Search

SUMMARYProtein isoforms generated by alternative splicing contribute to proteome diversity. Due to the lack of effective techniques, isoform-specific functions, expression, localization, and signaling mechanisms of endogenous proteinsin vivoare unknown for most genes. Here we report a genetic method, termedisoTarget, for blocking the expression of a targeted isoform without affecting the other isoforms and for conditional tagging the targeted isoform for multi-level analyses in select cells. ApplyingisoTargetto two mutually exclusive isoforms ofDrosophilaDscam, Dscam[TM1] and [TM2], we found that endogenous Dscam[TM1] is localized in dendrites while Dscam[TM2] is in both dendrites and axons. We demonstrate that the difference in subcellular localization between Dscam[TM1] and [TM2], rather than any difference in biochemical properties, leads to the two isoforms’ differential contributions to dendrite and axon development. Moreover, withisoTarget, we discovered that the subcellular enrichment of functional partners results in a DLK/Wallenda-Dscam[TM2]-Dock signaling cascade specifically in axons.isoTargetis an effective technique for studying how alternative splicing enhances proteome complexity.

SUMMARYElucidating cell lineages provides crucial understanding of development. Recently developed sequencing-based techniques enhance the scale of lineage tracing but eliminate the spatial information offered by conventional approaches. Multispectral labeling techniques, such asBrainbow, have the potential to identify lineage-related cellsin situ. Here, we reportLineage Tracker Bitbow, a “digital” version ofBrainbowthat greatly expands the color diversity, and a suite of statistical methods for quantifying the lineage relationship of any two cells. Applying these tools toDrosophilaperipheral nervous system, we determined lineage relationship between all neuronal pairs. Based on the refined lineage map, we explored whether distinctcis-regulatory elements are used in controlling the expression of a terminal selector gene in distinct lineage patterns. This study demonstratesLT-Bitbowas an efficient tool forin-situlineage mapping and its potential in studying molecular mechanisms in the lineage context.

SUMMARY Protein isoforms generated by alternative splicing contribute to proteome diversity. Because of the lack of effective techniques, the isoform-specific function, expression, localization, and signaling of endogenous proteins are unknown for most genes. Here, we report a genetic method, isoTarget, for multi-purpose studies of targeted isoforms in select cells. Applying isoTarget to two isoforms of Drosophila Dscam, Dscam[TM1] and [TM2], we found that, in neurons, endogenous Dscam[TM1] is in dendrites, whereas Dscam[TM2] is in both dendrites and axons. We demonstrate that the difference in subcellular localization, rather than biochemical properties, leads to the two isoforms’ functional differences. Moreover, we show that the subcellular enrichment of functional partners results in a DLK/Wallenda-Dscam[TM2]-Dock signaling cascade in axons. We further apply isoTarget to study two isoforms of a GABA receptor to demonstrate its general applicability. isoTarget is an effective technique for studying how alternative splicing enhances proteome complexity., Graphical Abstract, In Brief Liu et al. develop a genetic method that enables the investigation of isoform-specific function, expression, localization, and signaling of endogenous proteins in select cells. Using this method, they demonstrate that the difference in subcellular localization of two isoforms of Down syndrome cell adhesion molecule leads to functional differences between them.