Phenotyping Early Embryonic Lethal Knockout Mice to Identify Essential Genes with Previously Uncharacterized Roles in Pre-implantation Development, Gastrulation, Turning, and Placentation

 描述(由申请人提供)：在过去的十年中，基因组技术的进步产生了关于动物基因组的前所未有的信息水平。然而，这些细胞和组织功能的2D模型背后的许多信息仍然有待实验验证和在完整的活体中进行进一步的研究。限制全基因组方法应用的另一个因素是缺乏对所有注释基因中≥1/2的功能描述。由国际基因敲除小鼠联盟(IKMC)产生的靶向突变，包括KOMP，为从功能上注释哺乳动物基因组提供了一个革命性的资源。我们建议表征KOMP2产生的KO系突变的表型，这些突变导致E9.5或之前的发育智力停滞或异常形态。我们将对大约200个以前未描述的基因进行功能定义，这些基因的功能在植入前和植入后的早期发育中是必不可少的。该小组的研究人员先前已经研究了约200个胚胎致死小鼠突变的表型，导致了许多令人惊讶的发现，包括创造哺乳动物内胚层的新的细胞机制，刺猬信号通路中对初级纤毛的要求等等。我们的具体目标是(1)通过在e9.5或之前致死的≥200株系的第二级表型，来表征早期胚胎发育所必需的新基因的功能。我们将定义停滞的阶段，分析形态异常，评估增殖和细胞死亡。只有敲除基因还没有特征的基因才会被研究，那些编码未特征蛋白的基因将被优先考虑，这使这成为对新的必要基因的公正筛选，并为200个必要基因的生物功能提供了第一个证据。(2)利用我们的专业知识在细胞分辨率上表征以前未描述的基因在着床前发育、早期胚胎形态发生和胎盘发育中的作用。我们将描述早期胚胎谱系多能性的新调节因子，原肠形成上皮-间充质转化，早期中胚层迁移和腹侧折叠，这些都是反复使用的形态发生程序，对人类发育的许多方面都是必不可少的。胎盘形成调节因子的细胞和发育功能的表征对胎儿和儿童的健康至关重要。我们的具体目标是：目标1。-建立表型管道，该管道将在三个交易日开始 NIH KOMP2生产和表型中心。目的对E9.5之前导致致死的约200个突变进行2-Tier-Two表型分析。目的对影响胚泡发育、植入后早期形态发生和胎盘形成的致死性突变进行3-Tier-3表型分析。KOMP和IKMC产生的胚胎致死性KO突变为扩展和丰富哺乳动物基因组的功能注释提供了前所未有的机会。这样确定的胚胎致死基因不仅对小鼠的早期发育是不可或缺的，而且对人类发育和组织动态平衡的多个方面也至关重要。

 DESCRIPTION (provided by applicant): Advances in genomic technologies over the past decade have yielded an unprecedented level of information about animal genomes. However, much of the information underlying these 2D models of cell and tissue function stills awaits experimental verification and further investigation in intact living organisms. Another factor limiting the application of genome wide approaches is the absence of functional characterization for ≥ 1/2 of all annotated genes. The targeted mutations generated by the International Knockout Mouse Consortium (IKMC), including KOMP, provide a revolutionary resource for functionally annotating the mammalian genome. We propose to characterize the phenotypes of mutations in the KOMP2-generated KO lines that result in develop- mental arrest or abnormal morphology at or prior to E9.5. We will functional define ~200 previously uncharacterized genes whose functions are essential during pre- and early post-implantation development. The investigators of this group have previously studied the phenotypes of ~200 embryonic lethal mouse mutants, leading to many surprising findings, including the novel cellular mechanisms that create the mammalian endoderm, the requirement for primary cilia in the Hedgehog signaling pathway and many more. Our specific goals are to (1) Characterize the functions of new genes essential for early embryonic development, by second tier phenotyping of ≥200 lines lethal at or before e9.5. We will define the stage of arrest, analyze morphological abnormalities, assess proliferation and cell death. Only genes in which knock- outs have not been characterized will be studied, and those encoding uncharacterized proteins will have highest priority, making this an unbiased screen for novel essential genes, and providing the first evidence on bio- logical function of 200 essential genes. (2) Use our expertise to characterize at cellular resolution the roles of previously uncharacterized genes in preimplantation development, early embryonic morphogenesis and placental development. We will characterize novel regulators of pluripotency of early embryonic lineages, the gastrulation epithelial-mesenchymal transition, early mesoderm migration and ventral folding, which are reiteratively used morphogenetic programs essential for many aspects of human development. Characterization of cellular and developmental functions of regulators of placentation is crucial for fetal and child health. Our specific Aims are: Aim 1. - Establishing a Phenotyping Pipeline, which will initiate at three trans NIH KOMP2 production and phenotyping centers. Aim 2 - Tier two phenotyping of ~200 mutations that cause lethality before E9.5. Aim 3 - Tier 3 phenotyping of lethal mutations that affect development of the blastocyst, early post-implantation morphogenesis and placentation. The embryonic lethal KO mutations generated by KOMP and IKMC provide an unprecedented opportunity to expand and enrich the functional annotation of the mammalian genome. The embryonic lethal genes thus identified will not only be indispensable for early mouse development, but also critical for multiple aspects of human development and tissue homeostasis.