Ganfornina M D, Sánchez D, Herrera M, Bastiani M J
Biology Department, University of Utah, Salt Lake City 84112, USA.
Dev Genet. 1999;24(1-2):137-50. doi: 10.1002/(SICI)1520-6408(1999)24:1/2<137::AID-DVG13>3.0.CO;2-7.
Gap junctions are membrane channels that directly connect the cytoplasm of neighboring cells, allowing the exchange of ions and small molecules. Two analogous families of proteins, the connexins and innexins, are the channel-forming molecules in vertebrates and invertebrates, respectively. In order to study the role of gap junctions in the embryonic development of the nervous system, we searched for innexins in the grasshopper Schistocerca americana. Here we present the molecular cloning and sequence analysis of two novel innexins, G-Inx(1) and G-Inx(2), expressed during grasshopper embryonic development. The analysis of G-Inx(1) and G-Inx(2) proteins suggests they bear four transmembrane domains, which show strong conservation in members of the innexin family. The study of the phylogenetic relationships between members of the innexin family and the new grasshopper proteins suggests that G-Inx(1) is orthologous to the Drosophila 1(1)-ogre. However, G-Inx(2) seems to be a member of a new group of insect innexins. We used in situ hybridization with the G-Inx(1) and G-Inx(2) cDNA clones, and two polyclonal sera raised against different regions of G-Inx(1) to study the mRNA and protein expression patterns and the subcellular localization of the grasshopper innexins. G-Inx(1) is primarily expressed in the embryonic nervous system, in neural precursors and glial cells. In addition, a restricted stripe of epithelial cells in the developing limb, involved in the guidance of sensory growth cones, expresses G-Inx(1). G-Inx(2) expression is more widespread in the grasshopper embryo, but a restricted expression is found in a subset of neural precursors. The generally different but partially overlapping expression patterns of G-Inx(1) and G-Inx(2) supports the combinatorial character of gap junction formation in invertebrates, an essential property to generate specificity in this form of cell-cell communication.
间隙连接是直接连接相邻细胞细胞质的膜通道，允许离子和小分子交换。两类类似的蛋白质家族，即连接蛋白和无脊椎动物连接蛋白，分别是脊椎动物和无脊椎动物中的通道形成分子。为了研究间隙连接在神经系统胚胎发育中的作用，我们在美洲沙漠蝗中寻找无脊椎动物连接蛋白。在此，我们展示了在蝗虫胚胎发育过程中表达的两种新型无脊椎动物连接蛋白G-Inx(1)和G-Inx(2)的分子克隆及序列分析。对G-Inx(1)和G-Inx(2)蛋白的分析表明它们具有四个跨膜结构域，这在无脊椎动物连接蛋白家族成员中表现出很强的保守性。对无脊椎动物连接蛋白家族成员与新的蝗虫蛋白之间系统发育关系的研究表明，G-Inx(1)与果蝇1(1)-食人魔蛋白直系同源。然而，G-Inx(2)似乎是昆虫无脊椎动物连接蛋白新群体的一员。我们使用G-Inx(1)和G-Inx(2) cDNA克隆进行原位杂交，并使用针对G-Inx(1)不同区域产生的两种多克隆血清来研究蝗虫无脊椎动物连接蛋白的mRNA和蛋白质表达模式以及亚细胞定位。G-Inx(1)主要在胚胎神经系统、神经前体细胞和神经胶质细胞中表达。此外，发育肢体中参与感觉生长锥引导的上皮细胞的一条受限条纹表达G-Inx(1)。G-Inx(2)在蝗虫胚胎中的表达更为广泛，但在一部分神经前体细胞中发现有受限表达。G-Inx(1)和G-Inx(2)通常不同但部分重叠的表达模式支持了无脊椎动物中间隙连接形成的组合特性，这是在这种细胞间通讯形式中产生特异性的一个基本特性。
