REGULATION OF MORPHOGENESIS IN EARLY MOUSE DEVELOPMENT

L)我们感兴趣的一个主要领域是确定哪个聚梳组(PcG)蛋白质相互作用，并确定在苍蝇和哺乳动物中的相互作用区域。我们已经证明了三种苍蝇PcG蛋白相互作用。Psc(后性梳)与ph(多同源异体)和Pc(多梳)相互作用，但ph和Pc不相互作用。它们的鼠标同源基因也是如此。RAE28(Ph)、BMI1(Psc)和M33(Pc)具有同源性。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(腿中段的性梳)相互作用。小鼠的PH和SCM同源物(RAE28和MSCM)也直接相互作用。布罗克博士进一步分析了更多不同蛋白质中的保守结构域2)我们发现，小鼠的三个PcG基因在胚胎发育期间和成年组织中有重叠但不相同的表达模式。这些基因产物从胚胎核提取液中免疫共沉淀。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作为大的多聚体复合体存在。免疫共沉淀物的异质性似乎是细胞类型特有的，因为M33和RAE28作为F9细胞大复合体的成员进行免疫共沉淀和共孵化，F9细胞表达BMI1的水平非常低。3)为了研究rae28基因在小鼠发育中的作用，我们通过在胚胎干细胞中进行基因打靶的方法产生了rae28缺陷小鼠。在28^-lt；-/-&gt；纯合子胚胎中，Hoxa-3、a-4、a-5、b-3、b-4和d-4表达的前边界向嘴方向移动，在菱形和/或咽弓中Hoxb-3和b-4的表达也发生了类似的变化。这些改变的HOX密码被推测与在RAE 28；-/-&gt；纯合子小鼠中观察到的后部骨骼变形和神经脊缺陷有关。这些结果表明，rae28基因参与了HOX基因表达的调控和旁轴中胚层和神经脊发育过程中的片段指定。4)我们正在确定rae28基因是否可以挽救相关的果蝇突变(正在进行中)。较少

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