Calcium-mediated repression of β-catenin and its transcriptional signaling mediates neural crest cell death in an avian model of fetal alcohol syndrome

Abstract

Fetal Alcohol Syndrome (FAS) is a common birth defect in many societies. Affected individuals have neurodevelopmental disabilities and a distinctive craniofacial dysmorphology. These latter deficits originate during early development from the ethanol-mediated apoptotic depletion of cranial facial progenitors, a population known as the neural crest. We showed previously that this apoptosis is caused because acute ethanol exposure activates a G protein-dependent intracellular calcium within cranial neural crest progenitors, and this calcium transient initiates the cell death. The dysregulated signals that reside downstream of ethanol’s calcium transient and effect neural crest death are unknown. Here we show that ethanol’s repression of the transcriptional effector β-catenin causes the neural crest losses. Clinically-relevant ethanol concentrations (22–78 mM) rapidly deplete nuclear β-catenin from neural crest progenitors, with accompanying losses of β-catenin transcriptional activity and downstream genes that govern neural crest induction, expansion and survival. Using forced expression studies we show that β-catenin loss of function (via dominant-negative TCF) recapitulates ethanol’s effects on neural crest apoptosis, whereas β-catenin gain-of-function in ethanol’s presence preserves neural crest survival. Blockade of ethanol’s calcium transient using Bapta-AM normalizes β-catenin activity and prevents the neural crest losses, whereas ionomycin treatment is sufficient to destabilize β-catenin. We propose that ethanol’s repression of β-catenin causes the neural crest losses in this model of FAS. β-Catenin is a novel target for ethanol’s teratogenicity. β-Catenin/Wnt signals participate in many developmental events and its rapid and persistent dysregulation by ethanol may explain why the latter is such a potent teratogen.

Fishing for Fetal Alcohol Spectrum Disorders: Zebrafish as a Model for Ethanol Teratogenesis

“…In both chicken and zebrafish, Flentke et al have shown that this ethanol-induced death is calcium and CaMKII dependent. 28,72 Collectively, these findings suggest that the mechanisms of facial deformity in FASD are conserved across vertebrates.…”

The avian embryo as a model for fetal alcohol spectrum disorder

“…Neural crest progenitors are induced immediately lateral to the neural plate, and thus a smaller prechordal and neural plate would also reduce the neural crest population size. Work in chick supports this mechanism, as gastrulation-stage exposure (50 mM, 20 mg/egg) reduces the expression of early neural crest markers including foxd3, wnt6 and snai2 (Flentke et al 2011), suggesting their suppressed induction and/or population shortfall.…”

“…Inhibition of other calcium-dependent kinases including protein kinase C and CaMKIV was ineffectual against alcohol, showing the pathway’s specificity. Ellies et al (2000) previously showed that the programmed cell death within a subset of neural crest progenitors depends on a non-canonical Wnt effector, and, following this lead, Flentke et al (2011) found that alcohol destabilizes β-catenin and its Wnt-dependent transcriptional activity within neural crest cells. The β-catenin destabilization is calcium-dependent and mediated not by known Wnt effectors including GSK3β and calpain, but rather by CaMKII (Flentke et al 2014).…”

“…Expression vectors work robustly in chick (apart from the thymidylate kinase promoter). Expression is readily manipulated using in ovo microinjection or electroporation of reporter plasmids, retrovirus constructs, morpholinos, and siRNA (Nakamura et al 2004; Flentke et al 2011), and most mammalian constructs produce equivalent outcomes in chick.…”

Bentonite protects neural crest stem cells from death caused by aflatoxin B1

“…Exposure to drugs or environmental chemicals can cause abnormal migration, differentiation, division or survival of NC cells and can contribute to multiple pathologies (Chen and Sulik, 2000;Garic-Stankovic et al, 2006;Wentzel and Eriksson, 2009;Flentke et al, 2011;Garic et al, 2011). Understanding the effects of bentonite on NC stem cells can help to prevent many NC-derived pathologies.…”