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

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.

Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy

“…We imaged the developing eye and the spinal tissue of zebrafish embryos, in which the cells undergo active cell division ∼17 h postfertilization. As with the BSC1 cells, we directly visualized the plasma membrane of cells, in this case marked by fluorescent citrine containing the palmitoylation and myristoylation sequence motifs from lyn kinase (Mosaliganti et al , 2012; Swinburne et al , 2015) expressed as a transgene in all cells. The z -stacks of the 3D time series, acquired for periods of 60–120 min, were obtained by translating the sample stage; they comprised ∼250 optical sections spaced at 400 nm along the s-axis, acquired with ∼40-ms exposures per frame.…”

“…The eggs were collected and screened for health before being transferred to a 28°C incubator and kept at this temperature, except during injection/dechorionation and imaging, which were performed at room temperature. Natural twitching of embryos when visualized beyond 20 h postfertilization was prevented by injection into 1-cell-stage embryos of 2.3 nl of 20 ng/μl mRNA encoding α-bungarotoxin used as anesthetic (Swinburne et al ., 2015). Injections were performed using a glass needle (15- to 25-μm diameter) with the Nanoinject system and a Leica stereoscope (Leica MZ12.5).…”

Sheath Cell Invasion and Trans-differentiation Repair Mechanical Damage Caused by Loss of Caveolae in the Zebrafish Notochord

“…To increase mechanical strain, we de-chorionated 24 hpf mz cavin1b − / − embryos and placed them in egg water or in 3% methylcellulose (MC) to increase the viscosity of the medium as previously shown [14]. To abrogate the effect of locomotion, we injected one-cell stage mz cavin1b − / − embryos with α-Bungarotoxin cRNA to paralyze them [20] and incubated them (without removing the chorion) in egg water. Then, at 72 hpf we used DIC microscopy to score the notochord phenotype into three categories: normal (no lesions), mild (one or more areas with limited vacuole collapse), and severe (one or more areas with extended vacuole collapse and debris) (Fig.1N).…”

Recovery of shape and size in a developing organ pair

“…To confirm our imaging, immobilization, and quantification methods give results that represent wild-type ear development accurately, considering that fish development can be affected by long-term immobilization methods (Swinburne et al, 2015), we compared our data with an established standard wild-type dataset (Mosaliganti et al, under review). To determine standard developmental trajectories for wild-type ears (Mosaliganti et al, under review), single ears were imaged at high resolution with a 40 Â 1.1 NA objective lens, imaging each embryo only once to avoid any effects of long-term immobilization (Mosaliganti et al, under review;Swinburne et al, 2015). Embryos were immobilized using tricaine (1.5%, in Danieau) only when imaging after the first twitch ($18 hpf) (Mosaliganti et al, under review), and 4-13 Standard wild-type dataset: means (black line and diamonds) and standard deviations (SDs) (light gray shading).…”

“…A Zeiss 710 confocal microscope (Plan‐Apochromat 20 × 1.0 NA and C‐Apochromat 40 × 1.2 NA objective lenses; 488 nm and 561 nm lasers) was used for imaging. Each embryo was in tricaine for a maximum of 2 hr continuously, with at least 2 hr unmounted in Danieau between successive image captures (for issues with tricaine use in long‐term imaging, see Swinburne et al, , Fig. ).…”