Horgan A M, Lagrange M T, Copenhaver P F
Department of Cell Biology and Anatomy L-215, Oregon Health Sciences University, Portland 97201, USA.
Dev Biol. 1995 Dec;172(2):640-53. doi: 10.1006/dbio.1995.8042.
The heterotrimeric G proteins are an extended family of guanyl nucleotide-binding proteins that serve essential functions in the mature nervous system but whose contributions to neuronal development remain poorly understood. We have investigated the potential role of one specific G protein, Go(alpha), in the control of neuronal migration. During embryogenesis of the moth, Manduca sexta, an identified population of undifferentiated neurons (the EP cells) migrate along sets of visceral muscle bands to form part of the enteric nervous system. Previously, immunohistochemical studies indicated the presence of Go(alpha)-related proteins in the EP cells during migration. We have now verified this result, using probes derived from the Go(alpha) gene in Manduca. A clone containing the full-length coding domain for Go(alpha) was sequenced from a Manduca cDNA library; digoxigenin-labeled probes were then made from this clone and used to examine the developmental expression of the Go(alpha) gene during embryogenesis. Go(alpha)-specific transcripts could first be detected in the EP cells several hours before the onset of their migration. The level of Go(alpha) expression in all of the EP cells continued to increase during migration, but subsequently was down-regulated in a subset of the postmigratory neurons at the time of their terminal differentiation. This pattern of regulated expression is consistent with the distribution of Go(alpha)-related protein in the EP cells. We also used a semi-intact culture preparation of staged embryos to investigate the effects of G protein-specific toxins on the migratory process. Intracellular injections of the wasp toxin mastoparan, a specific activator of Go(alpha)-and Gi(alpha)-related proteins, inhibited the migration of individual EP cells. Injections of pertussis toxin (an inhibitor of Go(alpha) and Gi(alpha)) or cholera toxin (a selective activator of Gs(alpha)) had no effect on migration, although pertussis toxin treatments did cause a measurable increase in the subsequent outgrowth of axonal processes. However, co-injection of mastoparan with pertussis toxin blocked the inhibitory effects of mastoparan alone. These results suggest that Go(alpha)-coupled signaling events within the EP cells may down-regulate their migratory behavior, possibly in response to inhibitory cues that normally guide migration in the developing embryo.
异源三聚体G蛋白是鸟苷酸结合蛋白大家族的成员，在成熟神经系统中发挥着重要功能，但其对神经元发育的作用仍知之甚少。我们研究了一种特定的G蛋白Go(α)在控制神经元迁移中的潜在作用。在蛾类烟草天蛾的胚胎发育过程中，一群已确定的未分化神经元（EP细胞）沿着内脏肌带迁移，形成肠神经系统的一部分。此前，免疫组织化学研究表明，在迁移过程中，EP细胞中存在与Go(α)相关的蛋白质。我们现在使用从烟草天蛾Go(α)基因衍生的探针验证了这一结果。从烟草天蛾cDNA文库中对一个包含Go(α)全长编码域的克隆进行了测序；然后从该克隆制备地高辛标记的探针，并用于检测胚胎发育过程中Go(α)基因的发育表达。在EP细胞开始迁移前几个小时，就可以首次检测到Go(α)特异性转录本。在迁移过程中，所有EP细胞中Go(α)的表达水平持续升高，但随后在一部分迁移后的神经元终末分化时被下调。这种表达调控模式与EP细胞中与Go(α)相关蛋白的分布一致。我们还使用了分期胚胎的半完整培养制剂来研究G蛋白特异性毒素对迁移过程的影响。向细胞内注射黄蜂毒素马斯托帕兰（一种与Go(α)和Gi(α)相关蛋白的特异性激活剂）可抑制单个EP细胞的迁移。注射百日咳毒素（Go(α)和Gi(α)的抑制剂）或霍乱毒素（Gs(α)的选择性激活剂）对迁移没有影响，尽管百日咳毒素处理确实导致随后轴突生长有可测量的增加。然而，将马斯托帕兰与百日咳毒素共同注射可阻断马斯托帕兰单独的抑制作用。这些结果表明，EP细胞内与Go(α)偶联的信号事件可能会下调其迁移行为，这可能是对正常引导发育中胚胎迁移的抑制性线索的反应。
