In teleosts, proper balance and hearing depend on mechanical sensors in the inner ear. These sensors include actin-based microvilli and microtubule-based cilia that extend from the surface of sensory hair cells and attach to biomineralized ‘ear stones’ (or otoliths)1. Otolith number, size and placement are under strict developmental control, but the mechanisms that ensure otolith assembly atop specific cells of the sensory epithelium are unclear. Here we demonstrate that cilia motility is required for normal otolith assembly and localization. Using in vivo video microscopy, we show that motile tether cilia at opposite poles of the otic vesicle create fluid vortices that attract otolith precursor particles, thereby biasing an otherwise random distribution to direct localized otolith seeding on tether cilia. Independent knockdown of subunits for the dynein regulatory complex and outer-arm dynein disrupt cilia motility, leading to defective otolith biogenesis. These results demonstrate a requirement for the dynein regulatory complex in vertebrates and show that cilia-driven flow is a key epigenetic factor in controlling otolith biomineralization.

Cardiac fibrillation, a form of cardiac arrhythmia, is the most common cause of embolic stroke and death associated with heart failure. The molecular mechanisms underlying cardiac fibrillation are largely unknown. Here we report a zebrafish model for cardiac fibrillation. The hearts of zebrafish tremblor (tre) mutants exhibit chaotic movements and fail to develop synchronized contractions. Calcium imaging showed that normal calcium transients are absent in tre cardiomyocytes, and molecular cloning of the tre mutation revealed that the tre locus encodes the zebrafish cardiac-specific sodium-calcium exchanger (NCX) 1, NCX1h. Forced expression of NCX1h or other calcium-handling molecules restored synchronized heartbeats in tre mutant embryos in a dosage-dependent manner, demonstrating the critical role of calcium homeostasis in maintaining embryonic cardiac function. By creating mosaic zebrafish embryos, we showed that sporadic NCX1h-null cells were not sufficient to disrupt normal cardiac function, but clustered wild-type cardiomyocytes contract in unison in tre mutant hearts. These data signify the essential role of calcium homeostasis and NCX1h in establishing rhythmic contraction in the embryonic zebrafish heart. calcium homeostasis ͉ cardiac arrhythmia ͉ heart T he heart is a muscular pump that drives circulation throughout the body. It is of the utmost importance to establish rhythmic and synchronized cardiac contraction early in development to ensure proper growth and survival of the embryo.Calcium plays an essential role in regulating cardiac cycles. As a wave of depolarization passes through the heart, a small amount of calcium is permitted to enter the cardiomyocytes through voltage-dependent L-type calcium channels. This small calcium influx then triggers the release of a larger amount of calcium from the sarcoplasmic reticulum via ryanodine receptors, resulting in an abrupt increase in cytosolic calcium levels and cardiac contraction. Relaxation is accomplished by resequestering of calcium to the sarcoplasmic reticulum by sarcoendoplasmic reticular Ca 2ϩ -ATPase2 (SERCA2) and extrusion of calcium from the cell by NCX1 and plasma membrane Ca 2ϩ -ATPase (PMCA). Abnormal calcium handling caused by altered expression levels or protein activities of NCX1 and SERCA2, or by mutations in ryanodine receptors, have been associated with cardiac diseases, such as heart failure and arrhythmia, and with sudden death in humans and animal models (1-6). In addition, genetic studies in the zebrafish demonstrate that L-type calcium channels and the sodium pump (a modulator of NCX activity) are indispensable for embryonic cardiac function (7,8). These findings underscore the critical roles of calcium in embryonic and adult cardiac physiology.Three NCX genes have been identified in mammals. NCX2 and NCX3 are expressed predominantly in the brain and skeletal muscle, respectively, whereas NCX1 is virtually ubiquitous (9). NCX1 is highly expressed in the heart and is considered to be the primary mechanism for calcium extrusion...

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The colonial ascidian Botryllus schlosseri continuously regenerates entire bodies in an asexual budding process. The germ line of the newly developing bodies is derived from migrating germ cell precursors, but the signals governing this homing process are unknown. Here we show that germ cell precursors can be prospectively isolated based on expression of aldehyde dehydrogenase and integrin alpha-6, and that these cells express germ cell markers such as vasa, pumilio and piwi, as well as sphingosine-1-phosphate receptor. In vitro, sphingosine-1-phosphate (S1P) stimulates migration of germ cells, which depends on integrin alpha-6 activity. In vivo, S1P signalling is essential for homing of germ cells to newly developing bodies. S1P is generated by sphingosine kinase in the developing germ cell niche and degraded by lipid phosphate phosphatase in somatic tissues. These results demonstrate a previously unknown role of the S1P signalling pathway in germ cell migration in the ascidian Botryllus schlosseri.

Leo1 is a component of the Polymerase-Associated Factor 1 (PAF1) complex, an evolutionarily conserved protein complex involved in gene transcription regulation and chromatin remodeling. The role of leo1 in vertebrate embryogenesis has not previously been examined. Here, we report that zebrafish leo1 encodes a nuclear protein that has a similar molecular structure to Leo1 proteins from other species. From a genetic screen, we identified a zebrafish mutant defective in the leo1 gene. The truncated Leo1LA1186 protein lacks a nuclear localization signal and is distributed mostly in the cytoplasm. Phenotypic analysis showed that while the initial patterning of the primitive heart tube is not affected in leo1LA1186 mutant embryos, the differentiation of cardiomyocytes at the atrioventricular boundary is aberrant, suggesting a requirement for Leo1 in cardiac differentiation. In addition, the expression levels of markers for neural crest-derived cells such as crestin, gch2, dct and mitfa, are greatly reduced in leo1LA1186 mutants, indicating a requirement for Leo1 in maintaining the neural crest population. Consistent with this finding, melanocyte and xanthophore populations are severely reduced, craniofacial cartilage is barely detectable, and mbp-positive glial cells are absent in leo1LA1186 mutants after three days of development. Taken together, these results provide the first genetic evidence of the requirement for Leo1 in the development of the heart and neural crest cell populations.