Many of the structural and functional abnormalities associated with Fetal Alcohol Spectrum Disorders (FASD)
have been uncovered, yet major gaps remain in our understanding of the associated pathogenesis and
mechanisms. For example, it is well known that ethanol exposure during gastrulation results in the classic
hypoteloric FAS face and midline forebrain dysgenesis; yet, exposure just slightly later, during neurulation,
induces expanded midline brain structures and hypertelorism. Interestingly, these abnormalities resemble
(phenocopy) those of many genetic ciliopathies, such as Joubert syndrome. The central pathogenic mechanism
of ciliopathies is a perturbation of the structure and/or function of primary cilia, hair-like organelles found on most
cells that integrate extra- and intra-cellular signals. The proposed research tests the overall novel hypothesis
that neurulation-stage ethanol exposure induces a “transient ciliopathy” (i.e., a temporary disruption of primary
cilia function) that is the basic cellular mechanism for the expansion of midline brain structures and hyperteloric
dysmorphologies. The proposed experiments are designed to meet the following integrated specific aims. Aim
1 will define the direct effects of early prenatal ethanol exposure on primary cilia structure and function.
For this, confocal microscopy and immunohistochemistry will be used to examine the effects of ethanol on
primary cilia number while gene expression assays will be used to assess cilia function. It is hypothesized that
ethanol exposure causes abnormal ciliary number and/or function, reducing activation of the Shh signaling
pathway. Aim 2 will characterize the secondary cellular pathogenic events in the neural tube resulting
from an ethanol-induced transient ciliopathy. The experiments in this aim will test the hypothesis that the
ethanol-induced transient ciliopathy and subsequent down-regulation of the Shh pathway will decrease
downstream cell proliferation genes in the ventral neural tube and expand morphogen gradients that pattern the
dorsal neural tube. Following ethanol exposure, genes with known roles in cell proliferation will be assessed
using qRT-PCR and the gradients of ventral and dorsal morphogens will be assessed using in situ hybridization.
These data will help to determine the precise mechanisms by which ethanol alters development. Aim 3 is to
determine the primary cellular mechanistic events underlying an ethanol-induced transient ciliopathy.
This final Aim will use RNA-seq to determine in an unbiased manner how ethanol disrupts normal ciliogenesis
by examining the total transcriptomic profile at several time points immediately following ethanol exposure. We
hypothesize that ethanol will alter key ciliogenesis genes; however, using this non-biased approach will aid in
identifying other potential changes. Finally, we test the alternative/complementary hypothesis that ethanol alters
tubulin post-translational modification, thereby disrupting normal cilia stability and function. Together, these novel
experiments will provide fundamental insights into the pathogenic mechanisms underlying the effects of ethanol
exposure during development, and propel alcohol research into new primary ciliary-related studies.