The major aim of this proposal is to determine whether in utero exposure to ?9-tetrahydrocannabinol (THC) inhibits Hedgehog (Hh) pathway signaling to cause defects in CNS development. Sonic (Shh) signaling regulates many aspects of CNS development, including defining the early midline of the forebrain, patterning of neuronal cell fates in the ventral neural tube and telencephalon, and axon guidance and synapse formation. Therefore, inhibition of Shh signaling, via genetic deficiency and/or teratogen exposure at specific times during development, may lead to a variety of CNS defects. For example, Shh signaling deficiency results in holoprosencephaly (HPE), the most common malformation of the forebrain and midface. HPE is associated with heterozygous mutations in components of the Hh pathway. Clinical presentation is extremely variable, encompassing a spectrum of phenotypes ranging from forebrain defects to mild deficits in the midfacial midline. Furthermore, many mutation carriers are unaffected, but offspring who inherit such mutations are at high risk of HPE. These observations have led to a multifactorial, ?mutation plus modifier? model, whereby HPE-associated mutations interact with more common modifiers, genetic and/or environmental, to produce severe phenotypes. We have developed models of HPE in mice that fit this paradigm. Cdon encodes a Shh coreceptor, and loss of function mutations in CDON have been found in HPE patients. Cdon mutant mice have a largely subthreshold defect in Shh signaling and are sensitive to additional insults that result in a broad spec- trum of HPE phenotypes. We have identified both genetic and environmental modifiers with these mice. It was recently reported that endocannabinoids and phytocannabinoids, including THC, inhibit Hh signaling. This raises the possibility that in utero THC exposure may promote HPE, particularly in genetically susceptible indiv- iduals. Indeed, our preliminary results show that THC induces HPE in Cdon mutant mice. Although HPE is the most common outcome of genetic deficiency in Shh signaling, this is because it results from disruption of a very early patterning event, and HPE is usually lethal. Exposure to Hh pathway inhibitors during development after the window of sensitivity for HPE would be predicted to disrupt later Shh-dependent processes, including neural patterning. Defects in such processes may result in cognitive or behavioral outcomes in later life. To determine if in utero exposure to THC inhibits Shh signaling to cause defects in CNS development, two specific aims are proposed: 1) to analyze THC-induced HPE; and 2) to analyze THC-induced defects in ventral neural tube development. These studies will test whether THC inhibits Shh signaling in vivo to contribute to neurodevelopmental defects. Marijuana is the illicit drug most commonly used by pregnant women, so the implications for public health are substantial, as behavior modification would be a preventive mechanism.