title: Transgenic zebrafish for ratiometric imaging of cytosolic and mitochondrial Ca2+ response in teleost embryo.
abstract: Intracellular Ca2+ imaging has widely been used to visualize intracellular signals, but the application in an intact animal is still limited due to difficulty of the indicator loading. In addition, the motion of the living animal produces artifacts. To investigate Ca2+ signaling at early embryonic stage, we established transgenic zebrafish line expressing a genetically encoded Ca2+ indicator, cameleon YC2.60, driven by a constitutively active promoter, hspa8. Although the embryo dynamically changes its morphology, the motion artifact could be canceled out by taking the advantage of YC2.60 as a ratiometric indicator. The transgenic zebrafish was used to visualize the propagation of cytosolic Ca2+ during the early embryonic stage upon fertilization and along cleavage furrow, and the rise in Ca2+ in the myocytes contracting spontaneously in the embryo. We also established a transgenic zebrafish line expressing YC2.60 targeted to the mitochondria. The rise in mitochondrial Ca2+ was rather sustained (≈2 min), which is consistent with the requirement of ATP refilling since the mitochondrial Ca2+ upregulates rate-limiting enzymes of Krebs cycle. This is in contrast with the transient rise in the cytosol Ca2+ that directly evokes the muscle contraction. These transgenic zebrafish lines are expected to serve as useful tools further Ca2+ imaging in vivo.
authors.affiliation: Cell Function and Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama 351-0198, Japan. hideaki.mizuno@chem.kuleuven.be
authors.forename: Hideaki
authors.initials: H
authors.lastname: Mizuno
authors.forename: Takayuki
authors.initials: T
authors.lastname: Sassa
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Higashijima
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Okamoto
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism*; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D032342:Muscle Cells / Q000502:physiology; D009119:Muscle Contraction / Q000502:physiology; D015027:Zebrafish / Q000254:growth & development / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D050883:HSC70 Heat-Shock Proteins; D029961:Zebrafish Proteins; C494374:hspa8 protein, zebrafish; D002118:Calcium
keywords: Calcium; Embryo; FRET; Fertilization; Mitochondrial calcium; Zebrafish