Stage‐Dependent Effects of Ethanol on Cranial Neural Crest Cell Development: Partial Basis for the Phenotypic Variations Observed in Fetal Alcohol Syndrome

Abstract

Fetal alcohol syndrome (FAS) is characterized by growth retardation, mental deficiencies, and numerous craniofacial and neuronal anomalies; the type and severity of these defects may be related to the time and dose of maternal ethanol exposure. Ethanol administered during presomitic stages results in the typical FAS craniofacial phenotype and is accompanied by a loss of cranial neural crest cells (CNCCs) through ethanol-induced cell death. However, the stage-specific effects of ethanol on the CNCC population is unknown. We examined the effects of ethanol on CNCC populations by treating in ovo chick embryos with a single ethanol dose (0.43 mmol/egg) at various stages of CNCC development, and corresponding to the first 3-4 weeks of human gestation. Ethanol treatment induced cell death and reduced CNCC populations in patterns consistent with observed dysmorphologies of CNCC-derived cranial structures. The precise population affected was dependent on the timing of ethanol exposure. Treatment at gastrulation or neurulation induced cell death and losses of CNCC populations, particularly those in rostral positions, and resulted in more severe craniofacial defects. In contrast, treatment at early somitic stages (4-16 somites) induced cell death, primarily within caudal CNCC populations, but resulted in less severe craniofacial defects, suggesting an increased capacity for recovery. These results suggest that there are distinct developmental windows during which the CNCCs may be particularly susceptible to ethanol-induced cell death. We conclude that ethanol exposure seems to affect specific events adversely during neural crest development. The timing of embryonic ethanol exposure relative to CNCC development could account, in part, for the heterogenous craniofacial defects observed in FAS.

Sonic hedgehog rescues cranial neural crest from cell death induced by ethanol exposure

“…The reduction in head size and the loss of neural crest cells was previously attributed to apoptotic death of cranial neural crest cells (6). To confirm neural crest cell death after ethanol exposure, we measured the percentage of pyknotic nuclei in the Fig.…”

“…Both treatments result in a reduction in head size and selective apoptosis of cranial neural crest cells. The neural crest cell death observed after blocking Shh signaling in the chick (1) is similar to that observed after ethanol exposure in a number of species including mouse, chick, and quail, both in vivo and in vitro (6)(7)(8)(9)(10). However, the mechanisms by which alcohol affects selective target tissues remain unknown.…”

“…Adapting a previously established protocol as a model for fetal alcohol syndrome in the chick (6,7,19), eggs were injected at stage 9-10 with sufficient ethanol to achieve an 0.15% alcohol level to total egg volume (20 Table 1). Total growth inhibition did not reach statistical significance at early stages (i.e., 3 days post treatment; Table 1).…”

“…In preliminary experiments, unopened eggs were injected with 200 l of a solution of 10% ethanol in Ringer's solution, or with Ringer's solution alone, at 26 h of incubation. This procedure has been previously demonstrated to result in craniofacial abnormalities (6). We then modified this procedure, such that eggs were opened at stage 9-10, and 20 l of a 1% ethanol solution (in Ringers) was placed between the vitelline membrane and the embryo.…”

Effects of Alcohol During Secondary Neurulation in Chick Embryos

“…Ethanol-induced anomalies are closely related to the timing of exposure. Namely, in chick embryos for example, ethanol exposure at early gastrulation through neurulation stages induces cranial defects (24,25) . However, exposure at post neurulation stages causes significant growth retardation.…”