complete absence of cilia motility during high-speed videomicroscopy and (I) a WT control embryo.
(J) Kymograph showing an example of one motile cilium from a dnah7 knockdown embryo showing a consistent beat frequency over 2 s filmed with 500 fps.
(K) Kymograph of a cilium from a dnah7 knockdown embryo that shows no active motility over 2 s at 500 fps.
(L–N) Gut laterality at 50 hpf filmed from the ventral side after dissecting the yolk. z projection showing the liver, pancreas, and intestinal bulb of a (L) WT embryo tg(sox17:GFP)s870, (M) dld−/− mutant in the same genetic background and developmental stage showing a gut without lateralization, and (N) another dld−/− mutant showing reversed organ position. Li, liver; Pa, pancreas; IB, intestinal bulb.
(O) Box plot of flow speed in WT, dld−/− embryos, and MO-dnah7-injected embryos (∗p < 0.05, Wilcoxon test). Left, central, and right conditions refer to liver situs. The total number of tracks (nt) and embryos (ne) followed with native particles for each condition was as follows: for WT: nt = 847, ne = 7; for dld−/− mutants with left liver: nt = 7,877, ne = 21; for dld−/− mutants with centralized guts: nt = 1,458, ne = 2; for dld−/− mutants with right liver: nt = 858, ne = 3; for dnah7 knockdown embryos with left liver: nt = 1,105, ne = 3; for dnah7 knockdown embryos with central liver: nt = 505, ne = 1; for dnah7 knockdown embryos with right liver: nt = 1,860, ne = 4; and for dnah7 mismatch control-MO: nt = 1,700, ne = 5.
Notched box plots display a “notch” or narrowing of the box around the median. Box plot whiskers represent the minimum and maximum of all of the data.
See also Movie S1.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 2 Experimental Fluid Flow Measurements
(A, D, G, and J) Probability of finding and tracking native particles within the KV represented as rose diagrams. Data obtained from pooled embryos with 14 hpf of each experimental condition: in (A), for WT, number of tracks followed (nt) = 675, number of embryos (ne) = 7; in (D), for dld−/− mutants with left liver, nt = 4,742, ne = 21; in (G), for dld−/− mutants with central guts, nt = 895, ne = 2; and in (J), for dld−/− mutants with right liver, nt = 723, ne = 3. Rayleigh tests were performed considering a null hypothesis of a bias distribution toward a given KV quadrant, and p values are indicated as blue numbers. Note that only particles moving at a distance of r > 0.5 from the center of the KVs were considered (r is the normalized distance from the center (r = 0) to the wall (r = 1) of the KV). A, anterior; P, posterior; R, right; L, left.
(B, E, H, K, and N) Box plots for instantaneous flow speed measured at different locations of the KVs for pooled embryos in each experimental condition: in (B), for WT, nt = 847, ne = 7; in (E), for dld−/− mutants with left liver, nt = 7,877, ne = 21; in (H), for dld−/− mutants with central guts, nt = 1,458, ne = 2; in (K), for dld−/− mutants with right liver, nt = 858, ne = 3; in (N), for dnah7 morphants with situs inversus, nt = 1,297, ne = 2.
(C, F, I, L, M, and O) Heat maps of flow speed showing detailed regions within each KV based on the same data set used to generate the box plots of (B), (E), (H), (K), and (N). (M) Heat map for a dnah7 morphant without any motile cilia. The pseudocolor scale represents flow speed in μms −1, where red represents high speed versus low speed in blue.
Notched box plots display a “notch” or narrowing of the box around the median. Box plot whiskers represent the minimum and maximum of all of the data. Means are represented as small circles. ∗p< 0.05, Wilcoxon test.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

4. Figure 3 Characterization of the WT and dld−/− KV Cilia Populations
(A) Localization of the KV (squared region) in the body of the zebrafish embryo at 14 hpf.
(B) Snapshot image of a KV of a live embryo filmed from the dorsal side.
(C) Snapshot image of a KV beating cilium in a live embryo after image improved contrast for posterior kymograph analysis.
(D) KV of a live embryo at 14 hpf, showing the KV cells labeled with the transgene sox17:GFP; cilia are also labeled with arl13b-GFP so that immotile cilia are bright and sharp (arrow), whereas motile cilia appear as cones with a blurred GFP label caused by the ciliary movement (arrowhead).
(E) Kymograph of a cilium presenting one fundamental frequency.
(F) Time series over 2 s for the same beating cilium.
(G) Power spectrum analysis using FFT for the same cilium, showing that it has only one fundamental frequency of 31.5 Hz.
(H) Kymograph of a cilium with two major frequencies, which we called a wobbling cilium.
(I) Time series for the same beating cilium (2 s).
(J) Power spectrum analysis using FFT for the wobbling cilium in (H), showing that it has two fundamental frequencies (highlighted in red): one at 7 Hz and another at 30 Hz (the lower frequency peak is an FFT artifact equal to the background noise).
(K and L) Quantification of the different types of KV cilia according to motility and to CBF in (K) WT embryos, ne = 16, nc = 80, and in (L) dld−/− mutants; ne = 26, nc = 77. The class “other” includes all cilia that had a random beating pattern with no specific frequency peak after FFT analysis.
KV Kupffer’s vesicle; CBF cilia beat frequency; ne number of embryos, nc number of cilia.
See also Figure S1 and Movie S2.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

5. Figure 4 Generating a Detailed Computational Mesh of KV
(A and B) The beating patterns of (A) single frequency cilia and (B) wobbling cilia showing the locus of the cilium tip (red). All KV diagrams are as viewed from the dorsal roof, with anterior, right, posterior, and left corresponding to north, east, south, and west.
(C) Random cilia placement following the distribution of Kreiling et al. (2007). Dorsal cilia are marked by circles, and ventral cilia are indicated by plus symbols. Red markers denote single frequency cilia, green markers denote wobbling cilia, and blue markers denote immotile cilia.
(D and E) The computational mesh has been split into the dorsal and ventral halves. (D) shows the dorsal roof, which has been made semitransparent to allow the cilia, the locations of which correspond to the circles in (C), to be viewed. (E) shows the ventral floor, with cilia locations corresponding to the plus symbols in (C).
(F) The time-averaged fluid flow generated in the coronal plane of a model dld−/− embryo with 36 cilia, looking toward the anterior-dorsal corner, where the number density of cilia is highest. The computational mesh is also shown, with the posterior-dorsal sector removed for visualization.
See also Supplemental Experimental Procedures.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

6. Figure 5 In Silico Experiments
From individual cilium kinematics to cluster distribution and flow fields. These five simulations, among a total of 40, were the ones that better matched the experimental data of Figure 2.
(A, D, G, J, and M) Seeding of different types of cilia in the KV mesh. Filled dots represent cilia on the dorsal side, crosses label cilia on the ventral side of the KV, red represents motile cilia with single CBFs, green represents motile cilia with wobbling behaviors, and blue represents immotile cilia.
(B, E, H, K, and N) Simulated flow speed in the different halves of the KV is plotted exactly in the same way as in Figure 2. Notched boxplots display a “notch” or narrowing of the box around the median. Boxplot whiskers represent the minimum and maximum of all of the data. Means are represented as small circles.
(C, F, I, L, and O) Flow forces produced by simulated cilia are represented by vectors. Scale is 2 μms −1 for WT and 1 μms −1 for dld−/−.
See also Figure S2.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 6 Correlation between Fluid Flow and Early L-R Markers
(A–C) WT embryo with strong left-sided flow reported by flow heat map in (A) and by the quantification of flow in each KV half in (B) showing an asymmetric charon expression on the right side of the KV in (C).
(D–L) Embryos injected with dnah7-MO. (D) through (F) show embryo with very weak flow as reported by the flow heat map in (D) and the quantification of flow in each KV half in (E) showing a weak symmetric charon expression in (F). (G) through (I) show embryo with strong homogeneous flow as reported by the flow heat map in (G) and the quantification of flow in each KV half in (H) showing a strong symmetric charon expression (I). (J) through (L) show embryo with weak flow showing local elevated right-sided flow as reported by the flow heat map in (J) and the quantification of flow in each KV half in (K) showing a slight degradation of charon expression on the right side of the KV in (L).
(M–O) WT embryo with strong left-sided anterior flow reported by flow heat map in (M) and by the quantification of flow in each KV half in (N) showing an asymmetric spaw expression on the left side of the LPM in (O).
(P–R) WT embryo with no biased L-R flow as reported by flow heat map in (P) and by the quantification of flow in each KV half in (Q) showing absent spaw expression on the LPM in (R). N, number of particle tracks measured in each embryo. Boxplot whiskers represent the minimum and maximum of all of the data. Means are represented as small circles. ∗p< 0.05, Wilcoxon test.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions

8. Figure 7 Model for the Biological Significance of KV Flow
(A, F, K, and P) When ciliated cells positioned both anteriorly and on the left side of KV are stimulated by flow (A) that is greater at these locations, then charon expression (blue cells) will be stronger on the right side of the KV because charon expression is degraded by local strong flow (F). Consequently, spaw expression will be on the left LPM (K), generating situs solitus as the most likely outcome (P).
(B, G, L, and Q) When the flow is faster on the right side and anterior (B), then the reverse situation of gene expression occurs, as shown in (G) and (L) and situs inversus will predominate (Q).
(C, H, M, and R) In the case where flow is stimulating both KV left and right sides (C) with unequal forces, then charon expression will be also unequal (H), which will be reflected in uneven bilateral spaw expression in the LPM (M) and heterotaxia may arise (R) depending on the anterior and posterior range of spaw expression domain.
(D, I, and N) When the flow is homogeneous, stimulating left and right KV sides in the same way (D), then charon is symmetric (I), and lack of spaw expression may occur (N) with the appearance of situs solitus, as shown in (P); situs inversus, as shown in (Q); or heterotaxia, as shown in (R), in a randomized manner.
(E, J, and O) Last, when there is no flow due to complete absence of motile cilia (E), then charon is also symmetric (J) but slightly weaker in our study, likely allowing some bilateral spaw expression in the LPM (O). Again lack of laterality may result in randomization between situs solitus, as shown in (P), situs inversus, as shown in (Q), or any form of heterotaxia, as shown in (R).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions