Transgenic Zebrafish Expressing mCherry in the Mitochondria of Dopaminergic Neurons.
Genetic mutations and environmental toxins are known to affect mitochondrial health and have been implicated in the progressive degeneration of dopaminergic neurons in Parkinson's disease. To visualize mitochondria in dopaminergic neurons of live zebrafish, we used the regulatory elements of the dopamine transporter (dat) gene to target a reporter, mCherry, after fusion with the mitochondrial localizing signal (MLS) of Tom20. Immunoblot analysis of mitochondrial and cytosolic fractions from Tg(dat:tom20 MLS-mCherry) larvae shows that mCherry is efficiently targeted to the mitochondria. Confocal imaging of live fish was carried out from 1 day postfertilization (dpf) to 9 dpf. We also colocalized dat mRNA expression with the mCherry protein in the olfactory bulb (OB), subpallium (SP), pretectum (Pr), diencephalic clusters 2 and 3 (DC2/3), caudal hypothalamus (Hc), locus coeruleus (LC), anterior preoptic area (POa), retinal amacrine cells (RAC), caudal hypothalamus (Hc), and preoptic area (PO). Treating Tg(dat:tom20 MLS-mCherry) larvae with the dopaminergic neurotoxin MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) at 2 or 3 dpf resulted in a decrease in mCherry fluorescence in the pretectum, olfactory bulb, subpallium, diencephalic clusters 2 and 3, and the caudal hypothalamus. Labeling of mitochondria in nigrostriatal dopaminergic neurons of zebrafish could allow their visualization in vivo following genetic or pharmacological manipulations.
1 Department of Biology, Center for Advanced Research in Environmental Genomics, University of Ottawa , Ottawa, Canada .
Sandra
S
Noble
1 Department of Biology, Center for Advanced Research in Environmental Genomics, University of Ottawa , Ottawa, Canada .
Rafael
R
Godoy
2 TEFOR Infrastructure, UPR 3294 N&D Neurobiologie et Développement, CNRS, Institut de Neurosciences A. Fessard , Gif-Sur-Yvette, France .
Pierre
P
Affaticati
1 Department of Biology, Center for Advanced Research in Environmental Genomics, University of Ottawa , Ottawa, Canada .
Marc
M
Ekker
D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine / Q000494:pharmacology*; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D059290:Dopaminergic Neurons / Q000378:metabolism*; D005453:Fluorescence; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D009498:Neurotoxins / Q000494:pharmacology*; D009928:Organ Specificity; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism; D000097573:Red Fluorescent Protein
D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; D004298:Dopamine; D050483:Dopamine Plasma Membrane Transport Proteins; D008164:Luminescent Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D009498:Neurotoxins; D018160:Receptors, Cytoplasmic and Nuclear; D029961:Zebrafish Proteins; D000097573:Red Fluorescent Protein; C546868:SLC6A3 protein, zebrafish; C000609118:tomm20b protein, zebrafish