DEV020735 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

• Supplemental Figure S1 -

  Fig. S1. seahorse mutants have a low level of left-right patterning defects. (A) At 20 somites, the majority of sea mutants have normal left-sided expression of southpaw and lefty1/2 in the lateral plate mesoderm, and lefty1 in the diencephalon (asterisk). southpaw is expressed lateral (marked with arrows) to the darker stained lefty1/2-expressing cells in the lateral plate. (B-G) The majority of sea mutants had correctly looped hearts (B) with the ventricle (white V) looped to the right of the atrium (white A) and correctly placed visceral organs (E) with the liver (white L) on the left and pancreas (white P) on the right. A low level of sea mutants display either situs inversus (complete reversal of organ positioning; C,F) or heterotaxia (random organ positioning; D,G). (H-J) sea mutants also display a low level of asymmetric defects in habenular expression of lov at 4 dpf. Although most mutants have the typical greater level of expression on the left (H), some mutants have reversed (I) or isomeric (J) expression. (A) Dorsal view, anterior towards the top. (B-D) Ventral views, anterior towards the top. (E-G) Dorsal views of the same embryos in B-D, anterior towards the top. (H-J) Dorsal views of the diencephalon, anterior towards the top. In all panels, black R indicates right, black L indicates left.

• Movie 1 -

  Movie 1. Wild-type pronephric tubule cilia movement at 30 hpf in the anterior tubule. The broken lines outline the lumen of the pronephric tubule; posterior towards the top. At this time point, cilia are not bundled and do not display a coordinated movement (see Movie 5).

• Movie 2 -

  Movie 2. Wild-type pronephric tubule cilia movement at 30 hpf in the posterior tubule. The broken lines outline the lumen of the pronephric tubule; posterior towards the top. At this time point, cilia are not bundled and do not display a coordinated movement (see Movie 5).

• Movie 3 -

  Movie 3. Wild-type cloaca cilia movement at 48 hpf. The broken lines outline the lumen of the cloaca; anterior towards the bottom right.

• Movie 4 -

  Movie 4. Wild-type cilia movement in the floor plate and neural tube at 48 hpf. The broken line indicates the position of the floor plate; posterior towards the top.

• Movie 5 -

  Movie 5. Wild-type cilia movement in the pronephric tubule at 3 dpf. The broken lines outline the lumen of the pronephric tubule; posterior towards the left. At 3 dpf, cilia in the medial tubules bundle and begin to move in a coordinated fashion.

• Movie 6 -

  Movie 6. Lack of cilia movement in the anterior pronephric tubule of sea^fa20r at 30 hpf. The broken lines outline the lumen of the pronephric tubule; white arrows indicate immotile cilia; posterior towards the top. In this movie, we could not detect any cilia movement in the tubules. In other mutant embryos, occasional flickering of cilia could be detected, but this movement was always slower than observed in wild type.

• Movie 7 -

  Movie 7. Lack of cilia movement in the posterior pronephric tubule of sea^fa20r at 30 hpf. The dotted lines outline the lumen of the pronephric tubule; white arrows point to immotile cilia; posterior to the top. In this movie, we could not detect any cilia movement in the tubules. In other mutant embryos, occasional flickering of cilia could be detected, but this movement was always slower than observed in wildtype.

• Movie 8 -

  Movie 8. Cilia movement in the cloaca of sea^fa20r at 48 hpf. The dotted lines outline the lumen of the cloaca; posterior is bottom left. In this embryo, we detect movement of some cilia in the cloaca, but the number of cilia moving is consistently reduced compared to what we observe in wildtype (Movie 2). In other embryos, no cilia movement can be detected.

• Movie 9 -

  Movie 9. Cilia movement in the floorplate and neural tube of sea^fa20r at 48hpf. The dotted line denotes the position of the floorplate; posterior to the top. Black arrows point to cilia that are motile in this embryo. In mutants, we typically see a range of defects from completely immotile to slow and disorganized cilia movement.

• Movie 10 -

  Movie 10. Cilia movement in the pronephric tubule of sea^fa20r at 3 dpf. The broken lines outline the lumen of the pronephric tubule; posterior towards the left. Black arrows indicate two multiciliated cells with immotile cilia. In this embryo, we did not observe cilia movement. In other embryos, we could detect occasional cilia movement, but the number of motile cilia, and their speed, was consistently reduced compared with wild type.

• Movie 11 -

  Movie 11. Cilia driven counter-clockwise flow in a wild-type embryo. DIC imaging of beads injected into Kupffer’s vesicle to visualize flow. This embryo displays normal counter-clockwise flow.

• Movie 12 -

  Movie 12. Cilia driven counter-clockwise flow in sea^fa20r embryo at the six-somite stage. DIC imaging of beads injected into Kupffer’s vesicle to visualize flow. This embryo displays normal counter-clockwise flow indistinguishable from wild type.

• Movie 13 -

  Movie 13. Cilia driven counter-clockwise flow in sea^tg238a embryo at the six-somite stage. DIC imaging of beads injected into Kupffer’s vesicle to visualize flow. This embryo displays a lack of fluid flow.