中国科学院昆明动物研究所机构知识库

着床前胚胎发育是哺乳动物胚胎发育的最早期阶段，起始于成熟卵的受精，完成于孵化的囊胚植入母体子宫。早期胚胎经历了一系列的重要发育事件，从单个细胞的合子发育成为具有三个细胞谱系、能够植入子宫的孵化囊胚，这些发育事件包括减数分裂完成及卵子成熟、受精、基因组重编程、卵裂、合子基因组启动、致密化、第一次命运决定（桑葚胚分化为内细胞团和滋养外胚层）、和第二次命运决定（内细胞团分化为表胚层和原始内胚层）等，这一系列发育事件处于复杂而严格的基因调控之下，目前我们对这些过程的了解，特别是在人以及猴子等灵长类动物中，仍然有限。对于早期胚胎发育的研究不仅使我们深入了发育生物学相关领域的认识，而且促进了辅助生殖技术以及遗传操作技术的产生和发展。此外，目前再生医学中有重要治疗应用前景的胚胎干细胞来源于早期胚胎阶段囊胚的内细胞团。因此，对于早期胚胎发育过程，特别是这一过程中基因调控的深入研究，不仅有助于我们认识这一过程的本质并解决一系列生物学问题，更重要的是能够帮助我们改进人类辅助生殖技术，完善哺乳动物的遗传操作技术，以及获得质量更好的干细胞并应用于治疗领域。我们利用低起始量转录组测序技术从转录组水平对猕猴着床前胚胎进行了研究，我们首先收集了包含GV卵，MII卵，1-细胞原核时期胚胎，2-细胞，4-细胞，8-细胞，桑葚胚，囊胚各发育阶段的单个完整胚胎样品，同时也收集了相同阶段多个胚胎混合的样品。通过生物信息学分析我们找到了一系列母源以及合子基因，这些基因的表达分别与基因组重编程，合子基因组启动以及命运分化同时发生，可能参与了这些过程。通过比较猕猴，人和小鼠的着床前胚胎数据，我们发现灵长类动物猕猴比小鼠更适合研究人着床前胚胎发育过程。通过基因调控网络的研究我们寻找了一系列重要的调控模块和基因。此外通过合子基因组启动前后的表达调控网络的比较，我们发现灵长类动物与啮齿类动物相比保持基因组稳定性能力偏低，特别是在DNA损伤信号传导和同源重组修复方面，这一结果经过功能实验得到了证实。这一结果解释了灵长类动物中着床前胚胎发育成功率低的原因，此外，最近基于同源重组修复的基因编辑技术在猕猴上较差的表现可能也与猕猴卵中较低的同源重组修复能力相关。我们在超过100个猕猴内细胞团细胞中检查了第二次命运决定过程以及表胚层成熟过程中42个谱系基因的表达情况，这些细胞分别来自于早期、中期、晚期以及孵化期囊胚。我们发现早期囊胚阶段的内细胞团细胞处于一种双潜能/未分化的状态，第二次命运决定发生在囊胚中期，不同谱系的细胞逐步确定其分化命运。通过检测这些谱系基因的表达模式，我们还确定一些谱系标记基因，这些基因在其对应的谱系中持续高表达，这些标记基因包括UTF1, DPPA5, GDF3 和 GATA4。此外，我们还比较了不同发育阶段的表胚层细胞并且注意到一些重要多能性基因在表胚层细胞的分化和成熟过程中出现表达下调，这一现象可能暗示表胚层细胞成熟过程中多能性状态的变化。综上所述，本研究首先利用低起始量转录组测序技术对整个猕猴着床前胚胎发育过程进行了深入的研究，研究了母源基因和合子基因的特点及功能，并通过与人和小鼠的比较对灵长类发育的特点进行了深入研究，发现了灵长类动物卵和着床前胚胎在保持遗传物质稳定性方面能力相对较弱。为了解猕猴着床前胚胎的第二次命运决定过程，本研究利用单细胞基因表达分析技术，对相应发育阶段的细胞进行了分析，从而对第二次命运决定发生的动态过程以及表胚层的成熟过程进行了深入的研究，并寻找了相关的谱系基因，深入了我们对灵长类命运决定过程的认识与了解。 

Preimplantation embryo development is the first stage of the mammal development, it begins at fertilization of mature oocytes, and ends at the implantation of hatched blastocysts. During this time, the single cell zygote develops into hatched blastocyst which contains three cell lineages and is ready to implant into the uterus. There is a series of development events during preimplantation development, including meiosis resumption, oocyte maturation, fertilization, genome reprogramming, cleavage, zygote genome activation (ZGA), compaction, first fate determination (segregation of trophectoderm and inner cell mass), and second fate determination (segregation of epiblast and primitive endoderm). All these development events are under control of gene regulation, however, our understanding of these sophisticated processes is still quite limited, especially in primates like human and monkey. The better understanding of preimplantation development could not only answer the basic scientific questions in development, but also promote the emergence and development of assisted reproductive technology and gene editing technology. Moreover, studies on preimplantation development, especially on the genetic regulatory circuits involved, could greatly benefit the understanding and application of embryonic stem cells which are derived from inner cell of mass of blastocyst.To understand the overall gene expression pattern in monkey preimplantation development, we employed low-input transcriptome profiling technology, we generated single embryo transcriptome data from different development stages, including, GV-stage oocyte, MII-stage oocyte, one-cell embryo at PN stage, two-cell embryo, four-cell embryo, eight-cell embryo, morula and blastocyst. To validate our results, we also collected pooled embryo samples which contain 5-20 embryos of the same stage. We analyzed the single and pooled embryo transcriptome data and identified lists of maternal and zygotic genes. We proposed their possible functions in reprogramming, ZGA and cell fate determination. Our results showed rhesus monkey is more suitable than mouse in studying human preimplantation development by comparing our data with published human and mouse preimplantation embryo transcriptome data. Molecular network analysis uncovered important network modules and hub genes during MZT. Comparison of the MZT-related network modules among the three species suggested a looser surveillance of genome stability in primate oocytes and early embryos than in rodent, in particular in the pathways of DNA damage signaling and HR-directed DNA double strand break (DSB) repair, which was further experimentally validated. The reduced capacity of HR-mediated DNA DSB repair in primate oocytes and early embryos interpreted the observation of the higher frequency of chromosome instability and embryo wastage in primates than in any other species assessed. The lower ability of DNA homologous recombination also accounts for the inefficiency to perform precise genome editing in monkey. We studied the second fate determination and maturation of epiblast (The epiblast is pluripotent and would further contribute to the embryo proper) in rhesus monkey by profiling the expression of 42 genes in over 100 individual cells from ICM(Inner cell mass) of early, middle, late and hatched blastocysts. In our results we found ICM cells were initially bi-potential, and taking their fate asynchronously and gradually at middle blastocyst stage. We also found UTF1, DPPA5, GDF3 and GATA4 were constantly different expressed between epiblast and primitive endoderm cells, these genes could serve as lineage markers. Furthermore, we examined the gene expression dynamics during the maturation of epiblast, and found some core pluripotent genes were down regulated at late or hatched stage, which implied a dynamic transition of pluripotent states. To summarize, in this study, we investigated the preimplantation development in rhesus monkey at transcriptome level, identified the maternal and zygotic genes. By comparing to human and mouse data, we noticed low capacity of DNA double strand breaks repair in primate oocytes and early embryos. To better understand the second fate determination in rhesus monkey, we collected single cell data and analyzed the dynamic of genes expression.