Morphogenesis involves changes in cell shape regulated by complex networks that control cell polarity, cytoskeleton rearrangements and adhesive properties. Neural tube closure (NTC) is a morphogenetic event, and neural tube defects (NTDs), including spina bifida and anencephaly, are the second leading cause of congenital disorders. In vertebrates, mutations in approximately 200 genes lead to NTDs, but aside from a few important examples, most NTD genes have unknown functions during NTC. The diversity of NTD genes is striking, and studying these genes may uncover novel components of known molecular pathways, or indicate new networks that are required for NTC. One major challenge in determining the roles of NTD genes is defining the cellular mechanisms disrupted in mutant phenotypes. Gastrulation in the nematode, Caenorhabditis elegans (C. elegans), represents a simple model that shares similar cell shape changes and cellular behaviors that occur during NTC. Thus C. elegans will be used to rapidly disrupt the function of NTD homologs by RNA interference (RNAi), and screen for potential regulators of cell shape changes by scoring for defects in gastrulation. Molecular and cellular mechanisms will be dissected in C. elegans and in the frog, Xenopus laevis (X. laevis) using the complementary strengths of each model system. Through this novel approach, the transcription factor sp8 was discovered to be required for gastrulation in C. elegans, and for NTC in X. laevis. Mutations in mouse sp8 result in severe NTDs in mice (Bell et al., 2003;Treichel et al., 2003), yet the etiology of NTDs is unknown. In addition, mutations in sp8 cause limb truncations, and studies in the limb bud suggest that sp8 is positively regulated by canonical/ 2-catenin Wnt signaling (Bell et al., 2003 and Kawakami et al., 2004). Thus, we hypothesize that sp8 is a conserved regulator of cell shape changes, and that sp8 expression is regulated by Wnt signaling in the neural tube.
In Specific Aim 1, the roles of sptf-3, the C. elegans homolog to sp8, will be analyzed in mediating gastrulation movements. in vivo imaging of membrane movements and actomyosin dynamics will be used to dissect cellular mechanisms.
In Specific Aim 2, the NTDs associated with sp8 morphants will be studied using time-lapse microscopy, histology, immunofluorescence, and explants studies to determine if cell shape changes are affected.
In Specific Aim 3, the relationship between Wnt signaling and sp8 expression in the neural tube will explored by analyzing sptf-3/sp8 expression in Wnt deficient backgrounds. These studies may indicate that the transcription factor sp8 is an important downstream factor that links canonical/2-catenin Wnt signals to cellular behaviors that are critical for NTC.
Spina bifida is a congenital birth defect that is caused by failure of the neural tube to close properly. A challenge in studying genes known to cause this disease is the underlying complexity of neural tube development. Therefore, I will study gene mutations that cause neural tube defects by using a tiny soil-dwelling worm, C. elegans, and the frog, Xenopus laevis, as simple systems to understand complex human diseases of the neural tube.