Trophoblast cell lineages form the outer epithelial component of the placenta and provide the functional bridge between the fetus and the mother. Multiple transcription factors have been implicated in the development and function of trophoblast cell subtypes including trophoblast stem (TS) cells. For example, transcription factor caudal-type homeobox 2 (CDX2) is important for the derivation of trophectoderm and proliferation of TS cells. Interestingly, transcription factors, GATA2 and GATA3, have also been implicated in the transcriptional regulation of trophoblast cell-specific gene expression. However, molecular mechanisms of GATA factor function in trophoblast cell lineages are poorly understood. Although the expression of several placental genes is reduced in Gata2-/- and Gata3-/- mice, the lack of an overt placental phenotype led to the prediction that these two factors might function in a complementary fashion during placental development. However, experiments have not been done in a context where both GATA2 and GATA3 are limiting. In addition, GATA factor expression pattern during trophoblast lineage differentiation, their mode of function in endogenous chromatin domains, and their target genes in trophoblast cells are poorly understood. We identified a novel function of GATA3, in which it is selectively expressed in the trophectoderm (TE) of developing mouse embryo, regulates expression of key genes in TE lineage, and is involved in morula to blastocyst transformation. In addition, we demonstrated that, during trophoblast giant cell differentiation, switch in chromatin occupancy between GATA3 and GATA2 is an important mechanism for the transcriptional regulation of GATA target genes including Cdx2. Therefore, we will utilize novel strategies to elucidate role of GATA factors in trophoblast cell differentiation and function.
Three specific aims are proposed.
Aim 1 will test the hypothesis that GATA factors regulate Cdx2 expression during development of the trophoblast lineage. We will examine this by determining GATA2 and GATA3 expression pattern in pre-implantation mice embryos, by identifying GATA factor binding regions at the endogenous Cdx2 chromatin domain in trophoblast cells, and by generating a genomic Cdx2 reporter.
Aim 2 will determine the role of GATA factors during trophoblast cell development and differentiation both in vitro and in vivo. We will test the hypothesis that loss of both GATA2 and GATA3 will impair trophoblast lineage differentiation. We will test this by establishing a TS cell system, in which both GATA2 and GATA3 will be limiting (in vitro approach), and by knocking-down GATA2 and GATA3 by RNA interference in the trophoblast cell lineage in vivo.
In aim 3, using a chromatin immunoprecipitation coupled with genomic microarray approach, we will test whether GATA2 and GATA3 have common and unique target genes in trophoblast cells. We will also determine whether GATA3 have differentiation stage specific target genes.
Early pregnancy loss is a major public health concern. One of the major causes of early pregnancy loss is defective function of the trophectoderm. In addition, defects in placental development due to impaired trophoblast cell functions lead to pregnancies that are at risk for miscarriage and intrauterine growth retardation, and are associated with preeclampsia, a leading cause of maternal death and premature birth. Therefore, understanding the molecular mechanisms of development and function of trophoblast cell lineages is critical to gaining insights into important features of pregnancy loss and pregnancy associated disorders.
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