Calcium imaging. (A) The fluorescent signals of calcium green dextran are detected in 2-day-old wild-type heart (Left) and tre mutant heart (Right). (B–D) High-speed two-dimensional confocal imaging shows transients of elevated calcium levels in the wild-type heart (B) but only unsynchronized calcium fluctuations in the tre mutant (C) and NCX1h morphant heart (D). Data were collected at 30 frames per sec. The relative fluorescence intensities (y axis) from consecutive frames of images (x axis) are plotted in graphs, where the maximum fluorescence intensity of each data set is considered to be 100%.

The tre locus encodes NCX1h. (A) Schematic diagram of NCX1h protein.AGtoT transversion at codon 489 is detected in tre^tc318, which creates a premature stop codon and MseI polymorphism. (B) MseI restriction fragment-length polymorphism in wild type, tre heterozygotes, and tre homozygotes. (C) Na⁺ gradient-dependent uptake assay. HEK293 cells transfected with zebrafish NCX1h (NCX1h) showed significant ⁴⁵Ca²⁺ uptake in the presence of an outwardly directed Na⁺ gradient. ⁴⁵Ca²⁺ uptake in cells transfected with tre^tc318 (tc318) remained at basal levels similar to the vector transfected cells (control). The gray bars represent uptake activities in the absence of a Na⁺ gradient, and the white bars represent uptake in the presence of an outwardly directed Na⁺ gradient.

Zebrafish NCX1 homologues. (A) Phylogeny of NCX homologues in rat, mouse, canine, trout, and zebrafish. The tree was calculated in clustalx by using the neighbor-joining method. Numbers at branches are bootstrap values. The unit of the scale represents amino acid substitutions per site. (B) Both NCX1n and NCX1h share high identity to other NCX1 proteins throughout the entire peptide. (C–E) In situ hybridization analysis showed that NCX1n is expressed throughout the zebrafish embryo at 16S (C) and becomes more strongly expressed in the brain and the neural tube at 24 hpf (D). By 48 hpf, NCX1n expression is restricted to the brain, the neural tube, and the fin bud, but not in the heart (E). Inset in E shows the ventral view focusing on the heart. The arrow points to the heart. (F–H) NCX1h is a cardiac-specific gene. NCX1h is expressed in cardiac precursors at 16S (F). At 24 hpf, the transcripts of NCX1h are detected throughout the primitive heart tube (G). By 48 hpf, NCX1h transcripts are found prominently in the myocardium of the atrium and ventricle and are barely detectable in the outflow tract (H). a, atrium; v, ventricle; o, outflow tract. Arrows point to the heart.

Loss of function phenotypes of NCX1h. The NCX1h defect is cardiac-specific. (A–E) In situ hybridization with pax2 (A and B) and flk1 (C and D) probes show that the development of the brain, kidney, and vasculature are not affected in tre, and the overall morphologies of tre (Lower) is similar to wild-type embryos (Upper) at 24 h pf (E). The arrows point to the midbrain–hindbrain boundary, and the arrowheads point to the developing pronephric duct in A and B.(F and G) Histological sections showed that, whereas a well patterned and dilated atrium and ventricle were observed in the wild-type heart (F), the tre heart is small, and the posterior end of the atrium collapsed (G). (H) The expression level of NCX1h is prominent in tre mutant heart at 48 hpf. (I) The expression of SERCA2 is abundant in the ventricle and present at lower levels in the atrium of 2-day-old wild-type embryos. (J) In 2-day-old tre mutant hearts, the atrial expression of SERCA2 expression is slightly elevated. The cardiac expression patterns of amhc and vmhc are restricted to their respective chambers in both wild-type (K and M) and tre (L and N) embryos. The expression of versican extends to the atrium and ventricle in tre mutants (P), whereas the expression of this gene is restricted to the boundary of atrium and ventricle in wild-type embryos after 2 days of development (O). Lines mark the boundary of atrium and ventricle. a, atrium; v, ventricle.