Cellular And Molecular Mechanism Of R7 Target Selection

R7靶标选择的细胞和分子机制

• 批准号: 8149308
• 负责人: Chi-Hon Lee
• 金额: $ 48.86万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8149308
• 关键词: Molecular

The chromatic photoreceptor neurons, R7 and R8, project their axons to the M3 and M6 layers of the medulla neuropil, respectively, and each pair of R7 and R8 axons innervates a single medulla column. Together these axonal termini tile the entire medulla neuropil. These organizing features, called layer-specific targeting and retinotopic mapping, are the hallmark of all complex visual systems. We have shown previously that during development, R7 and R8 axons project to specific layers and columns in two distinct stages. The sequential targeting of R7 and R8 axons to columns and layers reduces the number of potential synaptic partners (from over sixty medulla neurons to several), effectively reducing wiring complexity. By combining forward genetic screens and behavioral assays, we identified a number of mutants in which layer-specific targeting or retinotopic mapping of R7s is disrupted. We are currently focusing on two loci: overshoot (osh) and premature extension (pex). The hypomorphic osh mutants exhibit a novel phenotype: osh mutant R7s overshoot the target layer M6 and form additional synapses in the deeper medulla layer. Null osh mutant R7 has a much stronger phenotype. The mutant R7s exhibit abnormally enlarged synaptic boutons with excessive presynaptic structural protein, Bruchpilot. The overshoot and enlarged synaptic bouton phenotype appear during the synpatogenesis stage around 80% after pupal formation. Both developmental staging and phenotype suggest that osh play a regulatory role in synaptogenesis. By positional cloning, we have located the osh locus to a genomic region of approximately 50 Kb on the right arm of the second chromosome, and identified a candidate gene by complementation. We are in the process of confirming the identify of the osh gene by genomic rescue approach. Premature extension or pex mutations affects the refinement of R7 retinotopic map. During the larval stage, a gross retinotopic map of R7/R8 termini first forms via a Wnt4-dependent mechanism. During the late pupal stage when the R7 and R8 growth cones regain motility and proceed to their final destined layer, the R7 and R8 growth cones are confined to their retinotopically appropriate columns to form synaptic connections, a process called retinotopic map refinement. Pex is a temperature-sensitive allele of Baboo which encodes for a type I TGF-beta/Activin receptor. Baboon mutant R7 growth cones invades their neighboring columns, forming inappropriate synapses, indicating that Activin signaling is required for confining R7 growth cones to their retinotopically appropriate columns. We further determined that other known components of the canonical Activin signaling pathway, such as the downstream transcription factor Smad2, are required cell-autonomously in R7s for retinotopic map refinement. Immunohistochemistry analysis revealed that Smad2 is physically located at the R7 growth cones for receiving Activin signaling. This raises a question of how the phosphorylated and hence activated Smad2 is transported from growth cones back to nuclear to activate transcription. We found that the transport of Smad2 requires Dynein/Dynactin-dependent retrograde axonal transport system. Disrupting retrograde transport by expressing a dominant negative mutant of p150glued phenocopies baboon. In addition, we found that Improtin-alpha3, a known component of the nuclear import machinery, likely serves an unexpected role as a carrier to transport activated Smad2 from growth cones to nucleus. Importin-alpha3 mutant R7s exhibit retinotopic map defects as seen in baboon mutants. Biochemical analysis further revealed that Importin-alpha3 co-localizes with Smad2 in the R7 growth cones and they form a physical complex. Finally, the nuclear accumulation of Smad2 is disrupted in the absence of Importin-alpha or baboon, indicating that the carrier Importin-alpha3 and Actin-signaling, hence the phosphorylation of Smad2 are both required for transporting Smad2 from growth cones to nucleus. We are now examining the expression profile of baboon and Importin-alpha3 mutants for identifying the transcription targets of Activin signaling in R7s.

彩色感光神经元 R7 和 R8 将其轴突分别投射到髓质神经毡的 M3 和 M6 层，每对 R7 和 R8 轴突支配单个髓质柱。这些轴突末端一起构成了整个髓质神经纤维。这些组织功能，称为特定层定位和视网膜专题映射，是所有复杂视觉系统的标志。我们之前已经表明，在发育过程中，R7 和 R8 轴突在两个不同的阶段投射到特定的层和柱。 R7 和 R8 轴突依次靶向柱和层，减少了潜在突触伙伴的数量（从 60 多个髓质神经元减少到几个），从而有效降低了布线复杂性。 通过结合正向遗传筛选和行为测定，我们鉴定了许多突变体，其中 R7 的层特异性靶向或视网膜定位图被破坏。我们目前关注两个基因座：过冲 (osh) 和过早延伸 (pex)。亚等位的 osh 突变体表现出一种新的表型：osh 突变体 R7 超出目标层 M6 并在更深的髓质层中形成额外的突触。空 osh 突变体 R7 具有更强的表型。突变体 R7 表现出异常增大的突触纽带和过量的突触前结构蛋白 Bruchpilot。超调和扩大的突触 bouton 表型出现在蛹形成后约 80% 的突触发生阶段。发育阶段和表型都表明 osh 在突触发生中发挥调节作用。通过定位克隆，我们将osh位点定位到第二条染色体右臂上约50Kb的基因组区域，并通过互补鉴定了候选基因。我们正在通过基因组救援方法确认 osh 基因的身份。 过早延伸或 pex 突变会影响 R7 视网膜专题图的细化。在幼虫阶段，R7/R8 末端的总体视网膜专题图首先通过 Wnt4 依赖性机制形成。在蛹期后期，当 R7 和 R8 生长锥恢复运动性并进入其最终的预定层时，R7 和 R8 生长锥被限制在其视网膜局部适当的柱内以形成突触连接，这一过程称为视网膜局部图细化。 Pex 是 Baboo 的温度敏感等位基因，编码 I 型 TGF-β/激活素受体。狒狒突变体 R7 生长锥侵入其邻近的柱，形成不适当的突触，表明需要激活素信号传导将 R7 生长锥限制在其视网膜局部适当的柱上。我们进一步确定，R7s 中细胞自主地需要经典激活素信号通路的其他已知组件（例如下游转录因子 Smad2）来细化视网膜专题图。免疫组织化学分析显示，Smad2 物理上位于 R7 生长锥上，用于接收激活素信号传导。这就提出了一个问题：磷酸化并因此激活的 Smad2 如何从生长锥转运回核​​以激活转录。我们发现Smad2的运输需要动力蛋白/动态蛋白依赖性逆行轴突运输系统。通过表达p150glued表型狒狒的显性失活突变体来破坏逆行运输。此外，我们发现 Improtin-alpha3（核输入机制的一种已知成分）可能发挥着意想不到的作用，作为将激活的 Smad2 从生长锥运输到细胞核的载体。 Importin-alpha3 突变体 R7 表现出视网膜专题图缺陷，如狒狒突变体中所见。生化分析进一步表明，Importin-alpha3 与 Smad2 在 R7 生长锥中共定位，并形成物理复合物。最后，Smad2 的核积累在没有 Importin-alpha 或狒狒的情况下被破坏，表明载体 Importin-alpha3 和肌动蛋白信号传导，因此 Smad2 的磷酸化都是将 Smad2 从生长锥转运到细胞核所必需的。我们现在正在检查狒狒和 Importin-alpha3 突变体的表达谱，以鉴定 R7 中激活素信号传导的转录靶标。