Dscam-mediated self-versus non-self-recognition by individual neurons
Dscam-mediated self-versus non-self-recognition by individual neurons
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DOI：
10.1101/sqb.2004.69.485
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发表时间：
2004-01-01
期刊：
影响因子：
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通讯作者：
Chess, A
中科院分区：
文献类型：
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作者：
Neves, G;Chess, A
我们在单个细胞上进行的实验已经发表在其他地方（Neves et al. 2004），这里将进行总结。我们设计了一个微阵列，包含三个簇中编码可变细胞外免疫球蛋白结构域（外显子4,6和9）的所有93个备选外显子的探针。利用微阵列，我们首先分析了不同发育时间点的整只苍蝇。这些初步实验表明，Dscam的剪接是发育调控的，特别是关于外显子9。例如，33个外显子9亚型中有5个在胚胎阶段的表达水平明显高于幼虫阶段。我们的结果也证实了先前的观察，即从胚胎到成人，外显子4.2的相对代表性增加（Celloto和Graveley 2001）。然后我们转向如何使用Dscam多样性的问题，询问Dscam亚型的子集是否定义不同的细胞类型。这是一种关于Dscam基因选择性剪接调控的有趣可能性：特定的细胞群各自表达所有可用同种异构体的特定小子集。我们分析了多种细胞类型，以寻找许多不同类型的神经元和非神经元细胞在使用Dscam曲目方面的显着差异。例如，我们使用GAL4-UAS系统（Brand and Perrimon 1993）标记发育中的眼睛中的特定细胞群，结果表明，光感受器的R3/R4和R7群体在所有三个外显子簇上表达大量不同的同工异型。每种类型的光感受器仍然表达一系列的Dscam形式，据计算，其复杂性远远超过10,000种不同的形式（在细胞外不同的外显子4,6和9的约19,000种可能的组合中）。这些结果排除了一种模型，即投射到特定大脑区域的特定类型的光感受器表达一种或少量不同的Dscam同种异构体。尽管观察到的广谱，聚类分析表明，两个种群表达的Dscam亚型在彼此之间以及在整个眼天线成像盘上都有显著差异。因此，每种特定的细胞类型表达广泛而独特的Dscam亚型谱。在不同的细胞群体中，功能库使用的差异可能在功能上很重要，但更有可能不是具有如此不同的功能库的主要原因。只有在我们对单细胞细胞的分析中，才可能发现大量的Dscam基因存在的原因（见下文）。微阵列的使用也使我们能够检测S2细胞系及其亚克隆中的Dscam表达。在S2细胞系中表达了一个非常引人注目的Dscam亚型。虽然外显子4和6的不同异构体的使用大致相同，但外显子9显然只使用了33个异构体中的5个。因此，在S2细胞群体中，存在某种程度上有限的Dscam形式。在其他两种细胞系中也观察到类似的特征，这些细胞系与S2细胞一样，被认为具有与血细胞相似的特性。荧光活化细胞分选仪（FACS）从三龄幼虫中分离出表达绿色的血细胞
The experiments we carried out on individual cells have been published elsewhere (Neves et al. 2004) and will be summarized here. We designed a microarray containing probes for all 93 alternative exons in the three clusters that encode the variable extracellular immunoglobulin domains (exons 4, 6, and 9). Using the microarray, we first analyzed whole flies at various developmental time points. These initial experiments showed that splicing of Dscam is developmentally regulated, particularly with respect to exon 9. For example, 5 of the 33 exon 9 isoforms are expressed at significantly higher levels in embryos than at the larval stages. Our results also confirm previous observations that the relative representation of exon 4.2 increases from embryos to adults (Celloto and Graveley 2001).We then turned to the question of how the Dscam diversity is used, asking whether subsets of Dscam isoforms define different cell types. This was an intriguing possibility regarding the regulation of alternative splicing of the Dscam gene: that particular cell populations would each express a specific small subset of all the available isoforms. We analyzed a variety of cell types to search for significant differences in use of the Dscam repertoire by a number of different types of neuronal and nonneuronal cells. For example, we used the GAL4-UAS system (Brand and Perrimon 1993) to label specific populations of cells in the developing eye, and showed that both R3/R4 and R7 populations of photoreceptors express a large number of different isoforms at all three exon clusters. Each type of photoreceptor still expressed a repertoire of Dscam forms that was calculated to have a complexity well over 10,000 different forms (out of~ 19,000 possible combinations for the extracellular diverse exons 4, 6, and 9). These results ruled out the model in which a specific class of photoreceptor that projects to a particular brain region expresses one or a small number of distinct Dscam isoforms. The observed broad spectra notwithstanding, clustering analysis indicates that the profiles of Dscam isoforms expressed by the two populations are significantly different from each other and from the entire eye-antennal imaginal disk. Thus, each specific cell type expresses a broad yet distinctive spectrum of Dscam isoforms. The differences in repertoire usage in the distinct cell populations may be functionally important, but more likely are not the primary reason for having such distinct repertoires. The likely reason for the enormous repertoire of Dscam emerged only from our single-cell analyses (see below). The use of the microarray also allowed us to examine Dscam expression in S2 cell lines and subclones thereof. A very striking profile of Dscam isoforms is expressed in S2 cell lines. While the usage of different isoforms of exons 4 and 6 is roughly equal, for exon 9 apparently only 5 out of the 33 isoforms are used. Thus, there is a somewhat restricted repertoire of Dscam forms present within the population of S2 cells. Similar profiles were also observed with two other cell lines that have, like S2 cells, been suggested to share similar properties with hemocytes. Hemocytes isolated by fluorescence-activated cell sorter (FACS) from third-instar larvae that express green