The retinoblastoma tumor suppressor (Rb) is one of the major inhibitors of cellular proliferation that functions Iargely through its binding to basic helix-loop-helix (bHLH) transcription factors of the E2F family, which it can convert from stimulators of transcription to repressors. Rb activity is regulated during the cell cycle through its phosphorylation by Cyclin/Cyclin dependent Kinase complexes. Rb has been linked through studies of transgenic and knockout mice to many cellular and developmental processes, including proliferation, apoptosis, hematopoesis, muscle differentiation, and numerous others. Our recent experiments indicate that Rb also functions in the differentiation of an embryonic organ - the placenta. Placental defects may be responsible for many of the phenotypes seen earlier in Rb mutant embryos, because when Rb-deficient embryos gestate with a normal placenta, they survive significantly longer than when they develop with an Rb-deficient one. We therefore propose to do the following:
Specific Aim 1. Carry out a mechanistic analysis of Rb functions in placentation. Rb mutant placentas evince developmental abnormalities. A histopathological analysis will be carried out to determine the molecular nature of these defects, and Rb-deficient trophoblast stem cells will be used to determine the function of Rb in the differentiation of placental cell types.
Specific Aim 2. To examine the impact of Rb-mediated placental dysgenesis on the development of Rb-deficient embryos. Further placental rescue experiments must be carried out to determine the function of Rb in the development of the mammalian embryo proper, both through tetraploid rescue and genetic experiments. Wild-type embryos will be produced with Rb mutant placentas to determine whether Rb is acting in a cell autonomous fashion within the embryo, or if the Rb-associated defects seen in embryonic development are actually due to the observed placental defects.
Specific Aim 3. Examine the genetic regulatory networks employed by Rb in the placenta. The bHILH factors E2F3, and 1d2 both exert functions in the placenta, and genetic deletion of either prevents the occurrence of many embryonic Rb phenotypes. Mutations in each of these genes will be bred onto an Rb mutant background, and doubly homozygous embryos will be generated. Their placentas will be examined to determine whether deletion of bHLH proteins remediates the Rb placental phenotype, in order to delineate the Rb-dependent genetic circuitry of placental development.
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| Li, Jing; Ran, Cong; Li, Edward et al. (2008) Synergistic function of E2F7 and E2F8 is essential for cell survival and embryonic development. Dev Cell 14:62-75 |
