Lectin teratogenesis II: Demonstration of increased binding of concanavalin a to limb buds of rabbit embryos during the teratogenically sensitive period

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Abstract
The plant lectin concanavalin A (con A) causes malformations of rabbit embryos when 160μg (in 40 μl) are injected into the exocoelom on gestational days 12–15 but does not cause malformations on days 10–11. The purpose of this study was to investigate the mechanism for increased susceptibility of day 12–15 embryos to con A teratogenicity. Light microscopy of day 11 embryos 15–20 hr after treatment with con A revealed no observable difference from controls. Day 13 embryos at similar times exhibited limb buds with large areas that were denuded of ectoderm. Concurrent addition of α‐methyl‐D‐mannoside (αMM), a specific inhibitor of con A, to the injection solution of day 13 embryos resulted in limb buds that appearded normal. The regions of con A binding to day 11 and day 13 embryos were visualized through epifluorescent microscopy of untreated embryos stained with fluorescein‐labelled con A. Day 11 embryos exhibited moderate fluorescence on the surface of limb buds and the pericardial region. Day 13 embryos exhibited strong fluorescence of limb bud surfaces; the pericardial region remained moderately fluorescent. Addition of αMM to the incubation medium resulted in no fluorescence above background. Visualization of con A receptors was accomplished by ultrastructural analysis of forelimb buds stained with ferritin‐labelled con A. Ferritin label was observed only on the surfaces of the ectoderm and was sparse over all regions of day 11 limb buds. In contrast, ferritin label was moderately heavy in all regions of the day 13 limb buds. No labelling occurred when the ferritin‐labelled con A was preincubated with αMM. These observations indicate that the number of exposed con A receptors on limb buds of teratogenically sensitive embryos (day 13) is increased, compared with the number of exposed receptors on limb buds of younger, insensitive (day 11) embryos. The increased number of exposed con A receptors on limb buds during the teratogenically sensitive period provides not only increased binding of the lectin to sensitive embryos but also a potential mechanism for the anomalous attachment of distal regions of the limb buds to the body wall.