Microbead Implantation in the Zebrafish Embryo

Abstract

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

“…Zebrafish Breeding, Embryo Collection, and mRNA Injection 36-38 Assemble breeding tanks with a barrier to separate the tank into two regions and fill each breeding tank with aquaculture system water used in the zebrafish facility.In order to prevent untimely breeding, place two male fish on one side of the barrier and two female fish on the other side the night before breeding.The next day, thaw the previously prepared mRNA mixture on ice. Replace the water in breeding tanks with fresh aquaculture system water and remove the barriers.…”

“…Replace the water in breeding tanks with fresh aquaculture system water and remove the barriers. Immediately after the barriers are pulled, warm an injection mold to 28.5 °C and set up the equipment for microinjection.Note: For information on injection molds, please refer to Gerlach 36 .Collect eggs every 10 - 15 min using a tea strainer and rinse the eggs into a clean 100 × 15 mm Petri dish with E3 Blue (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 , 0.33 mM MgSO 4 , 10 -5 % Methylene blue). E3 Blue is used to prevent fungal growth and ensure proper development of larval fish.…”

“…E3 Blue is used to prevent fungal growth and ensure proper development of larval fish. For more information on mating and embryo collection, refer to Gerlach 36 and Porazinkski 37 .Embed one-cell staged embryos in a warmed injection mold and inject the RNA into the yolk in the desired amount of embryos (Figure 1A). Account for natural embryonic death and unfertilized embryos by performing embryonic injections on 15% more embryos than needed for the experiment.…”

“…Account for natural embryonic death and unfertilized embryos by performing embryonic injections on 15% more embryos than needed for the experiment. For additional details on microinjection of zebrafish embryos, please refer to Gerlach 36 and Porazinkski 37 .NOTE: For first time use of mRNA, perform a dose-curve analysis to determine the optimal dose for fluorescence and viability (defined as no gross developmental defects up to 5 dpf) prior to performing 5D imaging. 150 - 200 ng/μl injected into embryos is often the optimal concentration, therefore it is a good starting point for the final concentration.…”

“…E3 Blue is used to prevent fungal growth and ensure proper development of larval fish. For more information on mating and embryo collection, refer to Gerlach 36 and Porazinkski 37 .…”