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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

Long-term imaging of embryos immobilized with α-bungarotoxin mRNA.(A) Montage of an immobilized embryo’s development from the 1-cell stage to 85 hpf after it had been injected with 50 pg of α-bungarotoxin mRNA into the 1-cell. Images are shown from every hour of development. (B) Quantification of the full time-course that included 153,452 images that were acquired every 2 seconds. The movement index was calculated as the maximum difference between each image and its subsequent image in the time-series. The index was normalized to the average maximum difference in the first 2,000 time points. Control embryos (red) and embryos in 200 μg/ml tricaine (blue) begin twitching at around 18 hpf and then may swim out of the field while α-bungarotoxin injected embryos (green) showed very little movement until 80 hpf.
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pone.0134005.g004: Long-term imaging of embryos immobilized with α-bungarotoxin mRNA.(A) Montage of an immobilized embryo’s development from the 1-cell stage to 85 hpf after it had been injected with 50 pg of α-bungarotoxin mRNA into the 1-cell. Images are shown from every hour of development. (B) Quantification of the full time-course that included 153,452 images that were acquired every 2 seconds. The movement index was calculated as the maximum difference between each image and its subsequent image in the time-series. The index was normalized to the average maximum difference in the first 2,000 time points. Control embryos (red) and embryos in 200 μg/ml tricaine (blue) begin twitching at around 18 hpf and then may swim out of the field while α-bungarotoxin injected embryos (green) showed very little movement until 80 hpf.

Mentions: Before the touch response develops, spontaneous twitching initiates around 18 hpf. As a proof-of-principle of the 1-cell stage α-bungarotoxin mRNA injection immobilization strategy, we imaged embryonic development from the 1-cell stage to 85 hpf, for injection controls and for 50 pg of α-bungarotoxin mRNA into the 1-cell (Fig 4A). Images were acquired every 2 seconds but only presented for every hour in Fig 4A, or every 40–160 seconds in S1 Movie. We quantified movement by calculating the maximum pixel intensity difference between successive time-points within the full data-set (2 second time resolution, Fig 4B). Notably, α-bungarotoxin mRNA injected embryos do not perform the first twitches at 18 hpf or anytime thereafter, allowing us to capture an unprecedentedly complete movie (Fig 4B, S1 Movie). In contrast, control embryos and embryos treated with 200 μg/ml tricaine still began to move at 18 hpf (Fig 4B). What movement can be observed in the α-bungarotoxin time-course and its quantification is minor and includes: embryo rolling during cleavage and epiboly (0.5–5.5 hpf), embryo slippage within the agarose mount during tail extension (~24 hpf and ~47 hpf), jaw movement (starting ~ 75 hpf), and recovery from paralysis (starting ~80 hpf). A movie is here as S1 Movie and is available at http://www.youtube.com/watch?v=4c-Kw4timVA&feature=youtu.be. An additional movie is available at https://www.youtube.com/watch?v=A1vun3ETAkE. We have imaged 9 embryos injected with 50 pg of BTX mRNA and all have lacked movement until >72 hpf. Thus, injection of α-bungarotoxin mRNA at the 1-cell stage allows the tracking of normally developing embryos through long periods of time including the period of first twitches, which were typically difficult to stop with previous immobilization approaches.


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

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

Long-term imaging of embryos immobilized with α-bungarotoxin mRNA.(A) Montage of an immobilized embryo’s development from the 1-cell stage to 85 hpf after it had been injected with 50 pg of α-bungarotoxin mRNA into the 1-cell. Images are shown from every hour of development. (B) Quantification of the full time-course that included 153,452 images that were acquired every 2 seconds. The movement index was calculated as the maximum difference between each image and its subsequent image in the time-series. The index was normalized to the average maximum difference in the first 2,000 time points. Control embryos (red) and embryos in 200 μg/ml tricaine (blue) begin twitching at around 18 hpf and then may swim out of the field while α-bungarotoxin injected embryos (green) showed very little movement until 80 hpf.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134005.g004: Long-term imaging of embryos immobilized with α-bungarotoxin mRNA.(A) Montage of an immobilized embryo’s development from the 1-cell stage to 85 hpf after it had been injected with 50 pg of α-bungarotoxin mRNA into the 1-cell. Images are shown from every hour of development. (B) Quantification of the full time-course that included 153,452 images that were acquired every 2 seconds. The movement index was calculated as the maximum difference between each image and its subsequent image in the time-series. The index was normalized to the average maximum difference in the first 2,000 time points. Control embryos (red) and embryos in 200 μg/ml tricaine (blue) begin twitching at around 18 hpf and then may swim out of the field while α-bungarotoxin injected embryos (green) showed very little movement until 80 hpf.
Mentions: Before the touch response develops, spontaneous twitching initiates around 18 hpf. As a proof-of-principle of the 1-cell stage α-bungarotoxin mRNA injection immobilization strategy, we imaged embryonic development from the 1-cell stage to 85 hpf, for injection controls and for 50 pg of α-bungarotoxin mRNA into the 1-cell (Fig 4A). Images were acquired every 2 seconds but only presented for every hour in Fig 4A, or every 40–160 seconds in S1 Movie. We quantified movement by calculating the maximum pixel intensity difference between successive time-points within the full data-set (2 second time resolution, Fig 4B). Notably, α-bungarotoxin mRNA injected embryos do not perform the first twitches at 18 hpf or anytime thereafter, allowing us to capture an unprecedentedly complete movie (Fig 4B, S1 Movie). In contrast, control embryos and embryos treated with 200 μg/ml tricaine still began to move at 18 hpf (Fig 4B). What movement can be observed in the α-bungarotoxin time-course and its quantification is minor and includes: embryo rolling during cleavage and epiboly (0.5–5.5 hpf), embryo slippage within the agarose mount during tail extension (~24 hpf and ~47 hpf), jaw movement (starting ~ 75 hpf), and recovery from paralysis (starting ~80 hpf). A movie is here as S1 Movie and is available at http://www.youtube.com/watch?v=4c-Kw4timVA&feature=youtu.be. An additional movie is available at https://www.youtube.com/watch?v=A1vun3ETAkE. We have imaged 9 embryos injected with 50 pg of BTX mRNA and all have lacked movement until >72 hpf. Thus, injection of α-bungarotoxin mRNA at the 1-cell stage allows the tracking of normally developing embryos through long periods of time including the period of first twitches, which were typically difficult to stop with previous immobilization approaches.

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