Isolation of Novel Mutations Affecting the Mouse Embryo

描述（由申请人提供）：组织哺乳动物胚胎身体计划的遗传途径和网络尚不清楚。在着床和妊娠中期，发生了轴决定、原肠胚形成、胚层形成、形态发生和器官系统早期分化等关键事件，但调控这些过程的大多数基因尚未被确定。这是一个继续使用乙基亚硝基脲（ENU）诱变来识别控制小鼠胚胎早期发育的基因的建议。隐性突变是确定的，因为它们在妊娠中期胚胎中引起明显的形态异常。定位克隆用于鉴定与发育表型有关的基因。这种方法已经确定了神经和中胚层模式所需的30个基因的突变，包括至少24个以前未表征的基因。已经确定了导致6个突变的分子病变，包括先前未在小鼠中研究过的4个基因的突变。现在的工作将集中在轴规范、模式和形态发生的早期事件上。最近发现的影响早期模式和形态发生的突变将被绘制和分析。影响这些过程的几个基因将被克隆并进行分子表征。为了提高识别控制着床后早期发育的基因的效率，包括轴规范和形态发生，将在e7.5-e8.5胚胎的特定细胞亚群中产生表达荧光蛋白的转基因系。这些报告细胞系将用于帮助确定已经确定的突变特征，并更有效地筛选影响早期模式和形态发生的新突变。新的突变将根据这些报告基因的不恰当表达来确定，并确定影响早期发育特定方面的基因组。

DESCRIPTION (provided by applicant): The genetic pathways and networks that organize the body plan of the mammalian embryo are not well understood. Between implantation and midgestation, the critical events of axis determination, gastrulation, germ layer specification, morphogenesis and early differentiation of organ systems take place, but the majority of genes that regulate these processes have not yet been identified. This is a proposal to continue to use ethylnitrosourea (ENU) mutagenesis to identify genes that control early development in the mouse embryo. Recessive mutations are identified because they cause clear morphological abnormalities in the midgestation embryo. Positional cloning is used to identify the genes responsible for the developmental phenotypes. This approach has identified mutations in 30 genes required for neural and mesodermal patterning, including at least two dozen previously uncharacterized genes. The molecular lesions responsible for 6 mutations have been identified, including mutations in 4 genes that had not been studied previously in the mouse. The work will now focus on the early events of axis specification, patterning and morphogenesis. Recently identified mutations that affect early patterning and morphogenesis will be mapped and analyzed. Several genes that affect these processes will be cloned and characterized molecularly. To increase the efficiency of identification of genes that control early post-implantation development, including axis specification and morphogenesis, transgenic lines will be generated that express fluorescent proteins in defined subsets of cells in the e7.5-e8.5 embryo. These reporter lines will be used to help characterize mutations that have already been identified and to screen for new mutations that affect early patterning and morphogenesis more efficiently. New mutations will be identified based on inappropriate expression of these reporters and define groups of genes that affect specific aspects of early development. Identification of the genes that control development of the mammalian embryo is crucial for the detection, prevention and treatment of human birth defects. The signaling pathways that act in early development also have critical roles in tumor development, so it is likely that these genes identified in this study will define new steps in tumor development and progression.