PUBLICATION

Improved Long-Term Imaging of Embryos with Genetically Encoded α-Bungarotoxin

Authors
Swinburne, I.A., Mosaliganti, K.R., Green, A.A., Megason, S.G.
ID
ZDB-PUB-150806-1
Date
2015
Source
PLoS One   10: e0134005 (Journal)
Registered Authors
Megason, Sean, Mosaliganti, Kishore, Swinburne, Ian
Keywords
Embryos, Anesthetics, Zebrafish, Messenger RNA, Edema, Vesicles, Blood flow, Toxicity
MeSH Terms
  • Aminobenzoates/pharmacology
  • Anesthetics/pharmacology
  • Animals
  • Base Sequence
  • Bungarotoxins/genetics
  • Bungarotoxins/metabolism*
  • Embryo, Nonmammalian/embryology
  • Embryo, Nonmammalian/metabolism*
  • Eugenol/analogs & derivatives
  • Eugenol/pharmacology
  • Microscopy, Confocal
  • Molecular Sequence Data
  • Movement/drug effects
  • RNA, Messenger/genetics
  • RNA, Messenger/metabolism
  • Time-Lapse Imaging/methods*
  • Zebrafish/embryology
  • Zebrafish/genetics
  • Zebrafish/metabolism*
PubMed
26244658 Full text @ PLoS One
Abstract
Rapid advances in microscopy and genetic labeling strategies have created new opportunities for time-lapse imaging of embryonic development. However, methods for immobilizing embryos for long periods while maintaining normal development have changed little. In zebrafish, current immobilization techniques rely on the anesthetic tricaine. Unfortunately, prolonged tricaine treatment at concentrations high enough to immobilize the embryo produces undesirable side effects on development. We evaluate three alternative immobilization strategies: combinatorial soaking in tricaine and isoeugenol, injection of α-bungarotoxin protein, and injection of α-bungarotoxin mRNA. We find evidence for co-operation between tricaine and isoeugenol to give immobility with improved health. However, even in combination these anesthetics negatively affect long-term development. α-bungarotoxin is a small protein from snake venom that irreversibly binds and inactivates acetylcholine receptors. We find that α-bungarotoxin either as purified protein from snakes or endogenously expressed in zebrafish from a codon-optimized synthetic gene can immobilize embryos for extended periods of time with few health effects or developmental delays. Using α-bungarotoxin mRNA injection we obtain complete movies of zebrafish embryogenesis from the 1-cell stage to 3 days post fertilization, with normal health and no twitching. These results demonstrate that endogenously expressed α-bungarotoxin provides unprecedented immobility and health for time-lapse microscopy.
Genes / Markers
Figures
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Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping