The possible existence of surface receptors that serve as molecular tags to direct neuronal connectivity, known as """"""""chemoaffinity theory"""""""", has been an intriguing hypothesis since it was proposed by Roger Sperry some 40 years ago. As the number of synaptic connections far exceeds the predicted number of surface receptors, it is unlikely that the correspondence between pre- and post-synaptic partners mediated by such surface receptors represents a simple one-to-one relationship. Combinatory use of surface receptors and their differential regulation have been proposed to serve as additional mechanisms for directing specific connections. Furthermore, receptor repertoire can be expanded by alternative splicing, as observed in several receptors, such as vertebrate protocadherin CNR (cadherin-related neuronal receptor) and insect Dscam (Down syndrome cell adhesion molecule). The identification of Ncad as the key regulator of R7 targeting and the discovery of multiple Ncad isoforms generated by alternative splicing allow us to directly examine how these mechanisms contribute to connection specificity. We identified striking molecular diversity of Ncad generated by alternative splicing. The genomic sequence analysis reveals that Ncad locus contains three pairs of exons (exon7-exon7p, exon13-exon13p and exon18-exon18p) in modular arrangement. RNA transcript analysis indicated that these exon pairs are used in a mutually exclusive manner -- i.e. each mature transcript contains one and only one of the two alternative exons. All six exons are utilized in the developing eye tissue, albeit with different frequence. The exons 7, 13 and 18p appear to be the predominant species in the eye disk. Using these three """"""""exon modules"""""""" in a combinatory fashion, the Ncad locus is capable of encoding for 8 isoforms, each of which shares the same molecular architecture but has unique amino acid sequence. Similar molecular diversity generated by modular arrangement of alternative exons has been observed in other receptors and might be a general mechanism for neuronal diversity and connection specificity. The notion that the different Ncad isoforms might have distinct function was first suggested by protein sequence analysis and later verified by cell aggregation assay. The exon7 and exon7p encode for the C-terminal half of the 8th cadherin repeat and N-terminal half of the 9th cadherin repeat, and the exon 13 and 13p encode for the 11th and 12th cadherin repeat in a similar manner. The exon 18 and 18p encode for one and half of EGF repeat and half of the transmembrane region. The sequence identity between alternative exons ranges from 37% (for exon18-18p) to ~50% (for exon7-7p and exon13-13p) at both DNA and protein levels. Interestingly, we found non-conservative amino acid changes in two regions that potentially mediate calcium binding and homophilic interactions, suggesting that these isoforms might have different adhesive activity. To test this possibility, we expressed individual isoform in S2 cells and assayed their ability to induce cell aggregation. Our preliminary data showed that the isoforms encoded by exon 7 but not exon 7p are capable of mediating homophilic interaction. On the other hand, region of Ncad encoded by exon13 and exon13p does not significantly affect Ncad's ability to induce cell aggregation. We are currently testing whether these isoforms can mediate heterphilic, or graded, interaction. Our current model depicts that combinatory use of different Ncad isoforms generates an """"""""adhesion code"""""""" that match pre- and post-synaptic partners.
| Lee, Roger C; Clandinin, Thomas R; Lee, Chi-Hon et al. (2003) The protocadherin Flamingo is required for axon target selection in the Drosophila visual system. Nat Neurosci 6:557-63 |
