Calcium-dependent cell adhesion receptors, or cadherins, have been proposed to mediate synaptic target recognition and synaptogenesis in vertebrate brains. In particular, cadherins are thought to match pre- and postsynaptic partners on the basis of their homophilic binding activity, synaptic localization, and regional and dynamic expression patterns in the brain. We recently found that the Drosophila N-cadherins, like the vertebrate protocadherin CNR (cadherin-related neuronal receptor), can expand their receptor repertoire by alternative splicing. The Ncad gene that contains three exon modules, each of which is composed of a pair of highly similar but distinct exons, designated exons 7a/7b, exons 13a/13b, and exons 18a/18b. By combinatorial use of these alternative exons and another small exon, the Ncad locus is capable of generating 12 isoforms, all of which share the same modular structure of a distinct type of classic cadherin receptors. The alternatively spliced cadherin isoforms could largely increase the complexity and specificity of the adhesive interaction in the developing nervous system. We investigate the in vitro and in vivo functions of the Ncad molecular diversity in the development of the Drosophila visual system. To test whether Ncad alternative splicing is regulated, we developed a Tagman-based real-time PCR assay that can reliably measure the amounts of Ncad alternative transcripts. Using this assay system, we found that Ncad alternative splicing is temporally and spatially regulated during development. The general picture emerged from these results depicts that the isoform 7b-13a-18a is prevalent in the adult stage while the other isoforms encoded by exons 7a, 13b, and 18b were expressed primarily during development. When examining Ncad expression profiles in eye discs at developmental stages, we found that the expression of exons 18a/18b is dynamically regulated: from the third instar larval to the adult stage, the exon 18a level gradually increases from 15% to 92%. In contrast, the relative level of the exons13a/13b remains constant, and the exons 7a/7b varies modestly throughout development. In collaboration with Andrew Chess at MIT, we examined the Ncad expression profiles in R3/4 and R7 photoreceptor neurons using a cell-sorting method. To our surprise, R3/4 and R7 neurons exhibit virtually identical expression profiles on exons 7a/b and 18a/b and a similar preference for exon 13a over 13b, indicating little difference between the expression profiles of the two distinct R-cell types. In summary, Ncad alternative splicing in the eyes is regulated in a temporal, but not cell-type-specific, manner. To determine the in vivo function of Ncad isoforms, we carried out transgene rescue and mis/overexpression experiments. We uncovered that expressing a single Ncad isoform, including 7a-13a-18a, 7b-13a-18a, 7b-13b-18a, and 7b-13a-18b, substantially if not completely, rescues the Ncad phenotypes in R7 axons. We found no significant difference in the abilities of different Ncad isoform transgenes to rescue the mistargeting or growth cones morphological defects at either 17 hr or 35 hr pupal stages. We next determined whether in the wild-type background, mis- or over-expressing a single Ncad isoform in R7s alters their target specificity. We found that expressing any of the isoforms in R7 axons causes modest mistargeting and growth cone morphological defects. However, both defects were reduced in the older R7 axons and were not observed at the later stages. These findings indicate that mis/overexpressing single Ncad isoforms in R7 neurons is insufficient to permanently change their target specificity. We next examined whether the promiscuous in vivo function of the Ncad isoforms correlates with their adhesive activities. Using an S2 cell-aggregation assay, we determined the specificity of the homo- and heterophilic interactions mediated by Ncad isoforms. We found that all tested Ncad isoforms, including Ncad 7b-13a-18a, 7a-13a-18a, 7b-13b-18a, and 7b-13a-18b induced cell aggregates in the presence of calcium, indicating that they mediate homophilic interaction. However, the Ncad isoform 7b-13a-18b is capable of inducing large aggregates while Ncad 7b-13a-18a induces mostly small cell aggregates, suggesting that they have different homophilic binding affinities. Next, we examined whether Ncad isoforms can mediate heterophilic interaction with one another and with another Drosophila classic cadherin, E-cadherin. We found that all tested Ncad isoforms induced mixed-cell aggregates, indicating that they can mediate heterophilic interactions. However, the Ncad-expressing S2 cells did not intermix with the S2 cells expressing E-cadherin; instead, they formed separate cell aggregates. In summary, Ncad isoforms mediate type-specific but not isoform-specific heterophilic interactions. The lack of isoform-specificity, revealed by the transgene rescue, overexpression experiments, and heterophilic interaction assays, argues against the hypothesis that the Ncad isoforms constitute an adhesion code to direct targeting specificity. Instead, we favor the idea that Ncad plays a permissive role in R7 layer-selection. Based on the dynamic regulation and the differential homophilic binding affinity, we speculate that the Ncad 7b-13a-18b isoform provides strong homophilic interactions between the axons during axon extension, while the 7b-13a-18a isoform mediates weak, but perhaps tunable, interactions between the R-cell growth cones and their temporary targets.
| 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 |
