Esch R K, Firtel R A
Department of Biology, University of California-San Diego, La Jolla 92093.
Genes Dev. 1991 Jan;5(1):9-21. doi: 10.1101/gad.5.1.9.
The Dictyostelium ras gene (Dd-ras) is expressed at a low level in vegetative cells, is not expressed between the onset of development and aggregation, and is then re-expressed in the multicellular aggregate stages from the distal, now cAMP-responsive, promoter and from two more proximal promoters. Expression of activated Dd-ras (G12----T12) (Reymond et al. 1986) results in an abnormal developmental phenotype with the formation of aggregates having multiple tips and an inhibition of further development. In this report we investigate the spatial expression of Dd-ras by fusing the 5'-flanking region to the Escherichia coli lacZ gene and by staining aggregates for beta-galactosidase (beta-gal) activity. We show that fusions using 5'-flanking sequences that include all promoters are expressed in approximately 10-20% of the cells randomly scattered within the early aggregate. Our data indicate that these beta-gal-expressing cells migrate to newly formed tips of aggregates and localize in the region that becomes the prestalk zone. Staining is also seen in the very posterior of the organism. The anterior staining appears to be specific for the prestalk A population, and beta-gal activity is subsequently present in stalk cells as developmental proceeds. When only the two more proximal promoters are used to drive lacZ expression, localized staining is seen in the anterior prestalk region, although it is weaker than with the construct carrying all promoters. Moreover, staining is not seen in the posterior domain in the first finger stage, suggesting differences in the spatial expression from the different promoters. Staining is also observed in some cells within the prespore region, which could be anterior-like cells. The pattern of Dd-ras/lacZ staining during tip formation suggests a directed, spiral pattern of cell migration, possibly in response to the proposed spiral gradient of cAMP within the developing aggregate. The pattern of Dd-ras is consistent with the abnormal developmental phenotype caused by expressing an activated Dd-ras Thr12 gene and suggests an essential role for Dd-ras in controlling spatial differentiation.
盘基网柄菌ras基因（Dd-ras）在营养细胞中低水平表达，在发育起始至聚集阶段不表达，然后在多细胞聚集阶段从远端的、现在对cAMP有反应的启动子以及另外两个近端启动子重新表达。活化的Dd-ras（G12----T12）（雷蒙德等人，1986年）的表达导致异常的发育表型，形成具有多个尖端的聚集体并抑制进一步发育。在本报告中，我们通过将5'侧翼区域与大肠杆菌lacZ基因融合并对聚集体进行β-半乳糖苷酶（β-gal）活性染色来研究Dd-ras的空间表达。我们表明，使用包含所有启动子的5'侧翼序列的融合体在早期聚集体中随机散布的约10 - 20%的细胞中表达。我们的数据表明，这些表达β-gal的细胞迁移到聚集体新形成的尖端并定位在成为前柄区的区域。在生物体的最后端也可见染色。前部染色似乎对前柄A群体具有特异性，并且随着发育进行，β-gal活性随后存在于柄细胞中。当仅使用另外两个近端启动子驱动lacZ表达时，在前部前柄区域可见局部染色，尽管比携带所有启动子的构建体弱。此外，在第一指状阶段的后部区域未见染色，表明不同启动子的空间表达存在差异。在孢子前区域的一些细胞中也观察到染色，这些细胞可能是类似前部的细胞。尖端形成过程中Dd-ras/lacZ染色模式表明细胞迁移呈定向螺旋模式，可能是对发育中的聚集体中推测的cAMP螺旋梯度的反应。Dd-ras的模式与表达活化的Dd-ras Thr12基因引起的异常发育表型一致，并表明Dd-ras在控制空间分化中起重要作用。
