Retinoic acid-induced spina bifida: evidence for a pathogenetic mechanism

ABSTRACT Treatment of C57B1/6J mice with three successive doses of all-trans retinoic acid (28 mg kg-1 body weight) on 8 day, 6h (8d,6h), 8d,12h, and 8d,18h of gestation resulted in a high incidence (79%, 31/39 fetuses) of spina bifida with myeloschisis (spina bifida aperta) in near term fetuses. Twelve hours following the last maternal dose (9d,6h), the caudal aspects of treated embryos, were abnormal, with eversion of the neural plate at the posterior neuropore, as compared to its normal concavity in comparably staged control specimens. This eversion persisted in affected embryos through the time that the posterior neuropore should normally close. The distribution of cell death in control and experimental embryos was determined using vital staining with Nile blue sulphate and with routine histological techniques. Twelve hours following the maternal dosing regimen, experimental embryos showed evidence of excessive cell death, predominantly in the mesenchyme associated with the primitive streak and in the endoderm of the tail gut, both of which are readily identifiable sites of physiological cell death at this stage of development. In addition, the presumptive trunk neural crest cells located in the dorsal midline, cranial to the posterior neuropore, exhibited a marked amount of cell death in the experimental embryos. We propose that the major factor in the generation of spina bifida in this model is excessive cell death in the tail gut and mesenchyme ventral to the neuroepithelium of the posterior neuropore. This causes a disparity in growth between the ventral and dorsal regions of the tail causing the relatively faster growing dorsal region (the neural plate) to evert, preventing caudal neural tube closure. Mesenchymal deficiencies resulting from excessive cell death in the gastrulating mesoderm would also account for the lack of tail formation and the abnormally formed lumbosacral vertebrae observed in this model. Previous investigations have suggested that the pathogenesis of a number of retinoic acid-induced malformations is related to excessive cell death in regions of physiological cell death. The current investigation explores the role of this phenomenon in the pathogenesis of experimental spina bifida.