Regulation of Differentiation in Slime Mold Development

在细胞黏菌的发育过程中，细胞聚集形成组织，其中两种类型的细胞(前柄和前孢子)在前部和后部分化。为了阐明这种模式的形成机制，我们做了以下研究：1.利用J.Williams提供的带有肌动蛋白启动子的载体，分析了前孢子特异基因Dp87的顺式作用调控区，发现4个正的前孢子特异的调控区和1个负的前孢子特异的调控区和1个正的细胞类型非特异区调控其转录。第二阶段需要环状AMP，第三阶段需要DIF，其中还需要一种未知的低分子质量分泌物。在第三阶段给予的环状AMP对茎前特异的ecma和ecmB基因都有相同程度的抑制作用，而激活蛋白激酶A的8-溴-cAMP则特异性地抑制…EcmB的表达增加，提示该酶参与了两个基因的差异表达。3.利用组成表达β-半乳糖苷酶的转化子研究了细胞周期时相与模式形成的关系。研究发现，在G2期的某一点(PS)开始聚集之前，细胞处于饥饿状态，但被分选到迁移性鼻涕的后前孢子区，而在该点之后饥饿的细胞聚集滞后，但被分选到柄前区域。4.以水母的Apoqequorin基因转化细胞为材料，研究了发育过程中细胞内游离钙浓度的变化和对cAMP聚集的反应。在聚集时，细胞表现出相当大的[(Ca)^&lt；2+&gt；]增加，并通过短暂增加[(Ca)^&lt；2+&gt；]而获得对cAMP的反应性。前柄细胞含有两倍于前孢子细胞的[(Ca)^&lt；2+&gt；]，对cAMP的反应是前孢子细胞的三倍。较少

In the development of cellular slime molds, cells aggregate to form a tissue, in which two types of cells(prestalk and prespore) differentiate in the anterior and the posterior parts. To elucidate mechanisms of the pattern formation, we made the following studies.1.By the use of a vector with the actin-basal promoter provided by J.Williams, we analyzed the cis-acting regulatory regions of prespore-specific gene Dp87 and found 4 positive, prespore-specific and 1 negative, non-prespore-specific and 1 positive, cell-type-non-specific regions regulating its transcription.2.The process of stalk differentiation was found to be divided into 4 different stages. Cyclic AMP was required at the second stage and DIF at the third stage, where an unidentified low molecular weight secretory substance was also required. Cyclic AMP given at the third stage rather inhibited both of prestalk-specific ecmA and ecmB genes to the same extent, while 8-Br-cAMP which activates protein kinase A specifically ind … More uced ecmB expression, suggesting involvement of this enzyme in differential expression of the two genes.3.Relationship between the cell-cycle phase and the pattern formation was investigated by using a transformat constitutively expressingbeta-galactosidase. It was found that cells starved before a particular point (PS) within the G2 phase initiated aggregation, but sorted out to the posterior prespore region of a migrating slug, while those starved after that point lagged behind in aggregation, but sorted out to the anterior prestalk region.4.Changes in intracellular free calcium concentration ([(Ca)^<2+>]) and responsiveness to cAMP stumulation during development were studied, by using cells transformed with apoqequorin cDNA of a jellyfish. Cells exhibited a considerable increase in [(Ca)^<2+>] and acquired responsiveness to cAMP by showing a transient increase in [(Ca)^<2+>], at the time of aggregation. Prestalk cells contained twice as much [(Ca)^<2+>] and showed three times as large a response to cAMP as prespore cells. Less