Primate-specific miRNAs in Embryonic and Placental Development

项目摘要 胎盘病理起源于早期胎盘发育不良，其特征是浅表侵袭 滋养层细胞，也与灵长类特有的miRNAs的异常表达有关 19号染色体miRNA簇(C19MC)。C19MC miRNAs被认为在滋养层侵袭和 然而，它们在灵长类胚胎和早期胎盘发育中的作用(S)还没有明确的定义。我 假设C19MC miRNAs将在滋养外胚层谱系中具有灵长类特有的作用 以及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在灵长类滋养层细胞谱系特征和恒河猴胎盘发育中的作用 模型，并揭示了在灵长类胎盘形成中受miRNAs调控的基因网络。为了实现这个目标，我建议 三个具体目标。 具体目标1(K99阶段)。为了确定miRNAs在灵长类胚胎中的表达， 滋养层干细胞(TSC)。这一目标将在胚胎发育过程中建立miRNA签名 通过确定滋养外胚层谱系并确定这些miRNAs是否 以特定阶段或特定细胞类型的方式表达。 具体目标2(K99阶段)。确定C19MC成员在台糖委员会中的职能作用(S) 分化的滋养层功能。本实验将直接过表达C19MC miRNA成员 在TSC中识别这些miRNAs调控的基因和途径，评估它们的功能作用。 具体目标3(R00阶段)。使用基因组编辑策略在全球范围内扰乱C19MC和 评估C19MC miRNA异常表达对胚胎发育和灵长类动物的影响 胎盘摆放。这一目标将1)利用基因组抑制和过度表达C19MC簇表达 TSC中的编辑工具，2)将C19MC基因组编辑应用于胚胎，以评估簇的作用 表达对灵长类植入前胚胎发育的影响 在体外植入培养模型中基因组编辑对滋养外胚层功能和分化的影响。 我最近用Okae等人描述的方法开发了猕猴TSCs。(2018)，并有经验 用恒河猴体外受精获得胚胎，用于基因组编辑和体外植入实验。TSC与胚胎 这些资源将用于确定胚胎发育和滋养层细胞中miRNA和mrna的表达。 在早期胎盘形成中指定谱系并鉴定miRNA调控的基因网络。整体而言，建议的 研究将建立对miRNA表达和miRNA靶基因调控的基本了解 胚胎向胎盘的转变。最终，我们设想非人灵长类动物模型将允许我们扩展 这些方法将编辑后的胚胎移植到受体DAMs，并开发体内策略来直接 靶向胎盘以改变miRNA的表达，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.