Cellular commitment to mortality: analyses of mouse early embryo

细胞对死亡的承诺：小鼠早期胚胎的分析

• 批准号: 8552430
• 负责人: David Schlessinger
• 金额: $ 95.58万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552430
• 关键词: Aging; Cell Lineage; Cells; Development; Down-Regulation; Embryo; Gene Activation; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Goals; Hour; Implant; In Situ Hybridization; Inner Cell Mass; Maintenance; Mediating; Meiosis; Molecular Profiling; Morula; Mus; Names; Organ; Pattern; Play; RNA Degradation; Replacement Therapy; Role; Staging; Stem cells; Techniques; Telomere Recombination; Time; Undifferentiated; Uterus; Work; base; blastocyst; embryo cell; embryonic stem cell; enhancing factor; experience; homologous recombination; induced pluripotent stem cell; knock-down; mortality; pluripotency; preimplantation; research study; self-renewal; small hairpin RNA; telomere

In our previous work, we carried out microarray-based global expression profiling of all preimplantation stages in mouse. They revealed and characterized distinctive patterns of maternal RNA degradation and two major transient waves of de novo transcription. The first wave corresponds to zygotic genome activation (ZGA); the second wave, named mid-preimplantation gene activation (MGA), precedes the dynamic morphological and functional changes from the morula to blastocyst stage. We also developed a technique to do large-scale Whole Mount In Situ Hybridization (WISH), which allows us to reveal the spatial expression patterns of 91 genes in mouse preimplantation embryos. Through these analyses, we identified a number of genes that show unique expression patterns. As an example of ZGA genes, we found Zscan4, which is expressed exclusively in 2-cell mouse embryos and ES cells and plays a critical role in the progression of preimplantation development. As an example of MGA genes, we found Trim43. We showed that the downregulation of Zscan4 expression in 2-cell embryos by shRNAs delays the normal transition from 2-cell embryos to 4-cell embryos for about 24 hours, resulting in abnormal blastocysts that failed to implant in the uterus. This indicates a critical role of Zscan4 in preimplantation development. We also found that Zscan4 is expressed only in late 2-cell embryos and in the subpopulation (1 - 5%) of ES cells maintained in the undifferentiated condition. We found that other 2-cell-specific genes are also highly expressed in Zscan4(+) cells. This indicates that the subpopulation of ES cells marked by Zscan4 expression shows some resemblance to 2-cell embryos, whereas the majority of ES cells show similarities to the Inner Cell Mass (ICM) cells in blastocysts. Zscan4 is thus associated with a unique transient state in undifferentiated ES cells, in which other 2-cell embryo-specific genes are activated. By carrying out cell lineage tracing experiments, we have found that, although Zscan4 is expressed only in 5% of ES cells in culture at a given time, essentially all ES cells experience a Zscan4-positive state by 9 passages. By knocking down Zscan4 activation with an shRNA, we have found that Zscan4 is essential for the long-term maintenance of genomic integrity and for mediating a regulated telomere recombination in normal undifferentiated ES cells, therefore making it indispensable for the proper long-term self-renewal in ES cells. We have also found that Zscan4 is involved in the induction and recruitment of the meiosis-specific homologous recombination machinery to telomeres. Recently we have shown that Zscan4 can be used as one of the reprogramming factors and enhances the quantity and quality of induced pluripotent stem (iPS) cells.

在我们之前的工作中，我们对小鼠植入前的所有阶段进行了基于微阵列的全局表达谱分析。他们揭示并描述了母体RNA降解的独特模式和两个主要的从头转录的瞬时波。第一波对应于合子基因组激活（ZGA）；第二波，称为植入前中期基因激活（MGA），发生在桑葚胚到囊胚阶段的动态形态和功能变化之前。我们还开发了一种大规模全载原位杂交（WISH）技术，该技术使我们能够揭示91个基因在小鼠着床前胚胎中的空间表达模式。通过这些分析，我们发现了一些表现出独特表达模式的基因。作为ZGA基因的一个例子，我们发现了Zscan4，它只在2细胞小鼠胚胎和ES细胞中表达，并在着床前发育的进展中起关键作用。作为MGA基因的一个例子，我们发现了Trim43。我们发现，shrna下调2细胞胚胎中Zscan4的表达会使2细胞胚胎向4细胞胚胎的正常转变延迟约24小时，导致胚泡异常，不能在子宫内着床。这表明Zscan4在着床前发育中起着关键作用。我们还发现Zscan4仅在后期2细胞胚胎和维持未分化状态的胚胎干细胞亚群（1 - 5%）中表达。我们发现其他2细胞特异性基因也在Zscan4（+）细胞中高度表达。这表明Zscan4表达标记的胚胎干细胞亚群与2细胞胚胎有一定的相似性，而大多数胚胎干细胞与囊胚中的内细胞团（Inner Cell Mass， ICM）细胞有相似性。因此，在未分化的胚胎干细胞中，Zscan4与一种独特的瞬时状态有关，在这种状态下，其他2细胞胚胎特异性基因被激活。通过细胞谱系追踪实验，我们发现，虽然Zscan4在特定时间内仅在5%的培养ES细胞中表达，但基本上所有的ES细胞在9代后都经历了Zscan4阳性状态。通过用shRNA抑制Zscan4的激活，我们发现Zscan4对于长期维持基因组完整性和在正常未分化的胚胎干细胞中介导受调节的端粒重组是必不可少的，因此对于胚胎干细胞中适当的长期自我更新是必不可少的。我们还发现Zscan4参与了端粒减数分裂特异性同源重组机制的诱导和募集。最近我们发现Zscan4可以作为重编程因子之一，提高诱导多能干细胞（iPS）的数量和质量。