Homozygous diploid zebra fish have been produced on a large scale by the application of simple physical treatments. Clones of homozygous fish have been produced from individual homozygotes. These clones and associated genetic methods will facilitate genetic analyses of this vertebrate.

Mutations of the gene (TNNT2) encoding the thin-filament contractile protein cardiac troponin T are responsible for 15% of all cases of familial hypertrophic cardiomyopathy, the leading cause of sudden death in young athletes 1,2 . Mutant proteins are thought to act through a dominant-negative mode that impairs function of heart muscle 3 . TNNT2 mutations can also lead to dilated cardiomyopathy, a leading cause of heart failure 4 . Despite the importance of cardiac troponin T in human disease, its loss-of-function phenotype has not been described. We show that the zebrafish silent heart (sih) mutation affects the gene tnnt2. We characterize two mutated alleles of sih that severely reduce tnnt2 expression: one affects mRNA splicing, and the other affects gene transcription. Tnnt2, together with α-tropomyosin (Tpma) and cardiac troponins C and I (Tnni3), forms a calcium-sensitive regulatory complex within sarcomeres 5 . Unexpectedly, in addition to loss of Tnnt2 expression in sih mutant hearts, we observed a significant reduction in Tpma and Tnni3, and consequently, severe sarcomere defects. This interdependence of thin-filament protein expression led us to postulate that some mutations in tnnt2 may trigger misregulation of thin-filament protein expression, resulting in sarcomere loss and myocyte disarray, the life-threatening hallmarks of TNNT2 mutations in mice and humans 6,7 .Forward genetics in zebrafish has led to the identification of several mutations affecting cardiac contractility 8,9 . The most severe and heart-specific of these is sih, which causes a non-contractile heart phenotype ( Fig. 1). Skeletal and smooth-muscle function remain intact in sih mutant embryos, as evidenced by their ability to hatch, swim and show gut peristalsis. One γ-ray-induced allele (sih b109 ) and one chemically induced allele (sih tc300b ) exist 8 . Both of these mutated alleles are fully penetrant and recessive lethal, and sih b109 and sih tc300b mutant embryos are phenotypically indistinguishable. As sih embryos age, pericardial edema develops, the endocardium peels away from the myocardium and the embryos die around seven days post fertilization. Until that time, embryos survive on diffused oxygen and are not dependent on circulating blood 10 .To determine whether the sih phenotype is due to defects in cellular excitation or excitation-contraction coupling, we devised a new assay using the fluorescent calcium indicator Ca 2+ green. In wildtype hearts, a wave of fluorescence representing Ca 2+ influx into cardiomyocytes precedes the contractile wave and progresses from the venous to the arterial end (see Web Movie A and Web Note A online). In mutant hearts, we also observed regular waves of fluorescence, but these were not followed by contraction (Fig. 2a-d). On the basis of this assay, cellular excitation seemed to be intact in mutant cardiomyocytes, indicating that the absence of contractility results from abnormalities downstream of calcium influx.The alignment of thick and thin filaments into sarcomeres creates a highly...

Mutations in the zebrafish knypek locus impair gastrulation movements of convergent extension that narrow embryonic body and elongate it from head to tail. We demonstrate that knypek regulates cellular movements but not cell fate specification. Convergent extension movement defects in knypek are associated with abnormal cell polarity, as mutant cells fail to elongate and align medio-laterally. Positional cloning reveals that knypek encodes a member of the glypican family of heparan sulfate proteoglycans. Double mutant and overexpression analyses show that Knypek potentiates Wnt11 signaling, mediating convergent extension. These studies provide experimental and genetic evidence that glypican Knypek acts during vertebrate gastrulation as a positive modulator of noncanonical Wnt signaling to establish polarized cell behaviors underlying convergent extension movements.

Adult zebrafish stripes are formed from stripes of gold iridophores alternating with stripes of black melanocytes lying beneath silver stripes. Analysis of defects in pigment pattern development caused by sparse (spa, rose (ros), and leopard (leo) single and double mutant combinations suggests that spa+ and ros+ functions are required for development of separate populations of pigment cells in the adult and that leo+ functions to control assembly of melanocytes into stripes. Thus, between 2 and 3 weeks of zebrafish development, spa-dependent melanocytes differentiate throughout the flank, followed by leo-dependent assembly of these cells into stripes. Beginning at 3 weeks of development, a distinct ros-dependent population of melanocytes differentiates in the stripe. Both early and late differentiating melanocytes then affect the formation of the silver stripes, ensuring registration of melanocyte and iridophore stripes.

The floor plate is a set of epithelial cells present in the ventral midline of the neural tube in vertebrates that seems to have an important role in the developmental patterning of central nervous system fibre pathways, and arrangements of specific neurons. The floor plate arises from dorsal ectodermal cells closely associated with the mesoderm that forms notochord, and it may depend on interactions from the notochord for its specification. To learn the nature of these interactions we have analysed mutations in zebrafish (Brachydanio rerio). We report here that in wild-type embryos the floor plate develops as a simply organized single cell row, but that its development fails in embryos bearing the newly discovered zygotic lethal 'cyclops' mutation, cyc-1(b16). Mosaic analysis establishes that cyc-1 blocks floor plate development autonomously and reveals the presence of homeogenetic induction between floor plate cells.