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

Swinburne IA, Mosaliganti KR, Green AA, Megason SG - PLoS ONE (2015)

Bottom Line: Unfortunately, prolonged tricaine treatment at concentrations high enough to immobilize the embryo produces undesirable side effects on development.We find evidence for co-operation between tricaine and isoeugenol to give immobility with improved health.These results demonstrate that endogenously expressed α-bungarotoxin provides unprecedented immobility and health for time-lapse microscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Prolonged immobilization with α-bungarotoxin mRNA does not grossly alter neural or cardiovascular development.(A-C) 3D reconstructions of confocal images of 72 hpf Tg(mnx1:gfp) centered at the sixth somite reveal no gross abnormalities in motor neuron patterning or development when embryos are immobilized with 50 pg of α-bungarotoxin mRNA injected at the 1-cell stage (B) or 200 μg/ml of tricaine from 24 to 72 hpf (C) (scale bar 50 μm). A stereotyped axon (red bracket, A, schematic, D) was used to quantify axon branching. (E-G) The distributions of distances between axon branches were not significantly altered in embryos immobilized with 50 pg of α-bungarotoxin mRNA injected at the 1-cell stage (F, Mann-Whitney-Wilcoxon two tailed P-value 0.58) or 200 μg/ml of tricaine from 24 to 72 hpf (G, Mann-Whitney-Wilcoxon two tailed P-value 0.36). (H) Representative laser-scanning velocimetry results reveal no gross difference in cardiovascular performance between control embryos and embryos immobilized with 50 pg of α-bungarotoxin mRNA. 200 μg/ml of tricaine from 24 to 72 hpf does grossly alter blood flow. Vertical scale bar 100 ms, horizontal scale bar 20 μm. (I-P) Quantitative image analysis of laser-scanning velocimetry reveals no significant difference in cardiovascular function between control and α-bungarotoxin mRNA injected embryos while prolonged tricaine treatment significantly reduces peak blood velocity and peak blood acceleration (Mann-Whitney-Wilcoxon two tailed P-values < 1e-12, tricaine relative to control peak velocities, and <1.4e-11, tricaine relative to control peak accelerations).
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pone.0134005.g003: Prolonged immobilization with α-bungarotoxin mRNA does not grossly alter neural or cardiovascular development.(A-C) 3D reconstructions of confocal images of 72 hpf Tg(mnx1:gfp) centered at the sixth somite reveal no gross abnormalities in motor neuron patterning or development when embryos are immobilized with 50 pg of α-bungarotoxin mRNA injected at the 1-cell stage (B) or 200 μg/ml of tricaine from 24 to 72 hpf (C) (scale bar 50 μm). A stereotyped axon (red bracket, A, schematic, D) was used to quantify axon branching. (E-G) The distributions of distances between axon branches were not significantly altered in embryos immobilized with 50 pg of α-bungarotoxin mRNA injected at the 1-cell stage (F, Mann-Whitney-Wilcoxon two tailed P-value 0.58) or 200 μg/ml of tricaine from 24 to 72 hpf (G, Mann-Whitney-Wilcoxon two tailed P-value 0.36). (H) Representative laser-scanning velocimetry results reveal no gross difference in cardiovascular performance between control embryos and embryos immobilized with 50 pg of α-bungarotoxin mRNA. 200 μg/ml of tricaine from 24 to 72 hpf does grossly alter blood flow. Vertical scale bar 100 ms, horizontal scale bar 20 μm. (I-P) Quantitative image analysis of laser-scanning velocimetry reveals no significant difference in cardiovascular function between control and α-bungarotoxin mRNA injected embryos while prolonged tricaine treatment significantly reduces peak blood velocity and peak blood acceleration (Mann-Whitney-Wilcoxon two tailed P-values < 1e-12, tricaine relative to control peak velocities, and <1.4e-11, tricaine relative to control peak accelerations).

Mentions: α-bungarotoxin inhibits postsynaptic activation of AChR’s by binding either the alpha1 or alpha7 subunits and could potentially interfere with developmental mechanisms that may require this activation. To help determine whether prolonged expression of α-bungarotoxin protein interferes with the path finding of motor neurons we imaged Tg(mnx1:gfp) embryos at 72 hpf that had been injected with a control or α-bungarotoxin mRNA at the 1-cell stage (Fig 3A–3C) and observed no obvious differences in the motor neuron patterning or path finding. We quantified distances between branches of a stereotyped dorsal axon (red bracket in Fig 3A, schematic in Fig 3D) and found no statistical difference between the distribution of axon branches between controls, embryos injected with 50 pg of α-bungarotoxin mRNA at the 1-cell stage, and embryos treated with 200 μg/ml Tricaine from 24 to 72 hpf (Mann-Whitney-Wilcoxon test, respective two tailed P-values of 0.58 and 0.36 indicate no significant differences between axon branch distributions relative to the control). This is consistent with the ability of paralyzed embryos to rapidly recover normal motility and the lack of muscle defects. This is also consistent with the nic1 mutant (AChR alpha 1 subunit) that lacks detectible motor neuron innervation defects or neuromuscular phenotypes [32]. However, there remains the possibility that these analyses miss more subtle differences arising from reduced feedback that can modulate synapse remodeling [33, 34]. Additionally, the subset of CNS neurons that use the AChR alpha 7 subunit could also be affected. Because α-bungarotoxin may have unidentified side effects, care should be taken when using α-bungarotoxin to study processes related to plasticity, wiring, and dynamics of neural networks.


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

Swinburne IA, Mosaliganti KR, Green AA, Megason SG - PLoS ONE (2015)

Prolonged immobilization with α-bungarotoxin mRNA does not grossly alter neural or cardiovascular development.(A-C) 3D reconstructions of confocal images of 72 hpf Tg(mnx1:gfp) centered at the sixth somite reveal no gross abnormalities in motor neuron patterning or development when embryos are immobilized with 50 pg of α-bungarotoxin mRNA injected at the 1-cell stage (B) or 200 μg/ml of tricaine from 24 to 72 hpf (C) (scale bar 50 μm). A stereotyped axon (red bracket, A, schematic, D) was used to quantify axon branching. (E-G) The distributions of distances between axon branches were not significantly altered in embryos immobilized with 50 pg of α-bungarotoxin mRNA injected at the 1-cell stage (F, Mann-Whitney-Wilcoxon two tailed P-value 0.58) or 200 μg/ml of tricaine from 24 to 72 hpf (G, Mann-Whitney-Wilcoxon two tailed P-value 0.36). (H) Representative laser-scanning velocimetry results reveal no gross difference in cardiovascular performance between control embryos and embryos immobilized with 50 pg of α-bungarotoxin mRNA. 200 μg/ml of tricaine from 24 to 72 hpf does grossly alter blood flow. Vertical scale bar 100 ms, horizontal scale bar 20 μm. (I-P) Quantitative image analysis of laser-scanning velocimetry reveals no significant difference in cardiovascular function between control and α-bungarotoxin mRNA injected embryos while prolonged tricaine treatment significantly reduces peak blood velocity and peak blood acceleration (Mann-Whitney-Wilcoxon two tailed P-values < 1e-12, tricaine relative to control peak velocities, and <1.4e-11, tricaine relative to control peak accelerations).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4526548&req=5

pone.0134005.g003: Prolonged immobilization with α-bungarotoxin mRNA does not grossly alter neural or cardiovascular development.(A-C) 3D reconstructions of confocal images of 72 hpf Tg(mnx1:gfp) centered at the sixth somite reveal no gross abnormalities in motor neuron patterning or development when embryos are immobilized with 50 pg of α-bungarotoxin mRNA injected at the 1-cell stage (B) or 200 μg/ml of tricaine from 24 to 72 hpf (C) (scale bar 50 μm). A stereotyped axon (red bracket, A, schematic, D) was used to quantify axon branching. (E-G) The distributions of distances between axon branches were not significantly altered in embryos immobilized with 50 pg of α-bungarotoxin mRNA injected at the 1-cell stage (F, Mann-Whitney-Wilcoxon two tailed P-value 0.58) or 200 μg/ml of tricaine from 24 to 72 hpf (G, Mann-Whitney-Wilcoxon two tailed P-value 0.36). (H) Representative laser-scanning velocimetry results reveal no gross difference in cardiovascular performance between control embryos and embryos immobilized with 50 pg of α-bungarotoxin mRNA. 200 μg/ml of tricaine from 24 to 72 hpf does grossly alter blood flow. Vertical scale bar 100 ms, horizontal scale bar 20 μm. (I-P) Quantitative image analysis of laser-scanning velocimetry reveals no significant difference in cardiovascular function between control and α-bungarotoxin mRNA injected embryos while prolonged tricaine treatment significantly reduces peak blood velocity and peak blood acceleration (Mann-Whitney-Wilcoxon two tailed P-values < 1e-12, tricaine relative to control peak velocities, and <1.4e-11, tricaine relative to control peak accelerations).
Mentions: α-bungarotoxin inhibits postsynaptic activation of AChR’s by binding either the alpha1 or alpha7 subunits and could potentially interfere with developmental mechanisms that may require this activation. To help determine whether prolonged expression of α-bungarotoxin protein interferes with the path finding of motor neurons we imaged Tg(mnx1:gfp) embryos at 72 hpf that had been injected with a control or α-bungarotoxin mRNA at the 1-cell stage (Fig 3A–3C) and observed no obvious differences in the motor neuron patterning or path finding. We quantified distances between branches of a stereotyped dorsal axon (red bracket in Fig 3A, schematic in Fig 3D) and found no statistical difference between the distribution of axon branches between controls, embryos injected with 50 pg of α-bungarotoxin mRNA at the 1-cell stage, and embryos treated with 200 μg/ml Tricaine from 24 to 72 hpf (Mann-Whitney-Wilcoxon test, respective two tailed P-values of 0.58 and 0.36 indicate no significant differences between axon branch distributions relative to the control). This is consistent with the ability of paralyzed embryos to rapidly recover normal motility and the lack of muscle defects. This is also consistent with the nic1 mutant (AChR alpha 1 subunit) that lacks detectible motor neuron innervation defects or neuromuscular phenotypes [32]. However, there remains the possibility that these analyses miss more subtle differences arising from reduced feedback that can modulate synapse remodeling [33, 34]. Additionally, the subset of CNS neurons that use the AChR alpha 7 subunit could also be affected. Because α-bungarotoxin may have unidentified side effects, care should be taken when using α-bungarotoxin to study processes related to plasticity, wiring, and dynamics of neural networks.

Bottom Line: Unfortunately, prolonged tricaine treatment at concentrations high enough to immobilize the embryo produces undesirable side effects on development.We find evidence for co-operation between tricaine and isoeugenol to give immobility with improved health.These results demonstrate that endogenously expressed α-bungarotoxin provides unprecedented immobility and health for time-lapse microscopy.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus