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

项目总结 大脑高效地处理信息，这要归功于其神经网络的精确组织。在大多数 根据神经元的空间组织，轴突投射被组织成地形图。 它们起源于Rons。这在视觉系统中尤其如此，在视觉系统中，视网膜投射传递的是一种预感 通过保持相邻关系，简洁而连续地将外部世界呈现给大脑- 它们起源于视网膜的视网膜神经节细胞(RGC)。过去几十年的研究表明 演示了地形图最初是粗略地建立的，然后根据活动进行了细化 态度变得更精确。MAP首先由特定的轴突-靶相互作用产生，因此轴突 通过一个独特的受体轮廓，解释在目标处以梯度分布的引导线索。在视觉上 系统中，引导线索Ephin及其受体Ephs已被确定为 视网膜定位图。例如，在前后轴线上，ePhinA和EphA以不同的形式表达。 在视网膜中的鼻颞轴和在大脑靶点中的嘴-尾轴上的TER梯度，从而 提供指导地图开发的空间代码。然而，包括我们在内的越来越多的研究表明- 这说明，电子短语并不是单独作用于生成视觉地图的。一些受体和黏附分子 确实在鼻部或颞部视网膜节细胞中被优先检测到，并被证明调节视网膜定位。 然而，到目前为止，技术限制阻碍了对信令路径的完全识别和表征- 产生视网膜定位图的方法不同于ePhin/Ephs。值得注意的是，我们缺乏关于 在视网膜节细胞和对照视网膜上差异表达的分泌因子、黏附分子和受体。 诺托普。在这里，我们建议通过识别不同的受体和配体来解决这一主要差距- 在鼻侧和颞侧视网膜节细胞中表达，并检测它们在视网膜原位地图形成和成熟中的功能。 活体内注射。我们将通过利用一种新的斑马鱼“视网膜”转基因来实现这些目标 我们最近创造的一条线，其中鼻部和颞部视网膜节细胞选择性地表达不同的荧光 记号笔。在目标1中，我们将使用单细胞RNA测序(scRNA-seq)来生成 分泌因子和质膜分子在鼻侧或颞侧视网膜节细胞中优先表达 随着时间的推移。我们还将使用原位杂交来测试这些候选基因是否确实存在差异。 按压在视网膜上，并量化它们在RGC层的表达。在AIM 2中，我们将使用CRISPR/ Cas9介导的突变筛选以检测识别的分泌因子和跨膜蛋白的功能 目标1。我们将重点分析鼻部和颞部视网膜投射在大脑中覆盖的区域 靶点随着时间的推移，以及突变体的视网膜定位图的锐化。总而言之，我们的创新AP- Proach将确定控制视网膜复制的新参与者，并重新审视地形图发展的经典模式- 从而为电路布线和成熟的潜在机制提供了新的视角。

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.