Cellular And Molecular Mechanism Of R7 Target Selection

R7靶标选择的细胞和分子机制

基本信息

批准号：
7734770
负责人：
Chi-Hon Lee
金额：
$ 33.31万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

In both vertebrates and invertebrates, the interneuronal connections are often organized into columns and layers, which facilitate information processing and propagating. We use the Drosophila visual system as a model to study circuit assembly and focus on the mechanisms guiding R7 axons into specific layers and columns during development. In a large genetic screen based on a R7-dependent behavior, we identified two loci, baboon and importin-alpha3, which are required for the establishment of a precise R7 retinotopic map. Baboon encodes for a type I Activin receptor and importin-alpha3 for a component of nuclear import machinery. Removing Baboon or Importin-alpha3 in single R7s resulted in their axons invading neighboring columns, indicating that Baboon and Importin-alpha3 are required cell-autonomously in R7s to restrict their growth cones to retinotopically appropriate columns. In addition, the synaptic boutons of baboon or importin-alph3 mutant R7s appeared to be smaller and more irregular than those of the wild-type, suggesting that these two gene products are involved in synaptogenesis. Examining other known components of the Activin pathways, including the ligand Activin, the downstream transcription factor Smad2, revealed that the canonical Activin signaling pathway is required for restricting R7 growth cones to their retinotopically appropriate columns. Interestingly, Activin is functionally required in R7s, suggesting that Activin serves as an autocrine ligand it is secreted from and act on R7 growth cones. Several lines of evidence indicate that Importin-alph3 is a new component of the Activin signaling pathway. First, Smad2 and Importin-alph3 form a physical complex in the growth cones and axons. Second, nuclear accumulation of Smad2 depends on Importin-alpha3. Most importantly, these observations raise the intriguing possibility that Importin-alph3 plays a role in the retrograde axonal transport of Smad2. A similar role for Importins in transporting transcription factors from axons/dendrites to nuclei has been recently proposed in vertebrate neurons, suggesting this function of Importins is conserved in both flies and vertebrates. In summary, our results support a novel model for Activin signaling in R7s: Autocrine Activin activates Baboon on R7 growth cones and results in the phosphorylation of the downstream transcription factor Smad2, which together with Importin-alpha3 is transported from the growth cones back to the nuclei to regulate transcription. This model is further supported by our observation that blocking retrograde axonal transport in R7s phenocopied baboon/importin-alpha3 phenotypes. Removing Importin-alpha3 or Baboon resulted in incomplete penetrance of R7 phenotypes: only 12-30% of mutant R7 axons invaded their neighboring columns. This suggests the existence of an additional mechanism that functions redundantly to the Activin signaling pathway in restricting R7 growth cones to their retinotopically appropriate columns. To test whether repulsive interactions among neighboring R7s play a role to restrict R7 termini in appropriate columns, we genetically ablated most of the R7s and examined the targeting of the remaining R7s. We found that wild-type R7 axons form normal synaptic boutons in retinotopically correct columns even in a largely empty R7 terminal field. By contrast, removing neighboring R7s greatly increased the tendency of importin-alpha3 or baboon mutant R7s to invade adjacent columns. These results suggest that importin-alpha3 and baboon mutant R7 are still responsive to repulsion by neighboring R7s and these repulsive interactions account for their incomplete penetrance of phenotype. To determine the molecular nature of the R7-R7 interactions, we examined a number of candidate genes, whose products are known to mediate repulsive interactions. Among these, we identified the protocadherin Flamingo. Removing Flamingo alone in single R7s did not cause any obvious phenotype but the invasiveness of baboon mutant R7s was greatly enhanced by the removal of Flamingo in the neighboring R7s. In summary, at least two redundant mechanisms restrict R7 termini to the correct columns: (i) an intrinsic mechanism mediated by autocrine Activin signaling; and (ii) an extrinsic mechanism by Flamingo-mediated repulsion among R7s.

在脊椎动物和无脊椎动物中，神经元间连接通常被组织成柱和层，这有利于信息处理和传播。我们使用果蝇视觉系统作为模型来研究电路组装，并重点研究在发育过程中引导 R7 轴突进入特定层和柱的机制。在基于 R7 依赖性行为的大型遗传筛选中，我们鉴定了两个基因座：狒狒和 importin-alpha3，这是建立精确的 R7 视网膜专题图所必需的。 Baboon 编码 I 型激活蛋白受体，并编码 importin-alpha3 作为核输入机制的一个组成部分。去除单个 R7 中的 Baboon 或 Importin-alpha3 会导致它们的轴突侵入邻近的柱，这表明 R7 中需要细胞自主地使用 Baboon 和 Importin-alpha3 将其生长锥限制在视网膜局部适当的柱上。此外，狒狒或importin-alph3突变体R7的突触纽扣似乎比野生型的突触纽扣更小且更不规则，表明这两种基因产物参与突触发生。检查激活素途径的其他已知成分，包括配体激活素、下游转录因子 Smad2，发现经典的激活素信号传导途径是将 R7 生长锥限制在其视网膜局部适当的柱上所必需的。有趣的是，激活素是 R7 功能上所必需的，这表明激活素作为一种自分泌配体，由 R7 生长锥分泌并作用于 R7 生长锥。多项证据表明 Importin-alph3 是 Activin 信号通路的新组成部分。首先，Smad2 和 Importin-alph3 在生长锥和轴突中形成物理复合体。其次，Smad2 的核积累依赖于 Importin-alpha3。最重要的是，这些观察结果提出了一个有趣的可能性，即 Importin-alph3 在 Smad2 的逆行轴突运输中发挥作用。最近在脊椎动物神经元中提出了 Importins 在将转录因子从轴突/树突转运到细胞核中的类似作用，这表明 Importins 的这种功能在果蝇和脊椎动物中都是保守的。总之，我们的结果支持 R7 中激活素信号传导的新模型：自分泌激活素激活 R7 生长锥上的狒狒并导致下游转录因子 Smad2 磷酸化，Smad2 与 Importin-alpha3 一起从生长锥转运回细胞核来调节转录。我们观察到阻断 R7s 表型狒狒/importin-alpha3 表型中的逆行轴突运输，进一步支持了该模型。去除 Importin-alpha3 或 Baboon 会导致 R7 表型的不完全外显：只有 12-30% 的突变 R7 轴突侵入其相邻柱。这表明存在一种额外的机制，该机制与激活素信号通路冗余地发挥作用，将 R7 生长锥限制在其视网膜局部适当的柱上。为了测试相邻 R7 之间的排斥相互作用是否起到限制 R7 末端在适当列中的作用，我们从基因上消除了大部分 R7，并检查了剩余 R7 的靶向性。我们发现，即使在很大程度上空的 R7 末端区域，野生型 R7 轴突也会在视网膜局部正确的列中形成正常的突触纽扣。相比之下，去除相邻的 R7 大大增加了 importin-alpha3 或狒狒突变体 R7 侵入相邻柱的趋势。这些结果表明 importin-alpha3 和狒狒突变体 R7 仍然对邻近 R7 的排斥反应有反应，这些排斥相互作用解释了它们表型的不完全外显。为了确定 R7-R7 相互作用的分子性质，我们检查了许多候选基因，已知这些基因的产物可介导排斥相互作用。其中，我们鉴定出了原钙粘蛋白火烈鸟。单独去除单个 R7 中的 Flamingo 不会引起任何明显的表型，但狒狒突变体 R7 的侵袭性通过去除邻近 R7 中的 Flamingo 大大增强。总之，至少有两种冗余机制将 R7 末端限制在正确的列上：（i）由自分泌激活素信号传导介导的内在机制； (ii) 火烈鸟介导的 R7 之间排斥的外在机制。