Placental pathologies stem from poor early placental development characterized by shallow invasion of trophoblasts, and are also associated with aberrant expression of miRNAs belonging to the primate-specific chromosome 19 miRNA cluster (C19MC). C19MC miRNAs are thought to have roles in trophoblast invasion and migration, however, their role(s) in primate embryonic and early placental development is not well-defined. I hypothesize that C19MC miRNAs will have primate-specific roles in trophectoderm lineage specification and early development of the primate placenta. The overall objective of my proposal is to determine the role of these miRNAs in primate trophoblast lineage specification and placental development in a rhesus macaque model, and reveal gene networks regulated by miRNAs in primate placentation. Towards this objective, I propose three Specific Aims.
Specific Aim 1 (K99-phase). To define the expression of miRNAs in the primate embryo and trophoblast stem cells (TSC).
This aim will establish the miRNA signature during embryo development through specification of the trophectoderm lineage and determine whether these miRNAs are expressed in a stage- or cell-type-specific manner.
Specific Aim 2 (K99-phase). To determine the functional role(s) of C19MC members in TSC and differentiated trophoblast function. This experiment will directly overexpress C19MC miRNA members in TSC to identify genes and pathways regulated by these miRNAs, assessing their functional roles.
Specific Aim 3 (R00-phase). To use genome editing strategies to globally perturb the C19MC and evaluate the impact of aberrant C19MC miRNA expression on embryo development and primate placentation.
This Aim will 1) repress and overexpress C19MC cluster expression utilizing genome editing tools in TSC, 2) apply C19MC genome editing to embryos to evaluate the role of cluster expression on primate preimplantation embryo development, and 3) determine the impact of embryonic genome editing on trophectoderm function and differentiation in an in vitro implantation culture model. I have recently developed macaque TSCs with methods described by Okae et al. (2018), and have experience with rhesus IVF to derive embryos for genome editing and in vitro implantation experiments. TSC and embryo resources will be used to define miRNA and mRNA expression during embryo development and trophoblast lineage specification and identify miRNA-regulated gene networks in early placentation. Overall, the proposed research will establish a basic understanding of miRNA expression and miRNA target gene regulation in the embryo to placenta transition. Ultimately, we envision that the nonhuman primate model will allow us to extend these approaches to transfer of edited embryos to recipient dams, and to develop in vivo strategies to directly target the placenta for modification of miRNA expression for experimental and therapeutic purposes.
The primate-specific miRNA cluster, C19MC, has roles in trophoblast invasion and migration, but the roles of these miRNAs in primate embryonic development and early placentation remains to be elucidated. Our proposal aims to determine the gene networks controlled by the primate- specific C19MC miRNA cluster in primate placental lineage specification and development utilizing rhesus macaque embryos, primary trophoblasts, and trophoblasts stem cells. Understanding the roles of the C19MC in early placentation may lead to the use of these miRNAs as diagnostic tools in maternal blood, and as therapeutic agents in developing placental therapies in experimental animal models.
