slc25a25b is required for left–right organ patterning in zebrafish.

(A,B) As a measure for left–right asymmetry, heart looping of slc25a25b-morphant fish was visualized by in situ hybridization for cmlc2. (C) Knockdown of slc25a25b caused randomization of heart looping (slc25a25b^MO; *P = 1.1 × 10⁻³⁴). This phenotype was rescued by injection (+) of slc25a25b mRNA in a concentration-dependent fashion (+slc25a25b). (D-F) southpaw (nodal) expression in 15-somite stage slc25a25b-morphant zebrafish embryos was visualized by in situ hybridization. (G) In contrast to control fish, in which southpaw expression was largely restricted to the left side, slc25a25b-morphant fish showed randomized southpaw expression (*P = 0.00008). Numbers of embryos are indicated above bars. L = left; S = symmetric; R = right. This denomination for heart looping is equivalent to wt d-loop, symmetric no-loop, and reversed, sinistral s-loop [33]. For numerical values, see S1 Data. cmlc2, cardiac myosin light chain 2; hpf, hours post fertilization; wt, wild type.

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Loss of pkd2 expression in zebrafish caused randomization of left–right patterning.

(A) In situ hybridization of pkd2 mRNA in wild-type zebrafish 24 (B) and 48 hpf. (C,D) Left–right asymmetry was visualized by in situ hybridization for cmlc2 to evaluate heart looping. Knockdown of pkd2 expression caused a randomization of heart looping [25]. cmlc2, cardiac myosin light chain 2; hpf, hours post fertilization.

Loss of slc25a25b expression in zebrafish caused randomization of left–right patterning.

(A) SCaMC has 3 homologs in vertebrates SLC25A23, SLC25A24, and SLC25A25. In zebrafish, these proteins are encoded by slc25a23a, slc25a23b, slc25a24, slc25a25a, and slc25a25b. MO-induced knockdown of slc25a25b caused randomized heart looping in zebrafish (slc25a25b^MO; *P = 1.1 × 10⁻³⁴). (B) Similar to splice-blocking slc25a25b^MO, translation-blocking slc25a25b^ATG MOs induced randomization of heart looping in zebrafish embryos (*P = 3.2 × 10⁻¹³). Similar to D. melanogaster (Fig 2E), this phenotype was rescued by injection (+) of human SLC25A25 mRNA in a concentration-dependent fashion (+SLC25A25). (C) In situ hybridization of slc25a25b mRNA in wild-type zebrafish 24 hpf, (D) 48 hpf, and (E) at 10-somite stage. (F) Lateral pancreas placement—visualized by in situ hybridization of preproinsulin (ins)—was altered in slc25a25b morphants, highlighting a general heterotaxy defect 52 hpf (n = 28; left = 10; center = 14; right = 4; in comparison to Control^MO: n = 24; left = 2; center = 10; right = 12; *P = 0.002). Numbers of embryos are indicated above bars. L = left; S = symmetric; R = right. For numerical values, see S1 Data. hpf, hours post fertilization; MO, Morpholino-oligonucleotide; SLC25A25, solute carrier 25 A 25.

Kupffer’s vesicle morphology in control and slc25a25-morphant fish.

Number and overall morphology of cilia in zebrafish Kupffer’s vesicle appeared normal in (A) control and (B) slc25a25b-morphant embryos, as visualized in transgenic arl13b^GFP fish (representative images, n ≥ 20). Scale bars = 10 μm. slc25a25, solute carrier 25 A 25.

Structure and motility of cilia in zebrafish Kupffer’s vesicle appeared normal at the 8-somite stage.

(A) Still image of S2 Movie from Kupffer’s vesicle of pkd2-morphant embryos shows cilia in 1 focal plane. It has been shown previously that knockdown of pkd2 does not affect cilia number and motility [25]. (B) slc25a25b-morphant Kupffer’s vesicle cilia resembled pkd2-morphant cilia in (C) number, (D) beating, and (E) length. Numbers of cilia are indicated in bars. pkd2^MO: n = 20; mean number of cilia / Kupffer’s vesicle = 25.05 (standard deviation = 8.69); percentage of beating cilia = 44.525 (standard deviation = 12.55); average length of cilia = 5.05 μm (standard deviation = 1.33). slc25a25b^MO: n = 21; mean number of cilia / Kupffer’s vesicle = 17.32 (standard deviation = 8.2); percentage of beating cilia = 40.154 (standard deviation = 17.5); average length of cilia = 5.77 μm (standard deviation = 1.61). Similar to (F) wild type, (G) pkd2^MO and (H) slc25a25b^MO fish generate effective directional flow in the Kupffer’s vesicle as visualized by particle tracking at the 8-somite stage [86,87]. No significant differences were observed in flow velocities (wild type: n = 6; mean velocity = 10.2 μm/s; standard deviation = 2.4; pkd2^MO: n = 9; mean velocity = 10.4 μm/s; standard deviation = 2.2; slc25a25b^MO: n = 10; mean velocity = 7.9 μm/s; standard deviation = 2.2). Scale bars = 20 μm. For numerical values, see S1 Data.

slc25a25b acts upstream of the southpaw (nodal) cascade.

(A-C) southpaw expression in pkd2-morphant zebrafish embryos. Loss of pkd2 caused left–right randomization of southpaw expression. southpaw mRNA in 15-somite stage zebrafish embryos was visualized by in situ hybridization. (D) Comparison of southpaw expression patterns in control and pkd2-morphant (*P = 0.0005) zebrafish. (E-G) Expression of dand5 in 6-somite stage slc25a25b-morphant zebrafish embryos. Asymmetry of dand5 expression was impaired in 6-somite slc25a25b-morphants (wild type: n = 27; left = 4; symmetric = 11; right = 12; Control^MO: n = 28; left = 5; symmetric = 12; right = 11; slc25a25b^MO: n = 18; left = 10; symmetric = 5; right = 3; in comparison to Control^MO *P = 0.03) as well as in (H) 8-somite slc25a25b-morphants (*P = 0.007). (I,J) lefty2 expression in slc25a25b-morphant zebrafish embryos 22 hpf. (K) lefty2 expression was randomized in pkd2- and slc25a25b-morphant zebrafish (*P = 2.2 × 10⁻¹² and *P = 3.4 × 10⁻⁸, respectively). Numbers of embryos are indicated above bars. L = left; S = symmetric; R = right. For numerical values, see S1 Data. hpf, hours post fertilization.