Microinjection of cultured rat embryos: a new technique for studies in chemical dysmorphogenesis

The utility of a new technique for exposure of cultured whole rat embryos to potential dysmorphogens was demonstrated with nitrosofluorene (NF), a cytotoxic and mutagenic metabolite of 2-acetylaminofluorene (AAF). At an initial concentration in the culture medium of 41 microM, NF produced a 100% incidence of defects in axial rotation with no significant effect on prosencephalic development, consistent with previous reports. This route of exposure was also associated with a significant decrease in yolk sac vasculature and protein content. However, when 2 to 20 ng of NF was microinjected directly into the amniotic space, the predominant malformation observed was prosencephalic hypoplasia. Injection of 10 ng NF resulted in approximately equivalent decreases in viability as 41 microM NF dissolved in the culture medium, but produced only a 41% incidence of rotation defects and a 27% incidence of open neural tubes in the rhombencephalic region. The protein content of injected conceptuses was significantly reduced in the embryo, but not in the visceral yolk sac. When 10 ng of NF was injected inside the yolk sac but outside the amnion, the incidence of abnormal rotation was increased to 75%, and the severity of prosencephalic hypoplasia as well as the incidence of neural tube abnormalities was attenuated. The protein content of both the embryo and yolk sac was significantly decreased relative to that of the controls. The data are consistent with the suggestion that NF elicits defects in axial rotation primarily via its effects on the visceral yolk sac and demonstrate the capacity of this technique to provide insights into mechanistic aspects of chemical dysmorphogenesis.