Primate-specific miRNAs in Embryonic and Placental Development

项目摘要 胎盘病理源于早期胎盘发育不良，其特征是胎盘浸润浅， 滋养细胞，也与属于灵长类特异性的miRNAs的异常表达有关。 19号染色体miRNA簇（C19 MC）。C19 MC miRNA被认为在滋养层侵袭中起作用， 然而，它们在灵长类动物胚胎和早期胎盘发育中的作用并不明确。我 假设C19 MC miRNA在滋养外胚层谱系特化中具有灵长类特异性作用 和灵长类胎盘的早期发育我的建议的总体目标是确定 这些miRNAs在灵长类滋养层谱系特化和恒河猴胎盘发育中的作用 模型，并揭示了灵长类胎盘形成中miRNA调控的基因网络。为了实现这一目标，我建议 三个具体目标。 具体目标1（K99-阶段）。为了确定miRNAs在灵长类动物胚胎中的表达， 滋养层干细胞（TSC）。这一目标将建立胚胎发育过程中的miRNA签名 通过滋养外胚层谱系的特化，并确定这些miRNAs是否 以阶段或细胞类型特异性方式表达。 具体目标2（K99阶段）。确定C19 MC成员在TSC中的职能作用， 分化的滋养层功能。本实验将直接过表达C19 MC miRNA成员 在TSC中鉴定由这些miRNA调控的基因和途径，评估其功能作用。 具体目标3（R 00阶段）。为了使用基因组编辑策略来全局扰动C19 MC， 评估异常C19 MC miRNA表达对胚胎发育和灵长类动物的影响 胎座式该目的将1）利用基因组抑制和过表达C19 MC簇表达， TSC中的编辑工具，2）将C19 MC基因组编辑应用于胚胎以评估簇的作用 表达对灵长类动物植入前胚胎发育的影响，以及3）确定胚胎发育的影响。 基因组编辑对体外植入培养模型中滋养外胚层功能和分化的影响。 我最近用Okae等人（2018）描述的方法开发了猕猴TSC，并有经验 与恒河猴体外受精（rhesus IVF）一起获得用于基因组编辑和体外植入实验的胚胎。TSC和胚胎 这些资源将用于确定胚胎发育和滋养层中miRNA和mRNA的表达 谱系特化和鉴定早期胎盘中miRNA调控的基因网络。总体而言，拟议 这项研究将建立对miRNA表达和miRNA靶基因调控的基本认识， 胚胎到胎盘的过渡。最终，我们设想非人类灵长类动物模型将允许我们扩展 这些方法将编辑的胚胎转移到受体母鼠，并开发体内策略， 靶向胎盘修饰miRNA表达用于实验和治疗目的。

PROJECT ABSTRACT 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.