The overall objective of this proposal is to analyze the mechanisms regulating cell lineage determination and cell commitment during mammalian development. Specifically, we propose to explore the role of imprinted genes and the role of cell surface molecules in initiation and maintenance of specific embryonic lineages. Genomic imprinting, which occurs during gametogenesis in mammals, imposes different functional states upon maternally and paternally derived genomes. Accordingly, completion of normal embryogenesis requires the presence of both a maternally derived and paternally derived nucleus. Zygotic transfer of pronuclei shows that the trophectodermal lineages fail to develop in gynogenetic embryos, which lack the male genome, while the inner cell mass derived lineages fail to develop in androgenetic embryos, which only contain the male genome. Analysis of gynogenetic <-> normal and androgenetic <-> normal chimeras has demonstrate the cell autonomous nature of lineage failure. In addition, analysis of parthenogenetic <-> normal chimeras has demonstrated an apparent need for the male genome in formation of skeletal muscle. Specific cell surface molecules, which appear in a distinct place and time during embryogenesis, serve as unique cell lineage markers and, very likely, also mediate cellula interactions necessary for lineage formation. In order to examine the role of imprinting in lineage formation we propose to isolate embryonic stem (ES) cells from androgenetic, parthenogenetic, an gynogenetic blastocysts, test the ability of these cells to contribute to chimeras, and compare this with the developmental capacity of androgenetic and gynogenetic embryos. In this way we will address the stability and extent of imprinting in cultured stem cell lines and determine whether changes occur in imprinting which alter the capacity for lineage allocation Chimeras between gynogenetic and normal embryos will be analyzed at progressive developmental stages using in situ hybridization in order to determine at which stage of muscle lineage development cells from parthenogenetic embryos fail to participate. Myoblast cultures from chimeric embryos will be initiated and clones derived from cells of parthenogenetic and normal embryonic origin isolated. Comparison of gene expression in normal v. parthenogenetic myoblasts will pinpoint the genes which are differentially expressed (i.e. imprinted). In order to approach the role of cell surface molecules in early lineage allocation we will analyze the control of synthesis and the function of early embryonic antigens present in embryonic ectoderm and endoderm. We propose to clone the gene for the specific fucosyl transferase involved in synthesis of stag specific embryonic antigen 1 (SSEA-1). This will be achieved by expression cloning using mammalian expression vectors and a monoclonal antibody. The cloned gene will be characterized and its function in development determine using gain and loss of function induced mutations.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
2R01HD023291-04
Application #
3323374
Study Section
Human Embryology and Development Subcommittee 1 (HED)
Project Start
1987-09-01
Project End
1993-08-31
Budget Start
1990-09-01
Budget End
1991-08-31
Support Year
4
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Wistar Institute
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Latham, K E; Garrels, J I; Solter, D (1993) Two-dimensional gel analysis of protein synthesis. Methods Enzymol 225:473-89
Latham, K E; Solter, D (1993) Transplantation of nuclei to oocytes and embryos. Methods Enzymol 225:719-32
Latham, K E; Beddington, R S; Solter, D et al. (1993) Quantitative analysis of protein synthesis in mouse embryos. II: Differentiation of endoderm, mesoderm, and ectoderm. Mol Reprod Dev 35:140-50
Lamb, B T; Satyamoorthy, K; Solter, D et al. (1992) A DNA element that regulates expression of an endogenous retrovirus during F9 cell differentiation is E1A dependent. Mol Cell Biol 12:4824-33
Latham, K E; Solter, D; Schultz, R M (1992) Acquisition of a transcriptionally permissive state during the 1-cell stage of mouse embryogenesis. Dev Biol 149:457-62
Latham, K E; Garrels, J I; Chang, C et al. (1992) Analysis of embryonic mouse development: construction of a high-resolution, two-dimensional gel protein database. Appl Theor Electrophor 2:163-70
Latham, K E; Garrels, J I; Chang, C et al. (1991) Quantitative analysis of protein synthesis in mouse embryos. I. Extensive reprogramming at the one- and two-cell stages. Development 112:921-32
Latham, K E; Solter, D (1991) Effect of egg composition on the developmental capacity of androgenetic mouse embryos. Development 113:561-8
Lamb, B T; Satyamoorthy, K; Li, L et al. (1991) CpG methylation of an endogenous retroviral enhancer inhibits transcription factor binding and activity. Gene Expr 1:185-96
Latham, K E; Solter, D; Schultz, R M (1991) Activation of a two-cell stage-specific gene following transfer of heterologous nuclei into enucleated mouse embryos. Mol Reprod Dev 30:182-6

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