Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system
Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system
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DOI：
10.1073/pnas.1506763112
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发表时间：
2015-06-02
影响因子：
11.1
通讯作者：
Rubin, Gerald M.
中科院分区：
文献类型：
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作者：
Nern, Aljoscha;Pfeiffer, Barret D.;Rubin, Gerald M.
我们描述了使用光学显微镜进行果蝇高通量神经解剖学方法的开发和应用。这些工具可以在低密度和高密度下对神经元进行有效的多色随机标记。多个膜靶向和不同表位标记蛋白的表达由转录驱动程序和重组酶介导的转录终止盒的随机切除控制。这种多色 FlpOut (MCFO) 方法可用于揭示细胞形状和相对细胞位置，并通过发育跟踪前体细胞的后代。使用两种不同的重组酶，可以单独控制标记的细胞数量和在这些细胞中观察到的颜色组合数量。我们展示了 MCFO 在对成人视觉系统中远端髓质 (Dm) 神经元、多柱局部中间神经元的多样性和变异性的详细研究中的实用性。与许多大脑区域类似，髓质具有重复的柱状结构，支持并行信息处理以及能够实现柱之间通信的细胞过程的正交层。我们发现，在髓质层内，给定 Dm 神经元类型的细胞过程形成不同的模式，反映单个细胞的形态及其乔木的相对位置。这些定型细胞排列在细胞类型之间有所不同，甚至对于不同髓质层中相同细胞类型的过程也可能不同。这种意想不到的覆盖模式多样性提供了多种独立的方式来整合视网膜专题列中的视觉信息，并暗示存在多种产生这些不同模式的发育机制。
We describe the development and application of methods for high-throughput neuroanatomy in Drosophila using light microscopy. These tools enable efficient multicolor stochastic labeling of neurons at both low and high densities. Expression of multiple membrane-targeted and distinct epitope-tagged proteins is controlled both by a transcriptional driver and by stochastic, recombinase-mediated excision of transcription-terminating cassettes. This MultiColor FlpOut (MCFO) approach can be used to reveal cell shapes and relative cell positions and to track the progeny of precursor cells through development. Using two different recombinases, the number of cells labeled and the number of color combinations observed in those cells can be controlled separately. We demonstrate the utility of MCFO in a detailed study of diversity and variability of Distal medulla (Dm) neurons, multicolumnar local interneurons in the adult visual system. Similar to many brain regions, the medulla has a repetitive columnar structure that supports parallel information processing together with orthogonal layers of cell processes that enable communication between columns. We find that, within a medulla layer, processes of the cells of a given Dm neuron type form distinct patterns that reflect both the morphology of individual cells and the relative positions of their arbors. These stereotyped cell arrangements differ between cell types and can even differ for the processes of the same cell type in different medulla layers. This unexpected diversity of coverage patterns provides multiple independent ways of integrating visual information across the retinotopic columns and implies the existence of multiple developmental mechanisms that generate these distinct patterns.