摘要:在發育過程中神經系統迴路的連結與建立,有賴於神經軸索分枝和突觸形成的精確調控。前人研究發現許多控制此過程之內源性機制與外來因子。Morphogen 為調控胚胎早期發育、形態生成和細胞分化的重要分泌性因子。近期的研究發現,這些早期的morphogen,如 Wnt,BMP 或 FGF,在神經系統發育的晚期會再度被用來促進神經軸索的分枝和突觸的發育。這些呈現梯度分布的胞外因子如何精確穩定地調控神經軸索分枝或突觸形成,目前了解仍然相當有限。 Wnt 蛋白可調控線蟲神經母細胞的分裂、神經細胞的遷移、軸索生長錐的導引,以及突觸形成的位置。為了更進一步了解 Wnt 蛋白如何控制神經軸索的分枝和突觸形成,我們以線蟲的 PLM 觸覺神經元做為研究對象。PLM 神經元在神經軸索上有單一的分枝,此分枝總是在幾乎固定不變的位置上出現,其末梢會和中間神經元形成抑制性的麩胺酸突觸,藉此調控線蟲對觸覺刺激的躲避反應。我們發現 Wnt 路徑的基因突變,包括Wnt,Wnt 的受體 Frizzled,β-catenin 以及 vang-1,都會造成 PLM 的分枝往更近細胞體處偏移,而且突觸也有明顯發育上的缺陷。 本研究計畫探討胞外的 Wnt 濃度梯度如何控制 PLM 分枝形成的位置和突觸的發育。在第一部分中,我們利用 optogenetics 來解析活體線蟲 PLM 神經突觸的功能。我們也將利用螢光激發剔除細胞的技術,來探究 PLM 鄰近細胞對其神經側枝的形成是否扮演重要角色。在第二部分中,我們進行完整的突變株分析,以解明許多 Wnt 路徑基因對 PLM 側枝和突觸發育的重要性。我們將藉由異位表達 Wnt 蛋白來探討不同方向性的Wnt 訊號是否可以決定 PLM 側枝形成的位置。在第三部分中,我們探討 Wnt 調控 PLM側枝形成和突觸發育的細胞生物學機轉。我們首先將利用活體神經造影技術找出決定神經側枝形成位置的重要細胞骨架或胞器,之後再利用突變株分析,佐以蛋白質體學技術來找出更多分子層面上的新機制。這一系列的研究將有助於吾人對於 Wnt 訊息調控神經側枝和突觸發育機轉上的了解。
Abstract: Axon branching and synapse formation are two critical developmental processes that establish the connectivity of neural circuits in the nervous system. Past studies had identified factors both intrinsic and extrinsic to the neurons that are essential for the formation of axon arbor and synapses. Morphogens are secreted molecules that govern early developmental pattern formation and cell fate specification in a concentration-dependent fashion. A recent conceptual advance is that many classical morphogens, such as Wnts, BMPs and FGFs, are reused later as organizers for axon branch arbors or synapses. How these graded extracellular signals pattern axon branching and synapse formation in a highly stereotyped, precise manner is not clear. In the nematode Caenorhabditis elegans, the Wnt secreted glycoproteins had been shown to regulate neuroblast division, neuronal migration, axon growth cone guidance and synapse localization. To investigate how Wnts pattern axon branches and promote synapse formation, we focus on the C. elegans PLM touch neuron. The PLM neuron sprouts a single axon branch at a stereotyped position along the neuronal process, forms inhibitory glutamatergic synapses with two interneurons, and regulates the avoidance response upon PLM activation. We found that mutations in the Wnt pathway genes, including Wnts, the Wnt receptor Frizzleds, the β-catenin and vang-1, a planar polarity gene, resulted in shifted axon branching sites and underdeveloped synapses in the PLM. PLM branches in these mutants were more proximal to the cell body, and they formed smaller synapses that were deficient for synaptic vesicles or active zone proteins. In this proposal, we address how the graded extracellular Wnt signals control the spatial placement of the PLM axon branch or the development of the PLM synapses. In Aim 1, we devise an optogenetic approach for non-invasive assessment of PLM synapse function in live animals. We also propose to determine the role of neighboring cells in regulating PLM branch placement by mutant analysis or chromophore-assisted cell ablation. In Aim 2, we first perform extensive mutant analysis to have a complete picture of the diverse Wnt pathways involved in PLM branch placement and synapse development. We then determine whether graded Wnt signals instruct the placement of the PLM branch, by altering in vivo Wnt distribution using site-specific ectopic expression. In Aim 3, we explore the cell biological basis of Wnt-dependent regulation of axon branching and synapse development. We propose to identify putative cytoskeletal or endosomal elements that mediate the effects of Wnt signals, first by live imaging and mutant analysis, followed by a proteomic approach. Together these experiments will provide novel mechanistic insights into the Wnt-dependent spatial control of axon branching and synapse development.