The proto-oncogenes are thought to play key roles in mammalian embryogenesis. However, data supporting this hypothesis are circumstantial and are based on investigations showing correlations between expression of genes and morphological events, and not on gene function. While homologous recombination in ES cells combined with transgenic mice technology has created gene """"""""knockouts"""""""" to determine proto-oncogene function; this technique is laborious and incapable of assessing gene function at specific points in time and space during development. Therefore we have developed a method to specifically inhibit the expression of individual genes in space and time using whole mammalian embryo culture. Normal development of mouse embryos is supported in vitro and antisense oligonucleotides are microinjected into the amniotic cavity and specific tissues. With this system, morphological phenotypes, including altered brain and cardiac development, associated with inhibition of Wnt-1 proto-oncogene expression have been determined. The defects are induced only by antisense probes and not by sense oligo's. Furthermore, their induction is restricted to injections made at the time of gene expression in the hindbrain region of neurulating embryos (5 somite stage: day 9; plug day = day 1). We propose to expand these results and to pursue the function of two other proto-oncogenes N-myc and ax1. These 3 proto-oncogenes were selected because their general pattern of expression has been determined in mouse embryos and suggests important roles for different aspects of organogenesis. Furthermore, the different spatial and temporal characteristics of their expression provide an opportunity to expand the technical capacity of antisense inhibition in studying murine organogenesis.
The specific aims will be: 1) to better define our understanding of when these 3 genes are expressed using in situ hybridization and immunocytochemistry; 2) to determine the effects of inhibiting expression of these 3 proto-oncogenes at specific stages of development using antisense probes. 3) to determine the biological effects of antisense inhibition of the proto-oncogene expression at the cellular level.
