Horgan A M, Lagrange M T, Copenhaver P F
Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland 97201, USA.
Development. 1994 Apr;120(4):729-42. doi: 10.1242/dev.120.4.729.
Directed neuronal migration contributes to the formation of many developing systems, but the molecular mechanisms that control the migratory process are still poorly understood. We have examined the role of heterotrimeric G proteins (guanyl nucleotide binding proteins) in regulating the migratory behavior of embryonic neurons in the enteric nervous system of the moth, Manduca sexta. During the formation of the enteric nervous system, a group of approx. 300 enteric neurons (the EP cells) participate in a precise migratory sequence, during which the undifferentiated cells populate a branching nerve plexus that lies superficially on the visceral musculature. Once migration is complete, the cells then acquire a variety of position-specific neuronal phenotypes. Using affinity-purified antisera against different G protein subtypes, we found no apparent staining for any G protein in the EP cells prior to their migration. Coincident with the onset of migration, however, the EP cells commenced the expression of one particular G protein, Go alpha. The intensity of immunostaining continued to increase as migration progressed, with Go alpha immunoreactivity being detectable in the leading processes of the neurons as well as their somata. The identity of the Go alpha-related proteins was confirmed by protein immunoblot analysis and by comparison with previously described forms of Go alpha from Drosophila. When cultured embryos were treated briefly with aluminium fluoride, a compound known to stimulate the activity of heterotrimeric G proteins, both EP cell migration and process outgrowth were inhibited. The effects of aluminium fluoride were potentiated by alpha toxin, a pore-forming compound that by itself caused no significant perturbations of migration. In preliminary experiments, intracellular injections of the non-hydrolyzable nucleotide GTP gamma-S also inhibited the migration of individual EP cells, supporting the hypothesis that G proteins play a key role in the control of neuronal motility in this system. In addition, once migration was complete, the expression of Go alpha-related proteins in the EP cells underwent a subsequent phase of regulation, so that only certain phenotypic classes among the differentiated EP cells retained detectable levels of Go alpha immunoreactivity. Thus Go may perform multiple functions within the same population of migratory neurons in the course of embryonic development.
定向神经元迁移有助于许多发育系统的形成，但控制迁移过程的分子机制仍知之甚少。我们研究了异源三聚体G蛋白（鸟苷酸结合蛋白）在调节烟草天蛾肠道神经系统中胚胎神经元迁移行为中的作用。在肠道神经系统形成过程中，一组约300个肠神经元（EP细胞）参与精确的迁移序列，在此期间，未分化细胞聚集在位于内脏肌肉表面的分支神经丛中。一旦迁移完成，这些细胞就会获得多种位置特异性神经元表型。使用针对不同G蛋白亚型的亲和纯化抗血清，我们发现在EP细胞迁移之前，没有任何G蛋白有明显染色。然而，与迁移开始同时，EP细胞开始表达一种特定的G蛋白，Goα。随着迁移的进行，免疫染色强度持续增加，在神经元的前端突起及其胞体中都可检测到Goα免疫反应性。通过蛋白质免疫印迹分析并与先前描述的果蝇Goα形式进行比较，证实了Goα相关蛋白的身份。当用已知能刺激异源三聚体G蛋白活性的氟化铝短暂处理培养的胚胎时，EP细胞迁移和突起生长均受到抑制。氟化铝的作用被α毒素增强，α毒素是一种形成孔的化合物，其本身不会对迁移造成明显干扰。在初步实验中，细胞内注射不可水解的核苷酸GTPγ-S也抑制了单个EP细胞的迁移，支持了G蛋白在该系统中神经元运动控制中起关键作用的假说。此外，一旦迁移完成，EP细胞中Goα相关蛋白的表达经历了随后的调节阶段，因此在分化的EP细胞中只有某些表型类别保留了可检测水平的Goα免疫反应性。因此，Go可能在胚胎发育过程中同一群迁移神经元内执行多种功能。
