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The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.

Summary: Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam1) can generate 38,016 different isoforms through largely stochastic, yet highly biased, alternative splicing. These isoforms are required for nervous functions. However, the functional significance of splicing bias remains unknown. Here, we provide evidence that Dscam1 splicing bias is required for mushroom body (MB) axonal wiring. We generate mutant flies with normal overall protein levels and an identical number but global changes in exon 4 and 9 isoform bias (DscamΔ4D−/− and DscamΔ9D−/−), respectively. In contrast to DscamΔ4D−/−, DscamΔ9D−/− exhibits remarkable MB defects, suggesting a variable domain-specific requirement for isoform bias. Importantly, changes in isoform bias cause axonal defects but do not influence the self-avoidance of axonal branches. We conclude that, in contrast to the isoform number that provides the molecular basis for neurite self-avoidance, isoform bias may play a role in MB axonal wiring by influencing non-repulsive signaling.

Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam1) can generate 38,016 different isoforms through largely stochastic, yet highly biased, alternative splicing. These isoforms are required for nervous functions. However, the functional significance of splicing bias remains unknown. Here, we provide evidence that Dscam1 splicing bias is required for mushroom body (MB) axonal wiring. We generate mutant flies with normal overall protein levels and an identical number but global changes in exon 4 and 9 isoform bias (DscamΔ4D−/− and DscamΔ9D−/−), respectively. In contrast to DscamΔ4D−/−, DscamΔ9D−/− exhibits remarkable MB defects, suggesting a variable domain-specific requirement for isoform bias. Importantly, changes in isoform bias cause axonal defects but do not influence the self-avoidance of axonal branches. We conclude that, in contrast to the isoform number that provides the molecular basis for neurite self-avoidance, isoform bias may play a role in MB axonal wiring by influencing non-repulsive signaling. [Display omitted] • Base pairing mediates Dscam1 variable exon 4 and 9 selection • Splicing bias is driven by diversifying base pairing architecture • Dscam1 splicing bias is essential for mushroom body axonal wiring • Isoform bias alteration does not affect the self-avoidance of MB axonal branches Hong et al. provide evidence that Dscam1 splicing bias is required for mushroom body (MB) axonal wiring. In contrast to the Dscam1 isoform number that provides the molecular basis for neurite self-avoidance, isoform bias may play a role in MB axonal wiring by influencing non-repulsive signaling. [ABSTRACT FROM AUTHOR]

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. • Translational stop cassettes allow isoform-specific manipulations of genes • isoTarget enables multi-purpose studies of a targeted isoform in select cells • The subcellular localizations of Dscam isoforms determine their cellular functions • isoTarget is generalizable for studying alternative splicing isoforms 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. [ABSTRACT FROM AUTHOR]