Abstract 936: Inhibition of the Endoplasmic Reticulum Calcium ATP-ase Affects the Early Steps of Left-Right Patterning in Zebrafish Embryos.
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 pitx2α 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.