A novel whole-embryo culture model for pharmaceutical and developmental studies.

INTRODUCTION

This report introduces a new vertebrate whole-embryo culture model for the direct application of pharmaceuticals and/or toxins into developing embryos. This method uses a terrestrial amphibian system and therefore has eliminated the problem of mammalian placental and uterine concerns. To test the utility and effectiveness of this method, we investigated the effect of fluoxetine on craniofacial development. Fluoxetine is one of the most commonly prescribed selective serotonin reuptake inhibitor (SSRI) on the market and treatment of depression during pregnancy is commonly deemed necessary. Previous studies have shown that SSRIs may promote developmental defects and congenital malformations of the heart.

METHODS

This model utilized the egg/embryos of the directly developing Puerto Rican coquí frog, Eleutherodactylus coqui. The E. coqui embryo clutches were placed on filter paper in a Petri dish and were directly exposed (chronically) to fluoxetine concentrations ranging from 0.10mM to 1.0mM. Traditional whole-mount bone (Alizarin red) and cartilage (Alcian blue) staining was utilized to show the effect of fluoxetine on craniofacial development.

RESULTS

Whole-mount staining revealed profound defects in cartilage development, particularly in the nasal capsule, mandible, and the brain case. Further, fluoxetine-treated embryos developed significantly slower compared to control animals.

DISCUSSION: We found that the E. coqui culture model was an effective and sensitive technique for pharmaceutical studies, particularly since it allows the direct application of drugs and toxins into the developing embryo without the hindrance of the uterus and placenta. Chromatographic analysis revealed that fluoxetine infiltrated and penetrated embryonic tissue. It was found that altering serotonergic activity during development, via fluoxetine, stunted craniofacial development and organization.