Karen E. Boschen, Ph.D. - US grants

Abstract Fetal Alcohol Spectrum Disorders (FASD) affects up to 5% of births in the US each year and results in life-long physical and behavioral impairments. Neurulation-stage ethanol exposure (~ 4th week of pregnancy in humans, gestational days 8-10 in mice), is associated with a widening of the face and brain, particularly the ventral midline structures (e.g. septum, pituitary, ventricles), and neurofunctional changes later in life. This expansion is similar to the CNS and craniofacial abnormalities observed in ciliopathic genetic disorders such as Joubert?s syndrome. Ciliopathies are a consequence of defects in primary cilia, immotile sensory organelles critical for sonic hedgehog (Shh) pathway transduction and cell proliferation during development. Cilia dysfunction in some ciliopathies results in an overactivation of Shh, leading to the observed CNS anomalies. Previous work has suggested that mutations in cilia-associated motor proteins lead to ciliopathic phenotypes and interact with ethanol to cause wider brains (unpublished data). In this proposal, we use a well-characterized mouse model of FASD to test the hypothesis that ethanol exposure during neurulation induces a ?transient? ciliopathy in the embryo, leading to the shared phenotype between ciliopathies and FASD. Aim 1 analyzes primary cilia number and morphology in the neural tube following neurulation-stage ethanol exposure. Aim 2 investigates the mechanisms of these ciliary defects by examining cilia-related gene expression changes. Finally, Aim 3 examines whether neurulation-stage ethanol exposure alters cilia stability and function through analysis of tubulin post-translational modifications and Shh pathway signaling. Preliminary data suggest that ethanol- exposed embryos have altered expression of key ciliogenesis genes and an increased number of cilia in the neural tube. Presence of more cilia would lead to upregulated Shh signaling and abnormal cell proliferation, causing the observed CNS abnormalities. Importantly, these experiments will provide evidence supporting alterations to primary cilia number and function in the neural tube as a novel pathway through which ethanol exposure causes symptoms of FASD.

Project Summary Fetal Alcohol Spectrum Disorders (FASD) affects up to 5% of live births in the US each year and results in life- long physical, cognitive, and behavioral impairments. Alcohol exposure during neurulation, the formation and closure of the neural tube (~ 4th week of pregnancy in humans, gestational days 8-10 in mice), is associated with abnormal growth of midline structures, such as the cortex, septum, pituitary, and ventricles, and neurofunctional changes later in life. My preliminary work suggested that neurulation-stage alcohol causes cell cycle arrest or delayed cell cycle progression, resulting in disrupted proliferation and, ultimately, anomalous tissue and organ development. Specifically, we performed whole transcriptome profiling of the rostroventral neural tube 6 hr after alcohol exposure and found that many genes and gene networks related to cell cycle regulation and cell proliferation were altered by alcohol. In addition, neurulation-stage alcohol caused significant dysregulation of the sonic hedgehog (Shh) pathway and cell cycle genes. These changes in morphogenic signaling were concomitant with smaller rostral neural tube volumes and fewer actively dividing cells in alcohol-exposed embryos. In this proposal, we use a well-characterized mouse model of FASD to test the hypothesis that neurulation-stage alcohol exposure alters cell cycle regulation in the rostral neural tube through disruption of processes that regulate cell cycle progression. Aim 1 analyzes cell cycle arrest and G1- specific processes in the neural tube following prenatal alcohol. Preliminary data suggest dysregulation of molecular mechanisms that control the successful transition between cell cycle stages and the DNA damage response, possibly leading to impaired DNA integrity and replication errors. Aim 2 investigates pathways that control protein degradation and trafficking during the cell cycle, following up on previous work showing downregulation of genes encoding ubiquitylation enzymes by prenatal alcohol. Finally, Aim 3 examines epigenetic marks associated with chromatin that regulate cell cycle progression, as pathways related to chromatin modifications were found to altered by neurulation-stage alcohol in our preliminary studies. These experiments will provide evidence that mechanisms of cell cycle progression represent an under-studied pathway through which prenatal alcohol causes symptoms of FASD.