Manipulated embryos develop normal LRO cilia evaluated by immunofluorescence.

Cilia number, length and anterior-posterior ratio of cilia distribution in WT and fluid extracted embryos – (A–B): Acetylated α-tubulin (in green) immunostaining examples showing LRO labelled cilia on WT and manipulated embryos. A number of 388 cilia in 8 WT embryos and 399 cilia in 8 manipulated embryos were measured in 3D (C). Mean cilia number (D) and A/P ratio (E) are displayed per embryo. Student’s t-test was used to assess differences between groups.

Figure 1—figure supplement 1—source data 1.

Manipulated embryos develop normal LRO cilia evaluated by immunofluorescence.

Cilia number, length and anterior-posterior ratio of cilia distribution in WT and fluid extracted embryos – (A–B): Acetylated α-tubulin (in green) immunostaining examples showing LRO labelled cilia on WT and manipulated embryos. A number of 388 cilia in 8 WT embryos and 399 cilia in 8 manipulated embryos were measured in 3D (C). Mean cilia number (D) and A/P ratio (E) are displayed per embryo. Student’s t-test was used to assess differences between groups.

Figure 1—figure supplement 1—source data 1.

Cilia Beat Frequency and motile/ immotile cilia ratio do not change in manipulated embryos evaluated by live imaging.

(A) Diagram highlighting motile and immotile cilia intercalated in the LRO cells. (B) CBF of the motile beating cilia that could be visualized by bright field live microscopy in the Sham control embryos versus manipulated embryos (C) Total number of motile and immotile cilia located anteriorly and posteriorly between sham controls and embryos without LR defects. (D) Motile to immotile cilia ratio in the anterior and posterior LRO of Sham control embryos versus embryos that later developed without LR defects. Fisher’s exact test was used to assess differences between treatments using pooled data from 6 embryos per treatment. Embryos were manipulated for LRO fluid extracted at 5 ss and were imaged at 6 ss. CBF: cilia beat frequency (relates to Figure 1—video 3).

Figure 1—figure supplement 2—source data 1.

LRO areas from recovering embryos that develop left-right defects are not different from embryos that have a normal development.

(A) LRO areas at 6–8 ss following extraction at 5 ss (B) Quantifications of LRO area of the three different groups (‘Sham’ control, ‘No Defects’ and ‘Defects’ group) in manipulated embryos during LRO lumen area recovery from 6 ss to 8 ss. (C) Area change per somite. Mann-Whitney U test p-value <0.05.

Figure 1—figure supplement 3—source data 1.

Angular velocity during LRO fluid flow recovery. Angular velocity polar plots for 6 ss, 7 ss, and 8 ss for the three different groups (A) 'Sham’ control embryos colour coded in green, (B) ‘No LR Defects’ group of embryos colour coded in blue and (C) 'LR Defects’ group of embryos colour coded in red. LR defects refer to misplacement of heart or liver encountered at the larval stage after fluid extraction was performed at 5 ss; number of tracks refers to the number of particle trajectories identified for the quantifications and respective angular velocity plots. Colour code on polar plots refers to the median angular velocity for pooled embryos. (D–E) Violin plots showing quantifications of angular velocities found for the tracks analysed (D) anterior-posterior and (E) left and right. Dots contained in the violin plots correspond to median values per embryo. A statistical linear mixed effects regression was applied (see results in Table 1).

Tangential velocity component of particle movement in the outer LRO luminal space.

Tangential velocity polar plots for 6 ss, 7 ss, and 8 ss for the three different groups (‘Sham’ control, ‘Defects’ and ‘No Defects’ group) – number of tracks refers to the number of particle trajectories identified for the quantifications and respective tangential velocity plots. Colour code on polar plots refers to the median angular velocity for all pooled embryos. LRO: left-right organizer; ss: somite stage.

Radial velocity components of particle movement in the outer KV luminal space during fluid recovery.

Radial velocity polar plots from the outermost area of the KV (radius > 0.5 for a maximum of 1) are at 6ss, 7ss. and 8ss are represented for the three different groups (‘Sham’ control, ‘Defects’ and ‘No Defects’ group) – number of tracks refers to the number of particle trajectories identified for the quantifications and respective velocity plots. Colors on circular plots refer to the median velocity component for all pooled embryos. Positive radial components indicate predominant flow movement towards the KV surface. ss: somite stage.

Directionality of vector fields changes in embryos that develop LR defects. (A) Diagram representing particle directionality. LRO area sections were delimited based on intervals of 30 degrees. Highlighted are regions (B) from 30 to 60 degrees and (C) 60–90 degrees, that showed significant differences in particle movement between the groups ‘Sham’ control embryos and embryos with ‘LR Defects’. Each density plot represents the pooled tracked trajectory of all moving particles at any given point in time. Respective area region analysed is represented on the top right corner of each plot. To assess differences upon fluid manipulation ‘Sham’ and ‘Defect’ groups were plotted for the 6 ss. Kolmogrov-Smirnov test was used for comparing trajectory distribution between the two groups. Full data can be found in Figure 3—figure supplements 1–6.

Particle movement direction at different regions of the LRO for ‘Sham’ and ‘No Defects’ groups at 6 ss.

Each plot represents the pooled tracked trajectory of a moving particle at even given point in time. Trajectories were plotted from the outermost area of the LRO (radius > 0.5 from a maximum of 1). Kolmogorov-Smirnov test was used from comparing trajectory distribution between the two groups. ss: somite stage. LRO: left-right organizer.

Particle movement direction at different regions of the LRO for ‘Sham’ and ‘No Defects’ groups at 7 ss.

Each plot represents the pooled tracked trajectory of a moving particle at even given point in time. Trajectories were plotted from the outermost area of the KV’s (radius > 0.5 from a maximum of 1). Kolmogrov-Smirnov test was used from comparing trajectory distribution between the two groups. ss: somite stage; LRO: left-right organizer.

Particle movement direction at different regions of the LRO for ‘Sham’ and ‘No Defects’ groups at 8ss.

Each plot represents the pooled tracked trajectory of a moving particle at even given point in time. Trajectories were plotted from the outermost area of the KV’s (radius > 0.5 from a maximum of 1). Kolmogorov-Smirnov test was used from comparing trajectory distribution between the two groups. ss: somite stage. LRO: left-right organizer.

Particle movement direction at different regions of the KV for ‘Sham’ and ‘Defects’ groups at 6 ss.

Each plot represents the pooled tracked trajectory of a moving particle at even given point in time. Trajectories were plotted from the outermost area of the KV’s (radius > 0.5 from a maximum of 1) Kolmogorov-Smirnov test was used from comparing trajectory distribution between the two groups. ss: somite stage. LRO: left-right organizer.

Particle movement direction at different regions of the LRO for ‘Sham’ and ‘Defects’ groups at 7 ss.

Each plot represents the pooled tracked trajectory of a moving particle at even given point in time. Trajectories were plotted from the outermost area of the KV’s (radius >0.5 from a maximum of 1). Kolmogorov-Smirnov test was used from comparing trajectory distribution between the two groups. ss: somite stage. LRO: left-right organizer.

Particle movement direction at different regions of the LRO for ‘Sham’ and ‘Defects’ groups at 8 ss.

Each plot represents the pooled tracked trajectory of a moving particle at even given point in time. Trajectories were plotted from the outermost area of the KV’s (radius >0.5 from a maximum of 1). Kolmogorov-Smirnov test was used from comparing trajectory distribution between the two groups. ss: somite stage. LRO: left-right organizer.

LRO flow dynamics is only affected when fluid dilution alters fluid viscosity (A) Angular velocity polar plots for 6 ss, 7 ss and 8 ss for the three different groups (A) 'Sham’ control, (B) 'DB dilution’ group and (C) 'MC dilution’ group.

Number of tracks refers to the number of particle trajectories identified for the quantifications and respective angular velocity plots. Colour code on polar plots refers to the median angular velocity for all pooled embryos. LRO: left-right organizer. DB: Danieau buffer. MC: methylcellulose.

LRO flow dynamics is only affected when fluid dilution alters fluid viscosity (A) Angular velocity polar plots for 6 ss, 7 ss and 8 ss for the three different groups (A) 'Sham’ control, (B) 'DB dilution’ group and (C) 'MC dilution’ group.

Number of tracks refers to the number of particle trajectories identified for the quantifications and respective angular velocity plots. Colour code on polar plots refers to the median angular velocity for all pooled embryos. LRO: left-right organizer. DB: Danieau buffer. MC: methylcellulose.

LRO fluid content does not affect left-right development.

(A) dand5 mRNA expression pattern in wild-type control embryos, embryos with LRO fluid diluted with DB and embryos with LRO fluid diluted with MC. Embryos were manipulated at 5 ss and fixed at 8 ss for dand5 whole mount embryo in situ hybridization. (B) Heart position (left, central or right sided) was scored at 30 hpf in wild-type control embryos, embryos with LRO fluid diluted with DB and embryos with LRO fluid diluted with MC. ss: somite stage; DB Danieau buffer; MC methylcellulose; hpf: hours post fertilization.

Figure 5—source data 1.

Mixed effects model fits for the distributions of anterior angular velocity to (A) data for sham intervention, (B) data for suction intervention at 5 ss.

Central line is fixed effect, outer pairs of lines are +/-1 and+/-2 standard deviations in normally distribution random effects. ss: somite stage.

Mixed effects model fits for the distributions of anterior angular velocity to (A) data for sham intervention, (B) data for suction intervention at 5 ss.

Central line is fixed effect, outer pairs of lines are +/-1 and+/-2 standard deviations in normally distribution random effects. ss: somite stage.

Anterior angular velocity distributions for all modelled experimental interventions.

(A) sham, (B) 3 ss intervention, (C) 4 ss intervention, (D) 5 ss intervention, (E) 6 ss intervention, (F) 7 ss intervention, (G) 8 ss intervention, (H) both 5 ss and 7 ss intervention. These distributions are combined with stage weightings to fit the normal/defect percentages for each experiment series. ss: somite stage.

3D smFISH and spatial analysis of dand5 mRNA transcripts in the KV.

(A) Time-course 3D analysis of dand5 mRNA transcripts by smFISH in Tg(sox17:GFP) embryos at 3, 5, 6, 7, and 8 somite stage (ss). 3D representations of dand5 mRNA (magenta), nuclei (cyan), and sox17:GFP (green) are shown. The KV surface (green) was used to mask both the dand5-Atto 565 and DAPI channels to remove nuclear staining. Spot detection was used to detect the dand5 cytoplasmic transcripts. Axes are indicated. Scale bar = 15 µm (B) The spot detection algorithm was validated in 8 ss dand5^-/- embryos raised on the Tg(sox17:GFP) background. (C–E) Quantification of differences in the number of dand5 mRNA transcripts detected on the left and right sides of the KV; total number (C), at the anterior (D), and posterior (E) sides of the KV. Gray and magenta represent the left and right sides, respectively. Paired t-tests were used. The bars represent the mean values, and the dots represent individual embryos. Asterisks denote statistical significance: * corresponds to a p-value <0.05, and ** indicates a p-value <0.005.

Figure 7—source data 1.