Characterizing Porcupine in Neural Tube Closure

Congenital birth defects such as spina bifida and anencephaly are two of the most common defects of the spine and brain respectively.  In fact, recent studies estimate that 1,500 babies are born with spina bifida in the United States annually.  These defects are caused by the incomplete closure of the embryonic neural tube.  Wnts are a family of secreted proteins involved in embryo development, and are expressed in the neural tube and surrounding tissues.  Porcupine is a member of the membrane-bound O-acyl transferase family and is involved in Wnt protein lipid modification, which is required for the secretion of Wnts.  Focal Dermal Hypoplasia, an X-linked dominant syndrome typified by multiple abnormalities, is caused by mutations in human Porcupine and includes neural tube defects.  These observations led to the hypothesis that pharmacological inhibition of Porcupine in the chick embryo model system will cause the neural tube to close improperly.  In order to explore and gain new insights into the role of Porcupine in neural tube closure, we will culture chick embryos in the absence (negative control) or presence of IWP-1, a pharmacological inhibitor of Porcupine, and will assay for the timing, extent, and location of neural tube defects.  We will also use a positive control ROCK inhibitor Y27632 due to its known effects on neural tube closure disruption.  Future experiments include the use of immunostaining and confocal microscopy to assess proliferation, axis of cell division, and cell morphology.