Vertebrate embryos generate striking Ca(2+) patterns, which are unique regulators of dynamic developmental events. In the present study, we used zebrafish embryos as a model system to examine the developmental roles of Ca(2+) during gastrulation. We found that gastrula stage embryos maintain a distinct pattern of cytosolic Ca(2+) along the dorsal-ventral axis, with higher Ca(2+) concentrations in the ventral margin and lower Ca(2+) concentrations in the dorsal margin and dorsal forerunner cells. Suppression of the endoplasmic reticulum Ca(2+) pump with 0.5 microM thapsigargin elevates cytosolic Ca(2+) in all embryonic regions and induces a randomization of laterality in the heart and brain. Affected hearts, visualized in living embryos by a subtractive imaging technique, displayed either a reversal or loss of left-right asymmetry. Brain defects include a left-right reversal of pitx2 expression in the dorsal diencephalon and a left-right reversal of the prominent habenular nucleus in the brain. Embryos are sensitive to inhibition of the endoplasmic reticulum Ca(2+) pump during early and mid gastrulation and lose their sensitivity during late gastrulation and early segmentation. Suppression of the endoplasmic reticulum Ca(2+) pump during gastrulation inhibits expression of no tail (ntl) and left-right dynein related (lrdr) in the dorsal forerunner cells and affects development of Kupffer's vesicle, a ciliated organ that generates a counter-clockwise flow of fluid. Previous studies have shown that Ca(2+) plays a role in Kupffer's vesicle function, influencing ciliary motility and translating the vesicle's counter-clockwise flow into asymmetric patterns of gene expression. The present results suggest that Ca(2+) plays an additional role in the formation of Kupffer's vesicle.

Vertebrates develop well defined and conserved left-right (L-R) asymmetries of the heart, gut and brain. Calcium signals play a role in the specification of L-R asymmetry, by translating cilia-dependent fluid flow into asymmetric patterns of gene expression. We aimed to determine the role of early calcium signals on the L-R patterning in zebrafish embryos. Calcium signals were manipulated in zebrafish embryos using thapsigargin, an inhibitor of the endoplasmic reticulum (ER) calcium ATP-ase pump. The embryos were treated with 0.5 μM thapsigargin during early gastrulation (4 – 6 hpf), mid-gastrulation (6 – 8 hpf), late gastrulation (8 –10 hpf) or early somitic stages (10 –12 hpf). The phenotype was analyzed with subtractive imaging, immunolabeling and in situ hybridization (ISH). At 30 hpf, the heart was centralized or reversed in 53% of the early thapsigargin-treated embryos (n=72) vs 5% of the DMSO-treated control embryos (n=75). The embryos were most sensitive to thapsigargin during early and mid-gastrulation with subsequent decrease in heart laterality defects. The incidence of heart laterality defects correlated with decreased or absent no tail expression in the dorsal forerunner cells and disruption of Kupffer’s vesicle formation in 73% of the early treated embryos (n=203), vs 2% of the control embryos (n=198) (p<0.01). Deviation from the normal morphological L-R asymmetry in the habenular nucleus of the diencephalon was found in 54% of the thapsigargin treated embryos (n=17) and in 88% of the embryos with L-looped heart (n=9) vs none control embryos. Analysis of the expression pattern of the “left-sided” marker pitx2 α in the dorsal diencephalon by ISH revealed this to be reversed or bilateral in 56% of the treated embryos (n=97) vs only 10% of the control embryos (p<0.05). In addition 47% of the thapsigargin treated embryo displayed reversed gut-looping (n=49) vs none control embryos. Our data suggest that inhibition of the ER calcium pump by thapsigargin during gastrulation impairs development of the Kupffer’s vesicle and disrupts the concordant heart, brain and gut L-R asymmetry. These results suggest an additional role of calcium in L-R asymmetry determination well before its previously recognized role in sensing fluid flow in the Kupffer’s vesicle.