Abstract
Fetal alcohol exposure is known to cause an array of deficits (fetal alcohol spectrum disorders, FASD) that may manifest as lifelong physical, cognitive, and behavioral anomalies of human development. However, when and how alcohol specifically affects the auditory system is still largely unknown. I hypothesized that embryonic alcohol exposure at specific time periods would affect the morphology and function of the octavolateral system (the inner ear and lateral line). In addition, I also hypothesized that biological processes occurring at the time of alcohol exposure, such as the neural crest and FGF signaling, would be adversely affected. This is the first study to investigate both the specific developmental timing and underlying molecular mechanisms that are most sensitive to alcohol, relative to the developing octavolateral organs. To model fetal alcohol exposure, I used zebrafish (Danio rerio) embryos. Transgenic Et(krt4:EGFP)sqet4 zebrafish that express green fluorescent protein in sensory hair cells were treated in 2% alcohol for 2, 3, and 5-hours to determine an alcohol sensitive period. Using confocal and light microscopy, I found that alcohol-exposed larvae had significantly smaller otic vesicles and saccular otoliths than control larvae at the time of hatching. In addition to these effects, only larvae that were alcohol-exposed from 12-17 hours post fertilization (hpf) had fewer saccular hair cells, neuromasts, and hair cells per neuromast, with some effects that persisted throughout the first week post-fertilization. Auditory function was also assessed by microphonic potential recordings from inner ear hair cells in response to 200-Hz stimulation. Hearing sensitivity was reduced for alcohol-exposed larvae from 7-12 and 12-17 hpf. These results show that 12-17 hpf is an alcohol-sensitive time window when morphology and function of zebrafish octavolateral organs are most vulnerable to alcohol exposure. To uncover the mechanisms that underlie alcohol-induced inner ear defects, I examined vital biological processes that contribute to inner ear development and occur during the alcohol-sensitive period from 12-17 hpf. I assessed the effects of alcohol exposure on the neural crest, by examining inner ear expression of sox10, a well-known neural crest marker. I report a reduction of sox10 expression patterns at the time of hatching in alcohol-treated larvae. As Fibroblast Growth Factor (FGF) is the major signaling required for inner ear development, I first compared effects of FGF-inhibition and alcohol-treatment. With the use of heat-inducible FGF-inhibition embryos, Tg(HSP70:XFD), I found similar inner ear phenotypes. I then examined the effects of embryonic alcohol exposure on FGF signaling with an antibody label specific for phospholyrized extracellular signal-related kinase (pERK; a FGF signaling marker). My results demonstrate a reduction inner ear pERK expression after alcohol exposure. In addition, in situ gene expression analyses also showed a decrease in both a FGF targetgene,sprouty4andaFGF-dependentgene,pairedbox8. Pharmacological drugs were utilized to elucidate the molecular level at which alcohol affects the FGF pathway. BCI (an FGF signaling enhancer) was administered with alcohol, but failed to rescue adverse effects of alcohol. Treatment with SU5402 (an FGF signaling inhibitor) and alcohol resulted in inner ear defects similar to alcohol treatment alone. These results suggest that alcohol, like SU5402, may have an effect at the receptor level. Finally, in situ results revealed that alcohol did not affect gene expression of fgf3, a critical ligand for initiation of FGF signaling. The molecular portion of these studies provides an initial platform from which further studies may continue to unravel the complex and intricate mechanisms of embryonic alcohol damage to octavolateral organogenesis. Overall, this is the first study to reveal a specific time period of embryonic alcohol exposure that can lead to a disarray of molecular mechanisms and cause decreased hearing capacity, a completely preventable known characteristic of FASD.