Regulation of early trophoblast differentiation and development
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[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Placenta is a transient organ which functions in multiple ways to support the growth and survival of the developing fetus. Placenta plays a critical role in nourishing the fetus throughout pregnancy, and also produces hormones that alter maternal physiology, and play a key role in maternal recognition of the developing conceptus. Maternal recognition of pregnancy requires a signal for the recognition of the conceptus by the mother and extension of the ovarian corpus luteum (CL) life-span. These events must occur in a coordinated manner if pregnancy is to be established. These studies discuss the development and differentiation of the early trophoblast cells of the placenta, and the regulation of their function in two different model systems. Interferon tau (IFN-[tau]) is the signal for maternal recognition of pregnancy in ruminants. IFN-[tau] is expressed as early as the blastocyst stage. Production is increased during the peri-implanation period and decreased at around the time of implantation. Hormones and growth factors secreted by the maternal endometrium may be required for the optimal production of IFN-[tau]. The expression of IFN-[tau] gene is controlled by a key transcription factor, Ets2. In the work presented in Chapter 2, I have examined the role of the protein kinase A (PKA) signal transduction pathway in regulating IFN-[tau] expression through the activation of Ets2. Although over-expression of the catalytic subunit of PKA or the addition of 8-bromo-cyclic AMP had a modest ability to up-regulate bovine IFN-[tau]1 reporter constructs (usually no more than 4-fold), co-expression of PKA and Ets2 led to a more than 500-fold increase in gene expression . Progressive truncation of reporter constructs was conducted to define the region of the IFN-[tau] promoter responsible for the synergism between PKA and Ets2. These experiments indicated that the responsive region lay between -126 and -67, which lacked a classical CRE but included the Ets2/AP1 element. Specific mutation of the Ets binding site reduced the PKA/Ets2 effects by more than 97 %, whereas mutation of the AP1 binding site adjacent to the Ets2 site led to a doubling of the combined Ets2/PKA effect (to nearly 1000 fold). Pharmacological inhibition of MEK2 kinase (PD98059) had a similar effect, suggesting there is competition between the Ras/MAPK pathway and the PKA signal transduction pathway for Ets2. I also investigated the role of the Dlx3 binding site identified upstream of the Ets binding site in mediating the effects of PKA. Mutation of the Dlx3 binding site reduced the PKA/Ets2 effects by about 87%, and activity was virtually abolished when both the Ets and Dlx3 binding sites were mutated. These results suggest that both Dlx3 and Ets2 must be bound to the proximal promoter for PKA to activate transcription. Subsequent work indicated that Ets2 itself is not a substrate for PKA and that the effect is indirect and involves one or more co-activators. Human embryonic stem cells provide a great model to study the development process including that of the placenta. The biggest obstacle to culture of hES cells is the problem of cells differentiating spontaneously under standard culture conditions which include 20% O[subscript 2]. In Chapter 3, I have collaborated with Dr. Ezashi to investigate the effects of culturing hES cells under low O[subscript 2] tension as the normal blastocyst from which these hES cells are derived develops in a low O[subscript 2] environment. I have used immunohistochemical staining for specific pluripotent markers Oct-4 and SSEA 3 and differentiation marker SSEA 1 to assess the effect of low oxygen tension on the development of hES cells. We observed that low O[subscript 2] tension prevents the spontaneous differentiation of human embryonic stem cells in culture. hES cells have the potential to develop into trophoblast with the addition of bone morphogenetic protein BMP4 in culture. Fibroblast growth factor FGF2 and low O[subscript 2] promote pluripotency in hES cells. In Chapter 4 I have demonstrated that the development of trophoblast from hES cells is faster in the absence of FGF2 and ambient O[subscript 2]. I have assessed the pattern of expression of pluripotent marker Oct-4, differentiation marker SSEA 1, trophoblast-specific markers such as cytokeratin 7 and 8, HLA-G, hCG α and ß subunits to assess the progress and extent of differentiation in response to BMP4. In addition, I have studied the two-dimensional distribution pattern of transcription factors, Cdx2 and GATA2, involved in trophoblast differentiation and development.and HIF 1a and 2a, which are considered to mediate changes in the expression of the oxygen-regulated transcription factors. I demonstrate that hES cells treated with BMP4 in absence of FGF2 differentiate unidirectionally towards trophoblast and, in presence of ambient O[subscript 2], form the syncytiotrophoblast sublineage. Therefore, hES cells in vitro mimic the pattern of trophoblast development and differentiation in vivo and can be used as a model to study the development of trophoblast.
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