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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

α-bungarotoxin immobilizes embryos while permitting normal development.(A) Percent of embryos immobile after injection of α-bungarotoxin protein (0.046–4.6ng) into the yolk at 24 hpf. (B) Percent of embryos immobile after injection of α-bungarotoxin mRNA (20–400 pg) into the yolk at 24 hpf. (C) Percent of embryos immobile after injection of of α-bungarotoxin mRNA (5–100 pg) into the 1-cell zygote. (D) Percent control OVD at 72 hpf for injection of α-bungarotoxin mRNA into the 1-cell zygote (green), into the yolk (yellow), and reference anesthetic treatments that permitted long-term immobilization (blue). (*) Not significantly different from control, Mann-Whitney-Wilcoxon two tailed P-value 0.87. (†) Significantly different from control, Mann-Whitney-Wilcoxon two tailed P-value 0.0011. (E, G) Control embryo at 72 hpf that was injected with 50 pg of membrane-citrine mRNA into the 1-cell zygote. (F, H) 72 hpf embryo that was injected with 50 pg of α-bungarotoxin mRNA into the 1-cell zygote. (I) Control larva at 8 days post fertilization (dpf) injected with 50 pg of membrane-citrine mRNA into the 1-cell zygote. (J) 8 dpf larva that was injected with 50 pg of α-bungarotoxin mRNA into the 1-cell zygote.
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pone.0134005.g002: α-bungarotoxin immobilizes embryos while permitting normal development.(A) Percent of embryos immobile after injection of α-bungarotoxin protein (0.046–4.6ng) into the yolk at 24 hpf. (B) Percent of embryos immobile after injection of α-bungarotoxin mRNA (20–400 pg) into the yolk at 24 hpf. (C) Percent of embryos immobile after injection of of α-bungarotoxin mRNA (5–100 pg) into the 1-cell zygote. (D) Percent control OVD at 72 hpf for injection of α-bungarotoxin mRNA into the 1-cell zygote (green), into the yolk (yellow), and reference anesthetic treatments that permitted long-term immobilization (blue). (*) Not significantly different from control, Mann-Whitney-Wilcoxon two tailed P-value 0.87. (†) Significantly different from control, Mann-Whitney-Wilcoxon two tailed P-value 0.0011. (E, G) Control embryo at 72 hpf that was injected with 50 pg of membrane-citrine mRNA into the 1-cell zygote. (F, H) 72 hpf embryo that was injected with 50 pg of α-bungarotoxin mRNA into the 1-cell zygote. (I) Control larva at 8 days post fertilization (dpf) injected with 50 pg of membrane-citrine mRNA into the 1-cell zygote. (J) 8 dpf larva that was injected with 50 pg of α-bungarotoxin mRNA into the 1-cell zygote.

Mentions: Combinations of tricaine and isoeugenol can minimize toxicity while still achieving immobility. We first examined heart edema as a read-out of embryo health and found it to be sensitive to both tricaine and isoeugenol (Fig 1E). We then focused on the size of an embryo’s otic vesicle as an indicator of developmental progress and health, because its morphogenesis begins early and is easily measured. Also, as illustrated here, reduced otic vesicle size correlates well with other health indicators such as pericardial edema while being a more sensitive and continuous readout of health (Fig 1E and 1F). Reduced otic vesicle size could be because of reduced lumen expansion or reduced proliferation. Tricaine and isoeugenol both act through ion channels in ways that could directly reduce lumen expansion. While the specific target of tricaine is not known, the scna gene family is believed to mediate a large portion of sodium conductance in zebrafish and some may be expressed in the developing otic vesicle [29, 30]. However, it is unclear if any are expressed in the developing heart so there are likely to be other targets that cause tricaine’s side effects. Additionally, off-target effects or overall poor health could reduce proliferation or indirectly influence complex morphogenetic processes. At 72 hpf we measured the diameter of the left otic vesicle along the anterior-posterior axis (Fig 1C, Otic Vesicle Diameter, OVD). Among 10 control embryos the OVD is 164 μm ± 4.0 μm (mean ± standard deviation; representative control embryo, Fig 1C). When soaked in 200 μg/ml of tricaine from 24 to 72 hpf the average OVD was significantly shorter at 148 ± 8.7 μm (representative embryo treated with 200 μg/ml tricaine, Mann-Whitney-Wilcoxon two tailed P-value < 0.0011, Figs 1D and 2D). We also observed an increased incidence of pericardial edema (asterisk, Fig 1D) and impaired tissue fusion during semicircular canal development (arrowhead, Fig 1D).


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

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

α-bungarotoxin immobilizes embryos while permitting normal development.(A) Percent of embryos immobile after injection of α-bungarotoxin protein (0.046–4.6ng) into the yolk at 24 hpf. (B) Percent of embryos immobile after injection of α-bungarotoxin mRNA (20–400 pg) into the yolk at 24 hpf. (C) Percent of embryos immobile after injection of of α-bungarotoxin mRNA (5–100 pg) into the 1-cell zygote. (D) Percent control OVD at 72 hpf for injection of α-bungarotoxin mRNA into the 1-cell zygote (green), into the yolk (yellow), and reference anesthetic treatments that permitted long-term immobilization (blue). (*) Not significantly different from control, Mann-Whitney-Wilcoxon two tailed P-value 0.87. (†) Significantly different from control, Mann-Whitney-Wilcoxon two tailed P-value 0.0011. (E, G) Control embryo at 72 hpf that was injected with 50 pg of membrane-citrine mRNA into the 1-cell zygote. (F, H) 72 hpf embryo that was injected with 50 pg of α-bungarotoxin mRNA into the 1-cell zygote. (I) Control larva at 8 days post fertilization (dpf) injected with 50 pg of membrane-citrine mRNA into the 1-cell zygote. (J) 8 dpf larva that was injected with 50 pg of α-bungarotoxin mRNA into the 1-cell zygote.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134005.g002: α-bungarotoxin immobilizes embryos while permitting normal development.(A) Percent of embryos immobile after injection of α-bungarotoxin protein (0.046–4.6ng) into the yolk at 24 hpf. (B) Percent of embryos immobile after injection of α-bungarotoxin mRNA (20–400 pg) into the yolk at 24 hpf. (C) Percent of embryos immobile after injection of of α-bungarotoxin mRNA (5–100 pg) into the 1-cell zygote. (D) Percent control OVD at 72 hpf for injection of α-bungarotoxin mRNA into the 1-cell zygote (green), into the yolk (yellow), and reference anesthetic treatments that permitted long-term immobilization (blue). (*) Not significantly different from control, Mann-Whitney-Wilcoxon two tailed P-value 0.87. (†) Significantly different from control, Mann-Whitney-Wilcoxon two tailed P-value 0.0011. (E, G) Control embryo at 72 hpf that was injected with 50 pg of membrane-citrine mRNA into the 1-cell zygote. (F, H) 72 hpf embryo that was injected with 50 pg of α-bungarotoxin mRNA into the 1-cell zygote. (I) Control larva at 8 days post fertilization (dpf) injected with 50 pg of membrane-citrine mRNA into the 1-cell zygote. (J) 8 dpf larva that was injected with 50 pg of α-bungarotoxin mRNA into the 1-cell zygote.
Mentions: Combinations of tricaine and isoeugenol can minimize toxicity while still achieving immobility. We first examined heart edema as a read-out of embryo health and found it to be sensitive to both tricaine and isoeugenol (Fig 1E). We then focused on the size of an embryo’s otic vesicle as an indicator of developmental progress and health, because its morphogenesis begins early and is easily measured. Also, as illustrated here, reduced otic vesicle size correlates well with other health indicators such as pericardial edema while being a more sensitive and continuous readout of health (Fig 1E and 1F). Reduced otic vesicle size could be because of reduced lumen expansion or reduced proliferation. Tricaine and isoeugenol both act through ion channels in ways that could directly reduce lumen expansion. While the specific target of tricaine is not known, the scna gene family is believed to mediate a large portion of sodium conductance in zebrafish and some may be expressed in the developing otic vesicle [29, 30]. However, it is unclear if any are expressed in the developing heart so there are likely to be other targets that cause tricaine’s side effects. Additionally, off-target effects or overall poor health could reduce proliferation or indirectly influence complex morphogenetic processes. At 72 hpf we measured the diameter of the left otic vesicle along the anterior-posterior axis (Fig 1C, Otic Vesicle Diameter, OVD). Among 10 control embryos the OVD is 164 μm ± 4.0 μm (mean ± standard deviation; representative control embryo, Fig 1C). When soaked in 200 μg/ml of tricaine from 24 to 72 hpf the average OVD was significantly shorter at 148 ± 8.7 μm (representative embryo treated with 200 μg/ml tricaine, Mann-Whitney-Wilcoxon two tailed P-value < 0.0011, Figs 1D and 2D). We also observed an increased incidence of pericardial edema (asterisk, Fig 1D) and impaired tissue fusion during semicircular canal development (arrowhead, Fig 1D).

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