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. 2023 Jul 21;12:e83861. doi: 10.7554/eLife.83861

Figure 1. Fluid extractions uncover a sensitive one-hour interval.

(A) Schematic representation on the micromanipulation setup developed for the zebrafish LRO fluid extraction throughout development. (B) Evaluation of organ patterning after 48 hr. Heart and liver position were scored for each embryo manipulated in the respective developing stage from 3 to 12 ss, situs inversus in zebrafish means both organs are localized on the right side and heterotaxia means that at least one organ is wrongly localized. (C) dand5 expression pattern at 8 ss after single intervention at 5 ss and double intervention at 5 and 7 ss for LRO liquid extraction. LRO. left-right organizer, ss. somite stage.

Figure 1—source data 1. Characterization of heart and gut situs in zebrafish larvae.

Figure 1.

Figure 1—figure supplement 1. Manipulated embryos develop normal LRO cilia evaluated by immunofluorescence.

Figure 1—figure supplement 1.

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 number for WT and manipulated embryos.
Figure 1—figure supplement 2. Cilia Beat Frequency and motile/ immotile cilia ratio do not change in manipulated embryos evaluated by live imaging.

Figure 1—figure supplement 2.

(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. Live imaging cilia motility status and localization.
Figure 1—figure supplement 3. LRO areas from recovering embryos that develop left-right defects are not different from embryos that have a normal development.

Figure 1—figure supplement 3.

(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. Left right organizer area quantification.
Figure 1—video 1. Close-up of fluid extraction technique employed in a 8 ss embryo.
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Sequence of processed bright-field images acquired at ×10 magnification, showing a micropipette extracting the liquid from the KV lumen (related to Figure 1A–B).
Figure 1—video 2. Close-up of fluid extraction technique employed in a 5 ss embryo.
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Sequence of processed bright-field images acquired at ×10 magnification, showing a micropipette extracting the liquid from the KV lumen (related to Figure 1A–B).
Figure 1—video 3. Evaluation of motile and immotile cilia distribution at 6 somite stage embryos.
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Injection of arl13b at one-cell stage allows for visualization of motile versus immotile cilia. Anterior-posterior distribution was scored in 15 embryos that were later grouped as sham controls (normal situs), embryos with LR defects and without LR defects (related to Supplementary file 1).