Beyond ephrins: unbiased discovery of novel signaling pathways regulating topographic map formation and maturation in vivo.

超越肝配蛋白：公正地发现调节体内地形图形成和成熟的新型信号通路。

• 批准号: 10330851
• 负责人: Fabienne Emmanuelle Poulain
• 金额: $ 40.75万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-08 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10330851
• 关键词: Address; Alleles; Area; Axon; Brain; CRISPR/Cas technology; Cell Adhesion Molecules; Cell membrane; Code; Cues; Development; Eph Family Receptors; Ephrins; Felis catus; Ganglion Cell Layer; Gene Expression Profile; Genes; Genetic; In Situ Hybridization; Integral Membrane Protein; Knowledge; Ligands; Maps; Mediating; Modeling; Nervous system structure; Neurodevelopmental Disorder; Neurons; Nose; Pathway interactions; Positioning Attribute; Problem Solving; Process; Retina; Retinal Ganglion Cells; Role; Signal Pathway; Stimulus; Tectum Mesencephali; Testing; Time; Transgenic Organisms; Visual system structure; Work; Zebrafish; adhesion receptor; base; differential expression; experience; high resolution imaging; imaging approach; in vivo; information processing; innovation; mutant; mutation screening; nerve injury; novel; novel therapeutic intervention; prevent; receptor; retinal axon; retinotectal; retinotopic; selective expression; single-cell RNA sequencing; vision development; visual map

PROJECT SUMMARY The brain efficiently processes information thanks to the precise organization of its neuronal networks. In most circuits, axonal projections are organized into topographic maps based on the spatial organization of the neu- rons they originate from. This is especially true in the visual system, where retinal projections transmit a pre- cise and continuous representation of the external world to the brain by maintaining the neighboring relation- ship of the retinal ganglion cells (RGCs) they originate from in the retina. Studies over the past decades have demonstrated that topographic maps are initially coarsely established and then refine in an activity-dependent manner to become more precise. Maps are first generated by specific axon-target interactions, whereby axons with a unique profile of receptors interpret guidance cues distributed in a gradient at the target. In the visual system, the guidance cues ephrins and their receptors Ephs have been identified as the “master regulators” of retinotopic mapping. Along the antero-posterior axis, for instance, ephrinAs and EphAs are expressed in coun- ter-gradients across the nasal-temporal axis in the retina and rostro-caudal axis in the brain target, thereby providing a spatial code instructing map development. Yet, an increasing number of studies including ours indi- cates that ephrins do not act alone to generate visual maps. Several receptors and adhesion molecules have indeed been preferentially detected in nasal or temporal RGCs and shown to regulate retinotopic mapping. Yet, technical limitations have so far prevented the full identification and characterization of the signaling path- ways other than ephrins/Ephs that generate retinotopic maps. We notably lack a comprehensive profile of the secreted factors, adhesion molecules and receptors that are differentially expressed in RGCs and control reti- notopy. Here, we propose to address that major gap by identifying the receptors and ligands that are differen- tially expressed in nasal vs temporal RGCs, and testing their function in retinotopic map formation and matura- tion in vivo. We will achieve these objectives by taking advantage of a new zebrafish “retinotopic” transgenic line we have recently generated, in which nasal and temporal RGCs selectively express different fluorescent markers. In Aim 1, we will use single-cell RNA sequencing (scRNA-seq) to generate a transcriptional profile of the secreted factors and plasma membrane molecules preferentially expressed in nasal or temporal RGCs over time. We will also use in situ hybridization to test whether these candidates are indeed differentially ex- pressed in the retina, and quantify their expression across the RGC layer. In Aim 2, we will use a CRISPR/ Cas9-mediated mutation screen to test the function of the secreted factors and trans-membrane proteins iden- tified in aim 1. We will notably analyze the area covered by nasal and temporal retinal projections at the brain target over time, as well as the sharpening of the retinotopic map in mutants. Altogether, our innovative ap- proach will identify new players controlling retinotopy and revisit the classical model of topographic map devel- opment, thereby providing new perspectives on the mechanisms underlying circuit wiring and maturation.

项目摘要 由于神经元网络的精确组织，大脑有效地处理信息。在大多数 回路、轴突投射根据神经元的空间组织被组织成地形图， 它们的起源。这在视觉系统中尤其如此，其中视网膜投射传输预- 通过保持相邻关系，外部世界对大脑的连续和连续的表征- 视网膜神经节细胞（RGC）是它们在视网膜中起源的细胞。过去几十年的研究 表明，地形图最初是粗略建立的，然后在活动依赖的细化 方式变得更加精确。地图首先由特定的轴突-靶相互作用产生， 具有独特的感受器轮廓，解释在目标处以梯度分布的引导线索。在视觉 系统中，引导信号ephrin及其受体Eph已被确定为“主调节器”， 视网膜定位图沿着前后轴，例如，ephrinAs和EphAs表达于coun-10中。 跨视网膜中的鼻颞轴和大脑目标中的喙尾轴的梯度，从而 提供指导地图开发的空间代码。然而，越来越多的研究，包括我们的indi- 表明肝配蛋白并不是单独产生视觉地图的。几种受体和粘附分子 事实上，优先在鼻或颞RGC中检测到，并显示出调节视网膜定位图。 然而，到目前为止，技术限制阻止了信令路径的完全识别和表征- 除了ephrins/Ephs之外的其他方法产生视网膜定位图。我们显然缺乏一个全面的概况 在RGCs和对照视网膜中差异表达的分泌因子、粘附分子和受体， notopy在这里，我们建议通过识别受体和配体来解决这一主要差距， 在鼻侧与颞侧RGC中表达，并测试它们在视网膜定位图形成和成熟中的功能。 在体内。我们将通过利用新的斑马鱼“视网膜定位”转基因来实现这些目标 我们最近产生的线，其中鼻和颞RGC选择性地表达不同的荧光 标记。在目标1中，我们将使用单细胞RNA测序（scRNA-seq）来生成以下转录谱： 分泌因子和质膜分子优先表达于鼻或颞RGC 随着时间我们还将使用原位杂交来测试这些候选人是否确实是差异表达的。 压在视网膜中，并量化它们在RGC层中的表达。在目标2中，我们将使用CRISPR/ Cas9介导的突变筛选，以测试分泌因子和跨膜蛋白iden的功能。 在目标1。我们将特别分析大脑中鼻侧和颞侧视网膜投射所覆盖的区域 随着时间的推移，以及突变体中视网膜定位图的锐化。总而言之，我们的创新AP- proach将确定控制视网膜病变的新玩家，并重新审视地形图开发的经典模型， opment，从而提供了新的观点的机制电路布线和成熟。