Calcium extrusion is critical for cardiac morphogenesis and rhythm in embryonic zebrafish hearts.

Calcium entry into myocytes drives contraction of the embryonic heart. To prepare for the next contraction, myocytes must extrude calcium from intracellular space via the Na+/Ca2+ exchanger (NCX1) or sequester it into the sarcoplasmic reticulum, via the sarcoplasmic reticulum Ca2+-ATPase2 (SERCA2). In mammals, defective calcium extrusion correlates with increased intracellular calcium levels and may be relevant to heart failure and sarcoplasmic dysfunction in adults. We report here that mutation of the cardiac-specific NCX1 (NCX1h) gene causes embryonic lethal cardiac arrhythmia in zebrafish tremblor (tre) embryos. The tre ventricle is nearly silent, whereas the atrium manifests a variety of arrhythmias including fibrillation. Calcium extrusion defects in tre mutants correlate with severe disruptions in sarcomere assembly, whereas mutations in the L-type calcium channel that abort calcium entry do not produce this phenotype. Knockdown of SERCA2 activity by morpholino-mediated translational inhibition or pharmacological inhibition causes embryonic lethality due to defects in cardiac contractility and morphology but, in contrast to tre mutation, does not produce arrhythmia. Analysis of intracellular calcium levels indicates that homozygous tre embryos develop calcium overload, which may contribute to the degeneration of cardiac function in this mutant. Thus, the inhibition of NCX1h versus SERCA2 activity differentially affects the pathophysiology of rhythm in the developing heart and suggests that relative levels of NCX1 and SERCA2 function are essential for normal development.

Growth and function of the embryonic heart depend upon the cardiac-specific L-type calcium channel alpha1 subunit.

The heart must function from the moment of its embryonic assembly, but the molecular underpinnings of the first heart beat are not known, nor whether function determines form at this early stage. Here, we find by positional cloning that the embryonic lethal island beat (isl) mutation in zebrafish disrupts the alpha1 C L-type calcium channel subunit (C-LTCC). The isl atrium is relatively normal in size, and individual cells contract chaotically, in a pattern resembling atrial fibrillation. The ventricle is completely silent. Unlike another mutation with a silent ventricle, isl fails to acquire the normal number of myocytes. Thus, calcium signaling via C-LTCC can regulate heart growth independently of contraction, and plays distinctive roles in fashioning both form and function of the two developing chambers.

The repertoire of Na,K-ATPase alpha and beta subunit genes expressed in the zebrafish, Danio rerio.

We have identified a cohort of zebrafish expressed sequence tags encoding eight Na,K-ATPase alpha subunits and five beta subunits. Sequence comparisons and phylogenetic analysis indicate that five of the zebrafish alpha subunit genes comprise an alpha1-like gene subfamily and two are orthologs of the mammalian alpha3 subunit gene. The remaining alpha subunit clone is most similar to the mammalian alpha2 subunit. Among the five beta subunit genes, two are orthologs of the mammalian beta1 isoform, one represents a beta2 ortholog, and two are orthologous to the mammalian beta3 subunit. Using zebrafish radiation hybrid and meiotic mapping panels, we determined linkage assignments for each alpha and beta subunit gene. Na,K-ATPase genes are dispersed in the zebrafish genome with the exception of four of the alpha1-like genes, which are tightly clustered on linkage group 1. Comparative mapping studies indicate that most of the zebrafish Na,K-ATPase genes localize to regions of conserved synteny between zebrafish and humans. The expression patterns of Na,K-ATPase alpha and beta subunit genes in zebrafish are quite distinctive. No two alpha or beta subunit genes exhibit the same expression profile. Together, our data imply a very high degree of Na,K-ATPase isoenzyme heterogeneity in zebrafish, with the potential for 40 structurally distinct alpha/beta subunit combinations. Differences in expression patterns of alpha and beta subunits suggest that many of the isoenzymes are also likely to exhibit differences in functional properties within specific cell and tissue types. Our studies form a framework for analyzing structure function relationships for sodium pump isoforms using reverse genetic approaches.

Zebrafish dracula encodes ferrochelatase and its mutation provides a model for erythropoietic protoporphyria.

Exposure to light precipitates the symptoms of several genetic disorders that affect both skin and internal organs. It is presumed that damage to non-cutaneous organs is initiated indirectly by light, but this is difficult to study in mammals. Zebrafish have an essentially transparent periderm for the first days of development. In a previous large-scale genetic screen we isolated a mutation, dracula (drc), which manifested as a light-dependent lysis of red blood cells [1]. We report here that protoporphyrin IX accumulates in the mutant embryos, suggesting a deficiency in the activity of ferrochelatase, the terminal enzyme in the pathway for heme biosynthesis. We find that homozygous drc(m248) mutant embryos have a G-->T transversion at a splice donor site in the ferrochelatase gene, creating a premature stop codon. The mutant phenotype, which shows light-dependent hemolysis and liver disease, is similar to that seen in humans with erythropoietic protoporphyria, a disorder of ferrochelatase.

Centromere-linked microsatellite markers for linkage groups 3, 4, 6, 7, 13, and 20 of zebrafish (Danio rerio).

A large number of interesting mutations affecting development and organogenesis have been identified through genetic screens in zebrafish. Mapping of these mutations to a chromosomal region can be rapidly accomplished using half-tetrad analysis. However, knowledge of centromere-linked markers on every chromosome is essential to this mapping method. Centromeres on all 25 linkage groups have been mapped on the RAPD zebrafish genetic map. However, species specificity and the lack of codominance make RAPD markers less practical for mapping than microsatellite-based markers. On the microsatellite-based genetic map, centromere-linked markers have been identified for 19 linkage groups. No direct evidence has been published linking microsatellite markers to the centromeres of linkage groups 3, 4, 6, 7, 13, and 20. Therefore, we compared the microsatellite-based genetic map with the RAPD map to identify markers most likely linked to the centromeres of these 6 linkage groups. These candidate markers were tested for potential centromere linkage using four panels of half-tetrad embryos derived by early-pressure treatment of eggs from four different female zebrafish. We have identified microsatellite markers for linkage groups 3, 4, 6, 7, 13, and 20 to within 1.7 cM of their centromeres. These markers will greatly facilitate the rapid mapping of mutations in zebrafish by half-tetrad analysis.

gridlock, an HLH gene required for assembly of the aorta in zebrafish.

The first artery and vein of the vertebrate embryo assemble in the trunk by migration and coalescence of angioblasts to form endothelial tubes. The gridlock (grl) mutation in zebrafish selectively perturbs assembly of the artery (the aorta). Here it is shown that grl encodes a basic helix-loop-helix (bHLH) protein belonging to the Hairy/Enhancer of the split family of bHLH proteins. The grl gene is expressed in lateral plate mesoderm before vessel formation, and thereafter in the aorta and not in the vein. These results suggest that the arterial endothelial identity is established even before the onset of blood flow and implicate the grl gene in assignment of vessel-specific cell fate.

A unique bFGF-responsive transcriptional element.

The mitogen-regulated protein/proliferin (mrp/plf) genes encode closely related proteins that stimulate cell proliferation and angiogenesis. Basic fibroblast growth factor (bFGF) increases mrp/plf mRNA and protein production by 3T3 cells. Although the three cloned mrp/plf gene promoters are over 97% identical, only mrp3 is transcriptionally activated by bFGF. A series of truncated mrp3 promoter sequences were tested to determine the minimal promoter sequence necessary for bFGF-responsive transcription. Within the minimal bFGF-responsive mrp3 promoter fragment, a putative FGF-regulatory element (FRE) was identified. Nuclear factors that bind the FRE are present in 3T3 cells. When present upstream of a thymidine kinase basal promoter, the FRE exhibits high transcriptional activity and responds to bFGF. Thus, the FRE is a strong transcriptional element that is regulated by bFGF and that may participate in regulating the mrp3 gene and perhaps other FGF-regulated genes.

Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo.

As part of a large-scale mutagenesis screen of the zebrafish genome, we have identified 58 mutations that affect the formation and function of the cardiovascular system. The cardiovascular system is particularly amenable for screening in the transparent zebrafish embryo because the heart and blood vessels are prominent and their function easily examined. We have classified the mutations affecting the heart into those that affect primarily either morphogenesis or function. Nine mutations clearly disrupt the formation of the heart. cloche deletes the endocardium. In cloche mutants, the myocardial layer forms in the absence of the endocardium but is dysmorphic and exhibits a weak contractility. Two loci, miles apart and bonnie and clyde, play a critical role in the fusion of the bilateral tubular primordia. Three mutations lead to an abnormally large heart and one to the formation of a diminutive, dysmorphic heart. We have found no mutation that deletes the myocardial cells altogether, but one, pandora, appears to eliminate the ventricle selectively. Seven mutations interfere with vascular integrity, as indicated by hemorrhage at particular sites. In terms of cardiac function, one large group exhibits a weak beat. In this group, five loci affect both chambers and seven a specific chamber (the atrium or ventricle). For example, the weak atrium mutation exhibits an atrium that becomes silent but has a normally beating ventricle. Seven mutations affect the rhythm of the heart causing, for example, a slow rate, a fibrillating pattern or an apparent block to conduction. In several other mutants, regurgitation of blood flow from ventricle to atrium is the most prominent abnormality, due either to the absence of valves or to poor coordination between the chambers with regard to the timing of contraction. The mutations identified in this screen point to discrete and critical steps in the formation and function of the heart and vasculature.