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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD029495-02
Application #
3330983
Study Section
Human Embryology and Development Subcommittee 1 (HED)
Project Start
1992-08-01
Project End
1995-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
2
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Sadler, T W; Merrill, Alfred H; Stevens, Victoria L et al. (2002) Prevention of fumonisin B1-induced neural tube defects by folic acid. Teratology 66:169-76
Burgoon, Jennifer M; Selhub, Jacob; Nadeau, Marie et al. (2002) Investigation of the effects of folate deficiency on embryonic development through the establishment of a folate deficient mouse model. Teratology 65:219-27
Fisher, M C; Zeisel, S H; Mar, M H et al. (2001) Inhibitors of choline uptake and metabolism cause developmental abnormalities in neurulating mouse embryos. Teratology 64:114-22
Sadler, T W (2000) Susceptible periods during embryogenesis of the heart and endocrine glands. Environ Health Perspect 108 Suppl 3:555-61
Foerst-Potts, L; Sadler, T W (1997) Disruption of Msx-1 and Msx-2 reveals roles for these genes in craniofacial, eye, and axial development. Dev Dyn 209:70-84
Sadler, T W (1997) Mouse embryos in culture: models for understanding diabetes-induced embryopathies and gene function. Int J Dev Biol 41:291-7
Augustine, K A; Liu, E T; Sadler, T W (1995) Antisense inhibition of engrailed genes in mouse embryos reveals roles for these genes in craniofacial and neural tube development. Teratology 51:300-10
Augustine, K A; Liu, E T; Sadler, T W (1995) Interactions of Wnt-1 and Wnt-3a are essential for neural tube patterning. Teratology 51:107-19
Sadler, T W; Liu, E T; Augustine, K A (1995) Antisense targeting of engrailed-1 causes abnormal axis formation in mouse embryos. Teratology 51:292-9
Augustine, K; Liu, E T; Sadler, T W (1993) Antisense attenuation of Wnt-1 and Wnt-3a expression in whole embryo culture reveals roles for these genes in craniofacial, spinal cord, and cardiac morphogenesis. Dev Genet 14:500-20