Arnone M I, Martin E L, Davidson E H
Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA.
Development. 1998 Apr;125(8):1381-95. doi: 10.1242/dev.125.8.1381.
CyIIa, a cytoskeletal actin gene of Strongylocentrotus purpuratus, is expressed specifically though transiently in the embryonic skeletogenic and secondary mesenchyme and, later in development, is permanently activated in the hindgut and midgut. CyIIa transcription follows, and is therefore downstream of, the initial specification of these embryonic domains. A detailed functional analysis of the cis-regulatory system governing the rate and the location of CyIIa expression during development was carried out using GFP expression constructs. About 4.4 kb of CyIIa sequence including a leader intron were examined for cis-regulatory function. Distal elements scattered over several kb account for 60% of the quantitative output of the expression construct and a strong amplifier of expression is located within the leader intron. However, the complex spatial pattern of CyIIa expression is completely reproduced by a compact upstream regulatory element <450 bp in length. We found no evidence anywhere in the 4.4 kb sequence examined for negative regulators required to repress ectopic expression. The specific site that mediates CyIIa expression in the midgut in late embryos and larvae was identified. This site is the same as that necessary and sufficient for midgut expression of the Endo16 gene late in development, and was shown to bind the same transcription factor. Except for some temporal and quantitative features, the S. purpuratus expression construct is expressed accurately and specifically in the same diverse cell types when introduced into embryos of Lytechinus pictus, which belongs to a different echinoid order. No ectopic expression was observed, in contrast to the result of a similar interspecific gene transfer experiment carried out earlier on a different cytoskeletal actin gene that is expressed much earlier in development. Presentation of the set of transcription factors that activate CyIIa in the differentiated cells in which it is expressed is apparently a conserved feature of these cell types.
紫球海胆的细胞骨架肌动蛋白基因CyIIa，在胚胎骨骼生成和次生间充质中特异性表达，不过是短暂表达，并且在发育后期，在后肠和中肠中被永久激活。CyIIa转录跟随这些胚胎区域的初始特化，因此处于其下游。利用绿色荧光蛋白（GFP）表达构建体，对发育过程中控制CyIIa表达速率和位置的顺式调控系统进行了详细的功能分析。对包括一个前导内含子在内的约4.4 kb的CyIIa序列进行了顺式调控功能检测。分布在几个kb上的远端元件占表达构建体定量输出的60%，并且一个强大的表达增强子位于前导内含子内。然而，CyIIa表达的复杂空间模式完全由一个长度小于450 bp的紧密上游调控元件重现。在所检测的4.4 kb序列中，我们没有发现任何抑制异位表达所需的负调控因子的证据。确定了在晚期胚胎和幼虫中肠介导CyIIa表达的特定位点。该位点与发育后期Endo16基因中肠表达所必需且充分的位点相同，并且已证明它能结合相同的转录因子。除了一些时间和定量特征外，当将紫球海胆表达构建体导入属于不同海胆纲的多棘海胆胚胎时，它能在相同的多种不同细胞类型中准确且特异性地表达。与之前对另一个在发育中更早表达的细胞骨架肌动蛋白基因进行的类似种间基因转移实验结果相反，未观察到异位表达。在其表达的分化细胞中激活CyIIa的一组转录因子的呈现显然是这些细胞类型的一个保守特征。
