The overall objective of this research is to analyze expression of class I products of the Major Histocompatibility Complex (MHC) in the developing mouse embryo. In addition to the MHC class I H chain and beta2-microglobulin, immunologists now appreciate that antigenic peptide is an essential structural component of the class I molecule. Our recent studies have shown that the peptide transporter molecules required for MHC class I assembly are developmentally regulated. A major aim of this proposal is to characterize expression of the components of the MHC class I peptide-loading machinery required for MHC class I surface expression in early embryos. In situ hybridization experiments will analyze the temporal and spatial pattern of expression of MHC-encoded transporter molecules and proteasome components. To examine the functional role of HAM1 in vivo, we will disrupt the HAM1 locus by homologous recombination in ES cells and introduce the null mutation into the germline. Our recent experiments demonstrated that MHC class I and beta2m gene expression is not coordinately regulated at early stages of post-implantation development. Indeed we found that the secondary trophoblast giant cells strongly express MHC class I but no detectable Beta2m mRNA. A major goal is to further characterize the individual class I transcripts and to determine which MHC class I gene products are expressed. In addition, recent studies indicate that over expression of H-2D-d transplantation antigens in ES cell chimeras results in prenatal lethalities. To further examine teratogenic effect(s) of ectopic H2D-d expression in early mouse embryos, we will determine whether embryonic lethalities are associated with aberrant assembly of class I molecules and/or can be attributed to over-expression of intracellular H chains. We will also examine the consequence(s) of MHC class I expression in specific target organ(s) using tissue-specific promoters. We will introduce the H-2D-d gene under control of the AFP and mPLII promoters to direct expression to the visceral yolk sac endoderm and secondary trophoblast giant cells respectively. Should our efforts to obtain transgenic strains be successful, the next goal will be to determine whether the H-2D-d chains are associated with beta2m and transported to the cell surface. Should the transgenic embryos fail to develop to term, we will investigate the possibility that ectopic MHC gene expression in these extra-embryonic cell lineages may trigger a maternal immune response directed against the allogeneic fetus. A particularly important point will be to test whether maternal lymphocytes infiltrate and disrupt the abnormal embryos. These experiments will hopefully lead to a clearer understanding of regulation of MHC gene expression during early mouse development and processes involved in maternal-fetal interactions.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD025926-06
Application #
2199761
Study Section
Human Embryology and Development Subcommittee 1 (HED)
Project Start
1989-08-01
Project End
1997-11-30
Budget Start
1993-12-01
Budget End
1994-11-30
Support Year
6
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Harvard University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
071723621
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Otten, G R; Bikoff, E; Ribaudo, R K et al. (1992) Peptide and beta 2-microglobulin regulation of cell surface MHC class I conformation and expression. J Immunol 148:3723-32
Jaffe, L; Robertson, E J; Bikoff, E K (1992) Developmental failure of chimeric embryos expressing high levels of H-2Dd transplantation antigens. Proc Natl Acad Sci U S A 89:5927-31
Bikoff, E K; Otten, G R; Robertson, E J (1991) Defective assembly of class I major histocompatibility complex molecules in an embryonic cell line. Eur J Immunol 21:1997-2004
Jaffe, L; Robertson, E J; Bikoff, E K (1991) Distinct patterns of expression of MHC class I and beta 2-microglobulin transcripts at early stages of mouse development. J Immunol 147:2740-9
Jaffe, L; Jeannotte, L; Bikoff, E K et al. (1990) Analysis of beta 2-microglobulin gene expression in the developing mouse embryo and placenta. J Immunol 145:3474-82