The primary goal of this research project is to delineate genetic controls of development of the embryonic axis and patterning of the neural tube in mammalian embryos. Genes postulated to play key roles in these processes, including Wnt-1 and 3a; Engrailed-1; nodal: hedgehog; and hepatic nuclear factors alpha and beta, will be disrupted in mouse whole embryo culture during gastrulation and neurulation stages of development using antisense oligonucleotides.
Specific Aims i nclude: 1. To determine the function of Wnt-1 and 3a, engrailed-1, Sonic hedgehog, nodal, and hepatic nuclear factors alpha and beta in axis formation and neural tube patterning. a. Antisense oligonucleotides targeted to each of the genes will be microinjected into the amnion of 3-5 somite stage mouse embryos undergoing the processes of neurulation and gastrulation. Embryos will be grown in whole embryo culture and the effects of the targeted gene disruptions on development will be monitored. b. Functional redundancies between genes and the interaction of genes in signalling pathways responsible for axis formation and neural tube patterning will be determined by targeting 2 or more genes simultaneously. c. Effects of targeted antisense exposures on gene expression will be monitored using quantitative PCR, quantitative in situ hybridization, and immunohistochemical procedures. 2. To determine the morphological alterations at the cell and tissue level that are altered by a targeted gene's disruption and that contribute to the abnormal phenotype. These studies will be completed using detailed histological and scanning electron microscopic techniques. 3. To determine the underlying biological effects produced by targeted gene disruption that may play a role in production of altered phenotypes. These studies will include analysis of cell death and cell proliferation patterns. Together these studies will provide information on the functional roles of these genes during embryogenesis, assist in defining genetic signals responsible for key morphogenic events, and identify cellular processes involved in development at stages when many birth defects are induced. In turn, identification of roles for these genes will provide data that will be important in defining the origins of birth defects, such as spina bifida and caudal dysgenesis.
