Imaging early embryonic calcium activity with GCaMP6s transgenic zebrafish.
Intracellular Ca2+signaling regulates cellular activities during embryogenesis and in adult organisms. We generated stable Tg[βactin2:GCaMP6s]stl351and Tg[ubi:GCaMP6s]stl352transgenic lines that combine the ubiquitously-expressed Ca2+indicator GCaMP6s with the transparent characteristics of zebrafish embryos to achieve superior in vivo Ca2+imaging. Using the Tg[βactin2:GCaMP6s]stl351line featuring strong GCaMP6s expression from cleavage through gastrula stages, we detected higher frequency of Ca2+transients in the superficial blastomeres during the blastula stages preceding the midblastula transition. Additionally, GCaMP6s also revealed that dorsal-biased Ca2+signaling that follows the midblastula transition persisted longer during gastrulation, compared with earlier studies. We observed that dorsal-biased Ca2+signaling is diminished in ventralized ichabod/β-catenin2 mutant embryos and ectopically induced in embryos dorsalized by excess β-catenin. During gastrulation, we directly visualized Ca2+signaling in the dorsal forerunner cells, which form in a Nodal signaling dependent manner and later give rise to the laterality organ. We found that excess Nodal increases the number and the duration of Ca2+transients specifically in the dorsal forerunner cells. The GCaMP6s transgenic lines described here enable unprecedented visualization of dynamic Ca2+events from embryogenesis through adulthood, augmenting the zebrafish toolbox.
Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Jiakun
J
Chen
Department of Anesthesiology, Division of Basic Research, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Biomedical Engineering, Washington University in St. Louis, St Louis, MO, 63105, USA.
Li
L
Xia
Department of Anesthesiology, Division of Basic Research, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Biomedical Engineering, Washington University in St. Louis, St Louis, MO, 63105, USA; Department of Neuroscience, Washington University in St. Louis, St. Louis, MO, USA.
Michael R
MR
Bruchas
Department of Developmental Biology, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: solnical@wustl.edu.
Lilianna
L
Solnica-Krezel
D000199:Actins / Q000235:genetics; D000818:Animals; D030801:Animals, Genetically Modified; D001757:Blastomeres / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D036703:Blastula / Q000737:chemistry / Q000648:ultrastructure; D019521:Body Patterning; D002118:Calcium / Q000032:analysis*; D020013:Calcium Signaling / Q000502:physiology*; D002147:Calmodulin / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics; D010455:Peptides / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics; D015027:Zebrafish / Q000196:embryology*
D000199:Actins; D002147:Calmodulin; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C065738:MLCK peptide; D049452:Green Fluorescent Proteins; D002118:Calcium
Calcium transients; Dorsal forerunner cells; Embryonic cleavages; Gastrulation; Nodal; β-catenin