07 Feb 2004

The analogy of a computer has often been used to explain the workings of the brain, and vice versa. In computers, however, each byte has a unique address that allows the processor to track where it stored snippets of information. The brain, on the other hand, is not so well cataloged. Or is it? In this month’s Nature Genetics, MIT researchers suggest that each of the billions of neurons in the human brain may also have its own unique address—courtesy of alternative splicing.

Alternative splicing of messenger RNA has been used to explain why a complement of 30,000 genes, rather than the 100,000 some experts had predicted, is sufficient to make us humans so complex. But as reported by Andrew Chess and colleagues, alternative splicing may be many orders of magnitude more sophisticated. Together with Chess, first author Guilherme Neves and colleagues focused their attention on Dscam, one of only a handful of proteins that have the potential to exist as thousands of different, alternatively spliced isoforms. In fact, Drosophila Dscam (for Down's syndrome cell adhesion molecule), a protein expressed in the central nervous system, has over 38,000 potential isoforms.

This apparent redundancy raises intriguing questions. Are all the possible isoforms actually translated into protein? And do all cells that express Dscam in an organism express the same isoform? It was these two questions that Neves and colleagues tried to answer.

To address the first, the authors used a microarray that recognizes all the possible splice forms of three of Dscam’s exons, numbers 4, 6, and 9. This array, though capable of quantitating only a subset of variants, revealed that most of them are expressed at some time in flies. Only variant 11 from exon 6 could never be detected. If extrapolated to the other exons, these data suggest that Drosophila expressed nearly all Dscam variants.

The scientists then tackled the question of differential expression. Would all cells express the same isoform or group of isoforms? First, they compared different stages of development and found that expression of Dscam isoforms is temporally regulated. Five of 33 variants coded by exon 9 are expressed at higher levels in the embryo as compared to larvae, for example, while several variants were almost undetectable in the embryo but were robustly expressed in adult flies. The finding suggested the expression of specific variants may be tightly controlled and that not all cells express the same isoform.

To address this issue more directly, the authors turned to spatial differences. First Neves and colleagues looked at groups of similar cells. R3/R4 photoreceptors, for example, were found to express one variant (No. 8) of exon 9 most strongly, whereas in the nearby R7 photoreceptors, a different variant (No. 9) was most abundant. To take this experiment a step further, the authors looked at expression in individual cells using a modified single-cell PCR assay (the assay was modified to ensure that one isoform in a pool of many was not preferentially amplified). Neves found that each of seven individual R3/R4 photoreceptors expressed a unique profile of isoforms. The same was found to be true for seven R7 photoreceptors. But significantly, when the authors determined how many different mRNAs are present in each cell, they found that it was between 14 and 50. But as there are over 38,000 potential isoforms, then mathematically it is possible that the Dscam repertoire for each cell in Drosophila, and for that matter in humans, is unique. Whether this has any functional significance is the next question in this evolving story.—Tom Fagan

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