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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

Tricaine and isoeugenol co-operate towards healthier immobilization.(A) Heat map of percent immobile for 48 combinations of tricaine (0–200 μg/ml) and isoeugenol (0–0.003% v/v). Embryos were dechorionated and soaked from 24–27 hpf when they were assayed for immobility. (B) Continuation of treatments from (A), embryos were assayed for immobility at 72 hpf. (C,D) Representative micrographs of control (C) and 200 μg/ml tricaine treated (D) embryos. Arrow in (D) shows failure of semicircular canal projection fusion. Asterisk in (D) shows pericardial edema. (E) Heat map of percent of embryos with pericardial edema at 72 hpf. (F) Percent control otic vesicle diameter (OVD) was calculated by dividing the average of 10–30 experimental embryos by the average of 10–30 control embryos. Heat map of percent control OVD for the combinatorial treatments. OVD was measured at 72 hpf using micrographs like those in (C, D). Percentage is based on normalization to untreated control. (G) Merge of heatmaps from (A, 27 hpf) and (F) that highlights the tradeoffs between embryo immobility and healthy development.
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pone.0134005.g001: Tricaine and isoeugenol co-operate towards healthier immobilization.(A) Heat map of percent immobile for 48 combinations of tricaine (0–200 μg/ml) and isoeugenol (0–0.003% v/v). Embryos were dechorionated and soaked from 24–27 hpf when they were assayed for immobility. (B) Continuation of treatments from (A), embryos were assayed for immobility at 72 hpf. (C,D) Representative micrographs of control (C) and 200 μg/ml tricaine treated (D) embryos. Arrow in (D) shows failure of semicircular canal projection fusion. Asterisk in (D) shows pericardial edema. (E) Heat map of percent of embryos with pericardial edema at 72 hpf. (F) Percent control otic vesicle diameter (OVD) was calculated by dividing the average of 10–30 experimental embryos by the average of 10–30 control embryos. Heat map of percent control OVD for the combinatorial treatments. OVD was measured at 72 hpf using micrographs like those in (C, D). Percentage is based on normalization to untreated control. (G) Merge of heatmaps from (A, 27 hpf) and (F) that highlights the tradeoffs between embryo immobility and healthy development.

Mentions: Embryo length, pericardial edema, and otic vesicle diameter (OVD) were determined at 72 hpf. Images of embryos and a stage micrometer (Ward’s) were captured with an Olympus MVX10 Macroview (Olympus) using either a 1x MVX plan apochromat, 0.25NA objective for 25x magnification or a 2x MVX plan apochromat, 0.5NA for 126x magnification. Embryo features were measured using ImageJ. OVD was measured at 72 hpf using micrographs like those in (Fig 1C and 1D). Percentage is based on normalization to controls.


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

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

Tricaine and isoeugenol co-operate towards healthier immobilization.(A) Heat map of percent immobile for 48 combinations of tricaine (0–200 μg/ml) and isoeugenol (0–0.003% v/v). Embryos were dechorionated and soaked from 24–27 hpf when they were assayed for immobility. (B) Continuation of treatments from (A), embryos were assayed for immobility at 72 hpf. (C,D) Representative micrographs of control (C) and 200 μg/ml tricaine treated (D) embryos. Arrow in (D) shows failure of semicircular canal projection fusion. Asterisk in (D) shows pericardial edema. (E) Heat map of percent of embryos with pericardial edema at 72 hpf. (F) Percent control otic vesicle diameter (OVD) was calculated by dividing the average of 10–30 experimental embryos by the average of 10–30 control embryos. Heat map of percent control OVD for the combinatorial treatments. OVD was measured at 72 hpf using micrographs like those in (C, D). Percentage is based on normalization to untreated control. (G) Merge of heatmaps from (A, 27 hpf) and (F) that highlights the tradeoffs between embryo immobility and healthy development.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134005.g001: Tricaine and isoeugenol co-operate towards healthier immobilization.(A) Heat map of percent immobile for 48 combinations of tricaine (0–200 μg/ml) and isoeugenol (0–0.003% v/v). Embryos were dechorionated and soaked from 24–27 hpf when they were assayed for immobility. (B) Continuation of treatments from (A), embryos were assayed for immobility at 72 hpf. (C,D) Representative micrographs of control (C) and 200 μg/ml tricaine treated (D) embryos. Arrow in (D) shows failure of semicircular canal projection fusion. Asterisk in (D) shows pericardial edema. (E) Heat map of percent of embryos with pericardial edema at 72 hpf. (F) Percent control otic vesicle diameter (OVD) was calculated by dividing the average of 10–30 experimental embryos by the average of 10–30 control embryos. Heat map of percent control OVD for the combinatorial treatments. OVD was measured at 72 hpf using micrographs like those in (C, D). Percentage is based on normalization to untreated control. (G) Merge of heatmaps from (A, 27 hpf) and (F) that highlights the tradeoffs between embryo immobility and healthy development.
Mentions: Embryo length, pericardial edema, and otic vesicle diameter (OVD) were determined at 72 hpf. Images of embryos and a stage micrometer (Ward’s) were captured with an Olympus MVX10 Macroview (Olympus) using either a 1x MVX plan apochromat, 0.25NA objective for 25x magnification or a 2x MVX plan apochromat, 0.5NA for 126x magnification. Embryo features were measured using ImageJ. OVD was measured at 72 hpf using micrographs like those in (Fig 1C and 1D). Percentage is based on normalization to controls.

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