Stochastic yet biased expression of multiple Dscam splice variants by individual cells

Abstract

The Drosophila melanogaster gene Dscam is essential for axon guidance and has 38,016 possible alternative splice forms. This diversity can potentially be used to distinguish cells. We analyzed the Dscam mRNA isoforms expressed by different cell types and individual cells. The choice of splice variants expressed is regulated both spatially and temporally. Different subtypes of photoreceptors express broad yet distinctive spectra of Dscam isoforms. Single-cell RT-PCR documented that individual cells express several different Dscam isoforms and allowed an estimation of the diversity that is present. For example, we estimate that each R3/R4 photoreceptor cell expresses 14-50 distinct mRNAs chosen from the spectrum of thousands of splice variants distinctive of its cell type. Thus, the Dscam repertoire of each cell is different from those of its neighbors, providing a potential mechanism for generating unique cell identity in the nervous system and elsewhere. To circumvent these technical difficulties, we used an approach that combined a customized oligonucleotide microarray, isolation of distinct populations of cells and sensitive RT-PCR that allowed examination of Dscam expression by single cells. Here we show that Dscam alternative splicing is differentially regulated both during development and in different tissues. A given cell type expresses a broad, yet distinctive, spectrum of splice variants. Individual cells express a unique repertoire of Dscam splice variants. We estimate that individual R3/R4 photoreceptors express 14-50 distinct mRNA molecules chosen from a subset of thousands of Dscam isoforms characteristic of the population. This would ensure that the Dscam repertoire of each cell is different from those of its neighbors. A R T I C L E S RESULTS A microarray for analyzing Dscam alternative splicingThe genomic structure of Dscam 1 is shown in Figure 1a. There are four alternatively spliced exons. In this study we focused on three alternatively spliced exons, which may create many possible isoforms (Fig. 1a): the cluster in exon 4 has 12 alternative exons, the cluster in exon 6 has 48 alternative exons and the cluster in exon 9 has 33 alternative exons. Each of these highly variable exons can form a portion of an immunoglobulin domain in the extracellular region of the protein. We designed a microarray containing probes for all 93 alternative exons in these three clusters. We amplified cloned DNAs (whose identities were confirmed by sequencing) by PCR to create a reference library that equally represents all the alternative exons present in the microarray. To test the specificity of the different probes in the microarray, we used combinations of RNAs in controlled amounts (Fig. 1b). RNA samples were in vitro transcribed from cloned cDNAs containing different combinations of exons 4, 6 and 9. We carried out a quantitative analysis of four independent experiments in which eight different samples were combined in different amounts spanning two orders of magnitude. Throughout this study, we expressed sample...

A regulator of Dscam mutually exclusive splicing fidelity

“…Is this process highly regulated or does it occur in a random or stochastic manner? Microarray experiments have revealed little if any regulation of exon 6 splicing 11,27 , which is consistent with the biology of Dscam. In the nervous system, it seems that the actual identity of the Dscam isoforms that are expressed is unimportant, provided that each neuron expresses a different set of isoforms than the neighboring neurons 11,[28][29][30][31] .…”

Reconstructing immune phylogeny: new perspectives

“…The possibilities for alternative spli cing increase markedly when exons have undergone tandem duplication 132 , such as for the D. melanogaster Down's syndrome cell-adhesion molecule (Dscam) gene, which functions in complex neuronal processing by generating tens of thousands of protein variants from a single gene through differential RNA processing 132,133 . Both the complexity and expression patterns of Dscam isoforms in haemocytes indicate that Dscam might have a role in immune function 133,134 . Recent studies have defined differences in the expression of Dscam isoforms in haemocytes, fat cells and brain tissue 10 .…”

Dscam-mediated repulsion controls tiling and self-avoidance

“…The immunoglobulin superfamily protein, Dscam1, was a good candidate for several reasons. First, the Dscam1 locus can generate tens of thousands of different recognition molecules through alternative splicing [5] and each neuron appears to express a unique combination of isoforms [6]. Second, each Dscam1 isoform mediates homophilic, but not heterophilic binding with other isoforms and thus, this single gene could generate the diversity required for self-recognition throughout the nervous system [7].…”

“…Although both of these scenarios would confer a discrete cell surface identity upon each neuron, the former scenario requires a complex alternative splicing mechanism, whereas the latter can be achieved through stochastic expression of many Dscam1 isoforms within each cell. Expression studies in mushroom body neurons (as well as several other subclasses) argue that many isoforms are expressed in each neuron and that the expression pattern is largely random [6]. In addition, genetic studies demonstrated that the type of isoform expressed in each MB neuron is not important; rather, what is crucial is that an individual MB neuron expresses different isoforms from its neighbors [9][10][11].…”