Hofherr et al., 2018 - The mitochondrial transporter SLC25A25 links ciliary TRPP2 signaling and cellular metabolism. PLoS Biology   16:e2005651 Full text @ PLoS Biol.

Fig. 4

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 (slc25a25bMO; *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.

EXPRESSION / LABELING:
Genes:
Fish:
Knockdown Reagent:
Anatomical Terms:
Stage Range: 14-19 somites to Long-pec

Fig. 7 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

PHENOTYPE:
Fish:
Observed In:
Stage: Long-pec

Fig. S4

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.

Fig. S5

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 (slc25a25bMO; *P = 1.1 × 10−34). (B) Similar to splice-blocking slc25a25bMO, translation-blocking slc25a25bATG 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 ControlMO: 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.

Fig. S6

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 arl13bGFP fish (representative images, n ≥ 20). Scale bars = 10 μm. slc25a25, solute carrier 25 A 25.

PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage: 5-9 somites

Fig. S7

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. pkd2MO: 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). slc25a25bMO: 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) pkd2MO and (H) slc25a25bMO 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; pkd2MO: n = 9; mean velocity = 10.4 μm/s; standard deviation = 2.2; slc25a25bMO: n = 10; mean velocity = 7.9 μm/s; standard deviation = 2.2). Scale bars = 20 μm. For numerical values, see S1 Data.

Fig. S8

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; ControlMO: n = 28; left = 5; symmetric = 12; right = 11; slc25a25bMO: n = 18; left = 10; symmetric = 5; right = 3; in comparison to ControlMO *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.

EXPRESSION / LABELING:
Genes:
Fish:
Knockdown Reagent:
Anatomical Terms:
Stage Range: 5-9 somites to 26+ somites
Acknowledgments:
ZFIN wishes to thank the journal PLoS Biology for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ PLoS Biol.