Diffusion of small molecules into medaka embryos improved by electroporation

Abstract

BackgroundDiffusion of small molecules into fish embryos is essential for many experimental procedures in developmental biology and toxicology. Since we observed a weak uptake of lithium into medaka eggs we started a detailed analysis of its diffusion properties using small fluorescent molecules.ResultsContrary to our expectations, not the rigid outer chorion but instead membrane systems surrounding the embryo/yolk turned out to be the limiting factor for diffusion into medaka eggs. The consequence is a bi-phasic uptake of small molecules first reaching the pervitelline space with a diffusion half-time in the range of a few minutes. This is followed by a slow second phase (half-time in the range of several hours) during which accumulation in the embryo/yolk takes place. Treatment with detergents improved the uptake, but strongly affected the internal distribution of the molecules. Testing electroporation we could establish conditions to overcome the diffusion barrier. Applying this method to lithium chloride we observed anterior truncations in medaka embryos in agreement with its proposed activation of Wnt signalling.ConclusionsThe diffusion of small molecules into medaka embryos is slow, caused by membrane systems underneath the chorion. These results have important implications for pharmacologic/toxicologic techniques like the fish embryo test, which therefore require extended incubation times in order to reach sufficient concentrations in the embryos.

Differential uptake of and sensitivity to diphenhydramine in embryonic and larval zebrafish

“…The channels, or pores, in the membrane have an average diameter of 1.5 mm and occur every roughly 1.5 to 3 mm [33,34]. Previous studies with fluorescent dyes in medaka suggest that the embryonic membrane may limit embryo exposure to some chemicals to a greater extent than the chorion [35]. To avoid potential experimental complications, methods for the removal of the chorion have been published [26] and proposed to improve the FET [36].…”

The function of tcf3 in medaka embryos: efficient knockdown with pePNAs

“…Such an anti-repressive effect would be equivalent to ectopic Wnt activation. Indeed, we have previously shown that induction of Wnt signaling leads to the formation of ectopic otic vesicles [ 38 , 53 ].…”

Toxicity Evaluation of Acrylamide on the Early Life Stages of the Zebrafish Embryos (<i>Danio rerio</i>)

“…The diffusion of the acrylamide in the zebrafish embryo was extremely slow in which slow infusion of a chemical is a protective mechanism of the outer chorion and membrane systems that protect the embryo from chemical insult [20].…”