An in ovo chicken model to study the systemic and localized teratogenic effects of valproic acid.
BACKGROUND
The antiepileptic valproic acid (VPA) is a teratogen whose embryopathic mechanism(s) remain uncertain. Elucidating potential cellular and molecular effects of VPA is complicated by systemic application paradigms. We developed an in ovo model to reproduce the teratogenic effects of VPA and a localized VPA application procedure to determine whether VPA can selectively effect abnormal development in one region of the embryo.


METHODS
VPA was applied topically to chicken embryos in ovo at different embryonic stages. Embryos were later evaluated for gross and skeletal anomalies. Pax-2 and Pax-6 protein expression in the developing eye was also evaluated because VPA-induced eye anomalies are similar to those seen by the disruption of Pax-2 and Pax-6. For localized application, a thin sheet of the synthetic polymer Elvax was impregnated with VPA. A small piece of the VPA-impregnated polymer was applied directly to the presumptive wing bud region in Stage 10-17 embryos. Embryos were examined for gross and skeletal anomalies. Sham controls were employed for all experiments.


RESULTS
Chicken embryos exposed to VPA in ovo demonstrated increased mortality, growth delay and anomalies similar to ones previously seen in humans: neural tube, cardiovascular, craniofacial, limb and skeletal. Pax-2 and Pax-6 protein expression was qualitatively diminished in the eye. Localized wing bud VPA exposure caused structural abnormalities in the developing wing in the absence of other anomalies in the embryos. These wing defects were similar to those observed after topical whole-embryo VPA application.


CONCLUSIONS
These results indicate that at least one mechanism for the teratogenicity of VPA involves a direct effect on developing tissue. The nature of the abnormalities observed implies that this effect may be mediated by disruption of genes that regulate pattern formation.
D000818:Animals; D001842:Bone and Bones / Q000002:abnormalities / Q000000981:diagnostic imaging / Q000196:embryology; D002642:Chick Embryo; D004396:Coloring Agents / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D004625:Embryo, Nonmammalian / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005136:Eye Proteins; D018398:Homeodomain Proteins / Q000096:biosynthesis; D007150:Immunohistochemistry; D051762:PAX2 Transcription Factor; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D011859:Radiography; D012097:Repressor Proteins; D013723:Teratogens; D045725:Tetrapyrroles; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis; D014635:Valproic Acid
D004396:Coloring Agents; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005136:Eye Proteins; D018398:Homeodomain Proteins; D051762:PAX2 Transcription Factor; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D013723:Teratogens; D045725:Tetrapyrroles; D014157:Transcription Factors; C044886:alcian green; D014635:Valproic Acid