REGULATION OF MORPHOGENESIS IN EARLY MOUSE DEVELOPMENT

l)我们感兴趣的主要领域之一是确定哪些Polycomb组（PcG）蛋白在果蝇和哺乳动物中相互作用，并确定相互作用域。我们已经证明了三种苍蝇PcG蛋白相互作用。Psc（后性梳）与ph（多同体）和Pc（多梳）相互作用，但ph和Pc不相互作用。它们的小鼠同系物也是如此。RAE28 （ph）、BMI1 （Psc）和M33 （Pc）具有同型相互作用。RAE28和M33都与BMI1相互作用，但不相互作用。此外，ph通过在许多蛋白质中发现的保守结构域与Scm（腿中部的性梳）相互作用。ph和Scm的小鼠同源物（RAE28和mScm）也直接相互作用。布洛克博士进一步分析了更多不同蛋白质的保守结构域。2)我们发现三个小鼠PcG基因在胚胎发生和成年组织中表达模式重叠但不相同。这些基因产物从胚胎核提取物中共免疫沉淀。凝胶过滤分析…更多的胚胎提取物表明这些蛋白质以多种形式存在，如大的多聚体复合物。共免疫沉淀的异质性似乎是细胞类型特异性的，因为M33和RAE28作为F9细胞中表达BMI1的大复合物的成员共同免疫沉淀和共固化，而F9细胞的表达水平非常低。这些研究表明，小鼠PcG蛋白受发育调控，并作为多种异质复合物发挥作用。3)为了研究rae28基因在小鼠发育中的作用，我们在胚胎干细胞中通过基因靶向培养rae28缺陷小鼠。Hoxb-3、a-4、a-5、b-3、b-4和d-4的前边界在rae28^<-/->纯合子胚的近轴中胚层中向吻侧方向表达，Hoxb-3和b-4的前边界在斜形体和/或咽弓中表达也发生了类似的改变。这些改变的Hox编码被认为与在rae28^<-/->纯合子小鼠中观察到的后骨骼转化和神经嵴缺陷有关。这些结果表明，rae28基因参与了近轴中胚层和神经嵴发育过程中Hox基因表达和片段规范的调控。4)我们正在确定rae28基因是否可以挽救相应的果蝇突变（正在进行中）。少

l) One of our major areas of interest has been to identify which Polycomb group (PcG) proteins interact, and to determine the interaction domains, both in flies and mammals. We have shown that three fly PcG proteins interact. Psc (Posterior sex combs) interacts with ph (polyhomeotic) and Pc (Polycomb), but ph and Pc do not interact. The same is true for their mouse homologues. RAE28 (ph), BMI1 (Psc), and M33 (Pc) interact homotypically. Both RAE28 and M33 interact with BMI1 , but not with each other. In addition, ph interacts with Scm (Sex comb on midleg) via a conserved domain found in many proteins. The mouse homologues of ph and Scm (RAE28 and mScm) also interact directly. Dr.Brock has further analyzed the conserved domain in more diverged proteins.2) We found that the three mouse PcG genes have overlapping but not identical expression patterns during embryogenesis and in adult tissues. These gene products coimmunoprecipitate from embryonic nuclear extracts. Gel filtration analysis … More of embryonic extracts indicate that these proteins exist in multiple forms as large, multimeric complexes. The heterogeneity of coimmunoprecipitates appears to be cell type-specific, as M33 and RAE28 coimmunoprecipitate and copurify as members of large complexes from F9 cells, which express BMI1 at very low levels. These studies indicate that murine PcG proteins are developmentally regulated, and function as multiple, heterogeneous complexes.3) To study the role of the rae28 gene in mouse development, we generated rae28-deficient mice by gene targeting in embryryonic stem cells. The anterior boundaries of Hoxa-3, a-4, a-5, b-3, b-4 and d-4 expression were shifted in the rostral direction in the paraxial mesoderms of the rae28^<-/-> homozygous embryos, and those of Hoxb-3 and b-4 expression were also similarly altered in the rhombomeres and/or pharyngeal arches. These altered Hox codes were presumed to be correlated with the posterior skeletal transformations and neural crest defects observed in the rae28^<-/-> homozygous mice. These results indicate that the rae28 gene is involved in the regulation of Hox gene expression and segment specification during paraxial mesoderm and neural crest development.4) We are determining if rae28 gene can rescue the corresonding fly mutations (in progress). Less