Primary cilia dysfunction as a novel pathogenic mechanism of prenatal alcohol-induced birth defects

Neurulation-stage alcohol exposure (NAE; gestational day [GD] 8-10) is associated with midline craniofacial and CNS defects that likely arise from disruption of morphogen pathways, such as Sonic hedgehog (Shh). We tested whether NAE alters Shh pathway signaling and the number and function of primary cilia, organelles critical for Shh pathway transduction. Notably, midline anomalies are also a hallmark of genetic ciliopathies such as Joubert9s syndrome. Female C57BL/6J mice were administered two doses of alcohol (2.9 g/kg/dose) or vehicle on GD9. Embryos were collected 6, 12, or 24 hr later, and changes to Shh, cell cycle genes, and primary cilia were measured in the rostroventral neural tube (RVNT). At 6 and 12 hr post-NAE, reductions in Shh pathway and cell cycle gene expression and the ratio of Gli3 isoforms in the full-length activator state were observed. RVNT volume and cell layer width were reduced at 12 hr. In addition, expression of multiple cilia-related genes were observed at 6 hr post-NAE. As a further test of cilia gene-ethanol interaction, mice heterozygous for Kif3a exhibited perturbed behavior during adolescence following NAE compared to vehicle-treated mice, and Kif3a heterozygosity exacerbated the hyperactive effects of NAE on exploratory activity. These data demonstrate that NAE downregulates the Shh pathway in a region of the neural tube that gives rise to ventral midline brain structures sensitive to alcohol and suggests primary cilia dysfunction, or a "transient ciliopathy", as a cellular mechanism of prenatal alcohol pathogenesis.