Specific Drosophila Dscam juxtamembrane variants control dendritic elaboration and axonal arborization

Abstract

Drosophila Dscam isoforms are derived from two alternative transmembrane/juxtamembrane domains (TMs) in addition to thousands of ectodomain variants. Using a microRNA-based RNA interference technology, we selectively knocked down different subsets of Dscams containing either the exon 17.1- or exon 17.2-encoding TM. Eliminating Dscam[TM1] reduced Dscam expression but minimally affected postembryonic axonal morphogenesis. In contrast, depleting Dscam[TM2] blocked axon arborization. Further removal of Dscam[TM1] enhanced the loss-of-Dscam[TM2] axonal phenotypes. However, Dscam[TM1] primarily regulates dendritic development, as evidenced by the observations that removing Dscam[TM1] alone impeded elaboration of dendrites and that transgenic Dscam[TM1], but not Dscam[TM2], effectively rescued Dscam mutant dendritic phenotypes in mosaic organisms. These distinct Dscam functions can be attributed to the juxtamembrane regions of TMs that govern dendritic versus axonal targeting of Dscam as well. Together, we suggest that specific Drosophila Dscam juxtamembrane variants control dendritic elaboration and axonal arborization.

Figure 1.

Silencing of endogenous Dscam expression by various transgenic miRNAs. Composite confocal images of wandering larvae CNS showing endogenous Dscam expression (magenta; as revealed by immunostaining with an anti-Dscam exon 18 peptide mAb), in wild-type (WT) and after asense-GAL4/GAL4-C155-dependent induction of various anti-Dscam miRNAs (B–E). A, Wild-type control. Note that UAS-18 miRNA alone (B) or only UAS-17.1 miRNA plus UAS-17.2 miRNA (C) could effectively eliminate the entire Dscam expression. In addition, regardless of the levels of Dscam, major neural structures, as revealed by coinduction of UAS-mCD8::GFP (green), remained comparable (insets). Scale bar: (here and in all figures) 20 μm.

Figure 2.

Dscam[TM2], but not Dscam[TM1], plays an essential role in MB axonal morphogenesis. A–E, Adult MB lobes visualized by 1D4 mAb. Compared with the wild type (A), induction of certain anti-Dscam miRNAs (see I) disrupted the formation of MB α/β lobes to various extents (B–E). F–H, Derivation of two chimeric Dscam TMs (F) and their effects on Dscam::GFP (green) protein targeting. After binary induction with GAL4–201Y, Dscam[17.1/17.2]::GFP, like Dscam[TM2]::GFP, is uniformly distributed in the larval MBs (G). In contrast, Dscam[17.2/17.1]::GFP, like Dscam[TM1]::GFP, is enriched in dendrites (H). Additionally, both Dscam[17.1/17.2]::GFP and Dscam[TM2]::GFP are preferentially targeted to axons after suppression of the induction by RNAi (data not shown) (similar to Fig. S1F,H, available at www.jneurosci.org as supplemental material). I, Quantitative analysis of MB lobe phenotypes, based on the above classification (A–E), after GAL4-OK107-dependent induction of various anti-Dscam miRNAs and in the absence or presence of distinct transgenic Dscam::GFP.

Figure 3.

Dscam[TM1] and Dscam[TM2] primarily govern dendritic and axonal morphogenesis, respectively. MARCM-labeled adult vPN Nb clones. Compared with the wild-type clone (A, green), induction of 17.1 miRNA, 17.2 miRNA, and 18 miRNA in vPN Nb clones (B–D, green) specifically disrupted dendritic elaboration (F, arrow), axonal arborization (K, arrow), and both (H, L; arrows), respectively. Adult fly brains were counterstained with nc82 mAb (magenta). The cropped images selectively show dendritic elaboration (E–H) or axonal arborization (I–L) of the clones.

Figure 4.

Rescue of Dscam mutant PN morphogenesis by transgenic Dscam with TM1 versus TM2. A–H, Adult single-cell clones of DL-1 PNs (green) of which the dendrite elaboration in the DL-1 glomeruli (as revealed by nc82 immunostaining; magenta) and axon arborization in the MB calyces and the LHs are, respectively, shown in A–D and E–H. A, E, Wild-type clones. B, F, Dscam mutant clones. C, G, Rescue of mutant clones with pDscam-Dscam[3.36.25.1-genomic 18–24]. D, H, Rescue with pDscam-Dscam[3.36.25.2-genomic 18–24]. Note partial coverage of DL-1 glomeruli by the green PN dendrites in B and D and the absence of bouton-like structures (arrowheads) in F and G. I–J, Quantitative analysis of the coverage of DL-1 glomeruli by single-cell PN clones (I) and the numbers of PN-derived bouton-like structures in the MB calyces (J). n = 25 in every condition.