Endodomain Diversity in the Drosophila Dscam and Its Roles in Neuronal Morphogenesis

miRNA-based RNA interference permits differential silencing of Dscam transcripts encoding distinct endodomains

Based on the analysis of RT-PCR products, we have previously suggested that four possible Dscam endodomains, extending from exon 18 to exon 24, could be generated from independent alternative splicing of exon 19 and exon 23 (Wang et al., 2004). The full-length Dscam endodomain, located on the carboxyl side of the exon 17-encoded transmembrane/juxtamembrane segment, consists of 367 aa. It carries several putative SH3 binding sites, two 33 aa repeats that contain two consensus SH2 binding motifs, a proline-rich motif, a motif similar to the immunoreceptor tyrosine-based activation motif (ITAM-like), and a putative postsynaptic density, disc large and zo-I protein (PDZ)-binding motif (Schmucker et al., 2000; Brites et al., 2008). Skipping exon 19 produces a Dscam without a proline-rich motif (PPVP) and the ITAM-like (YEELx₍₁₁₎YGHA) (Fig. 1A). Furthermore, due to use of a different splicing donor site, four additional amino acids (TVIS) are added at the junction where exon 18 connects with exon 20 (Fig. 1A). In contrast, omitting exon 23 makes a reading frame shift in exon 24 that results in the loss of the PDZ-binding motif (TMAV) (Fig. 1A).

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Figure 1.

Amino acid sequences of four Dscam endodomain variants and silencing of various UAS-Dscam transgenes by specific UAS-RNAis. A, Amino acid sequences of four Dscam endodomain variants, full length (+19 +23), missing exon 19 only (−19 +23), lacking exon 23 only (+19 −23), and omitting both exon 19 and exon 23 (−19 −23), are aligned within the cytoplasmic domain from exon 18 to exon 24. Common sequence between exon 20 and exon 22 is not shown. Four extra amino acids (TVIS) are in the Dscam variants which skip exon 19. Amino acids shown in italic result from a reading frame shift in exon 24 upon skipping of exon 23. Target sequences for different UAS-RNAis (18i, 18/20i, 19i, 22/24i and 23i) to eliminate different Dscam variants are indicated. B–I, Composite confocal images of wandering larval MBs show suppression of the GAL4–201Y-dependent induction of UAS-Dscam+19 +23::GFP (B–E) and UAS-Dscam−19 −23::GFP (F–I) by UAS-18/20RNAi (B, F), UAS-19RNAi (C, G), UAS-22/24RNAi (D, H) and UAS-23RNAi (E, I), respectively. Scale bar (in this and all other figures), 50 μm.

The variations in the Dscam endodomain occur because of the presence or absence of exon 19 and exon 23 (Fig. 1A). To determine whether Dscam uses different endodomains in support of different neural developmental processes, we selectively knocked down Dscam transcripts containing or lacking exons 19 and/or exon 23 using a miRNA-based RNAi technology that permits silencing of specific transcripts based on presence of only 22 nucleotide-long unique sequences (Chen et al., 2007; Shi et al., 2007).

By targeting exon 19- or exon 23-characteristic sequences or the unique sequences at the exon-exon junctions derived from skipping of exon 19 or exon 23 (Fig. 1A), we successfully silenced the expression of specific Dscam transgenes based on the presence or absence of exon 19 or exon 23. Multiple independent transformants carrying UAS-19RNAi, UAS-18/20RNAi, UAS-23RNAi, or UAS-22/24RNAi were obtained and individually examined to identify the most effective RNAi transgenes for silencing their target-sequence-containing Dscam::GFPs in mature larval MB neurons (Fig. 1) (data not shown). GAL4–201Y-dependent coinduction of distinct UAS-RNAis with a full-length Dscam (Dscam+19+23::GFP) (Fig. 1B–E) or its derivative that skips both exon 19 and exon 23 (Dscam−19–23::GFP) (Fig. 1F–I) further revealed that UAS-19RNAi and UAS-23RNAi effectively antagonized the expression of Dscam+19+23::GFP (Fig. 1C,E) while minimally affecting the induction of Dscam−19–23::GFP which lacks the target sequences for these transgenic miRNAs (Fig. 1G,I). In contrast, UAS-18/20RNAi and UAS-22/24RNAi effectively antagonized the expression of Dscam−19–23::GFP (Fig. 1F,H) while minimally affecting the induction of Dscam+19+23::GFP (Fig. 1B,D). The specificity of UAS-RNAis was further demonstrated by pan-neuronal induction of UAS-19RNAi and UAS-18/20RNAi to suppress the endogenous Dscam promoter-driven Dscam transgenes (supplemental Fig. S1A–F, available at www.jneurosci.org as supplemental material). UAS-18/20RNAi silenced the expression of Dscam promoter-driven Dscam−19+23::GFP (supplemental Fig. S1E, available at www.jneurosci.org as supplemental material) while minimally affecting the induction of Dscam+19+23::GFP (supplemental Fig. S1B, available at www.jneurosci.org as supplemental material). Conversely, UAS-19RNAi diminished the expression of Dscam promoter-driven Dscam+19+23::GFP (supplemental Fig. S1C, available at www.jneurosci.org as supplemental material) while hardly affecting the induction of Dscam−19+23::GFP (supplemental Fig. S1F, available at www.jneurosci.org as supplemental material). These observations justified the use of UAS-19RNAi, UAS-18/20RNAi, UAS-23RNAi, and UAS-22/24RNAi for selectively silencing exon 19-containing (Dscam+19), exon 19-lacking (Dscam−19), exon 23-containing (Dscam+23) and exon 23-lacking (Dscam−23) Dscam transcripts, respectively.

Differential expression of Dscams with different endodomains

We have made a UAS-18RNAi transgene targeting the exon 18 shared by all Dscam transcripts, and previously demonstrated that ubiquitous induction of UAS-18RNAi abolished endogenous Dscam expression in the developing nervous system (Shi et al., 2007). To determine the effectiveness of our new RNAi transgenes in silencing the endogenous expression of Dscam, we examined how induction of individual or multiple Dscam RNAis affected the endogenous patterns of Dscam expression at different developmental stages.

In control embryos, immunostaining with an anti-Dscam exon 18 monoclonal antibody (Ab18) revealed that endogenous Dscam is concentrated on the ladder-like axonal tracts of the ventral ganglion (Fig. 2A). Such a stereotyped pattern of Dscam distribution was completely eliminated by tubulin promoter (tubP)-GAL4-dependent ubiquitous induction of UAS-18RNAi (Fig. 2E), UAS-19RNAi plus UAS-18/20RNAi (Fig. 2D) or UAS-23RNAi plus UAS-22/24RNAi (Fig. 2H). These results confirmed the efficacy of these RNAi reagents in silencing the expression of endogenous Dscam variants. It was noted that pan-neuronal induction of UAS-18RNAi by GAL4-C155 plus asense-GAL4 could not deplete the ladder-like axonal expression of endogenous Dscam, possibly due to late or insufficient induction of RNAi at the embryonic stage (data not shown). In contrast, residual Dscam persisted following tubP-GAL4-dependent induction of UAS-19RNAi, UAS-18/20RNAi, UAS-23RNAi or UAS-22/24RNAi alone, even with multiple copies (data not shown). As to the induction of UAS-19RNAi (Fig. 2C) versus UAS-18/20RNAi (Fig. 2B), the ladder-like patterns of immunostaining, although weakened, looked morphologically indistinguishable from those seen in control embryos (Fig. 2A). In contrast, when endogenous Dscam was silenced by UAS-23RNAi (Fig. 2G) or UAS-22/24RNAi (Fig. 2F), we found that the whole Dscam distribution can be subdivided into two non-overlapping compartments that possibly exclusively contain Dscam+23 and Dscam−23, respectively. Residual Dscam was completely restricted to the midline of the ventral ganglion, following induction of UAS-22/24RNAi (Fig. 2F, arrows). Closer inspection revealed the midline localization of Dscam in control embryos as well (Figs. 2A, 3A,D, arrows); and the midline expression of Dscam was only abolished by induction of UAS-23RNAi (Fig. 2G) that otherwise did not affect the pattern or intensity of the endogenous Dscam expression (Fig. 2B,C,F). These phenomena indicate that, in embryos, Dscam+19 and Dscam−19 coexist in analogous structures while Dscam+23 and Dscam−23 reside in distinct compartments.

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Figure 2.

Elimination of specific Dscam endodoamin variants by transgenic miRNAs. A–P, Composite confocal images of embryonic and wandering larval CNS show endogenous Dscam expression (revealed by immunostaining with an anti-Dscam exon 18 monoclonal Ab), following tubP-GAL4-dependent (A–H) or GAL4-C155- plus asense-GAL4-dependent (I–P) induction of various anti-Dscam RNAis, including UAS-18RNAi (E, M), UAS-18/20RNAi (B, J), UAS-19RNAi (C, K), UAS-18/20RNAi plus UAS-19RNAi (D, L), UAS-22/24RNAi (F, N), UAS-23RNAi (G, O) or UAS-22/24RNAi plus UAS-23RNAi (H, P). Arrows point at the midline in F.

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Figure 3.

Stage-dependent expression of Dscam endodomain variants. A–F, An anti-Dscam exon 18 monoclonal antibody (Ab18) recognizing all Dscam proteins (A–C) and an anti-Dscam exon 19 polyclonal antibody (Ab19) recognizing Dscam+19 proteins (D–F) were used to reveal the endogenous expression pattern of all Dscam variants and Dscam+19 during development. Both Ab18 and Ab19 are specific for detecting Dscam endogenous proteins as evidenced by lack of immunostaining in Dscam null mutant embryos (B, E). Arrows point at the midline in A and D. G, RT-PCR analysis of Dscam transcripts revealed the usage of exon 19 and exon 23. Dscam+19 transcripts exist more abundantly than Dscam−19 transcripts at the embryonic stage, while Dscam−19 transcripts become dominant in postembryonic CNS. In contrast, Dscam−23 transcripts predominate over Dscam+23 transcripts throughout development.

Similar analysis revealed that most Dscams in the late larval CNS lack exon 19 and exon 23. Pan-neuronal induction of either UAS-19RNAi (Fig. 2K) or UAS-23RNAi (Fig. 2O) to deplete Dscam+19 or Dscam+23 minimally affected the expression pattern or level of endogenous Dscam. In contrast to these observations, depleting Dscam−19 (Fig. 2J) or Dscam−23 (Fig. 2N) drastically reduced the Dscam expression in the late larval CNS. Notably, distinct patterns of residual Dscam were detected. Upon induction of UAS-18/20RNAi to eliminate Dscam−19 (Fig. 2J), the residual Dscam, although barely detectable, existed in a similar pattern to that of control animals (Fig. 2I). In contrast, induction of UAS-22/24RNAi to silence Dscam−23 transcripts selectively depleted endogenous Dscam from those neuropil structures where Dscam is normally enriched (Fig. 2N). These results suggest that larval development of the Drosophila CNS is mainly supported by Dscam isoforms lacking exons 19 and 23. In addition, Dscam+23 or Dscam−23 exists in distinct patterns, although not as exclusive as in embryos, and potentially governs different morphogenetic processes through development of the Drosophila CNS.

To validate some of the above conclusions, we sought to detect Dscam+19 directly by immunostaining with exon 19-targeted antibody (Ab19), a polyclonal antibody raised against a peptide sequence characteristic of Dscam exon 19. The Ab19 selectively bound to ectopic Dscam+19::GFP (data not shown). Furthermore, a similar ladder-like pattern of immunocytochemical signals, which was eliminated in Dscam-null embryos, was detected following immunostaining of wild-type embryos with the Ab18 (Fig. 3A,B) or Ab19 (Fig. 3D,E). These results demonstrate that the Ab19 specifically binds with Dscam+19. When the Ab18 (Fig. 3C) or the Ab19 (Fig. 3F) were used to label all the Dscam isoforms or Dscam+19 isoforms in the late larval CNS, we detected abundant Dscam expression only with the Ab18. This substantiates the notion that Dscam+19 is largely absent during postembryonic development of the fly CNS. These results are also consistent using transgenic RNAis to silence alternative transcripts in determining the expression patterns of distinct isoforms in vivo (Fig. 2J,K).

Analysis of Dscam transcripts by RT-PCR revealed that the dynamic usage of exon 19 and exon 23 is evident even at the mRNA level (Fig. 3G). The relative abundance of Dscam transcripts encoding distinct endodomains varied at different developmental stages. The expression of Dscam+19 transcripts in the CNS drastically decreased from embryos (65.8%) to larvae (15.8%), became negligible by puparium formation (0%), and returned after eclosion (31.6%). In contrast, Dscam+23 transcripts rarely existed in embryos (2.6%), and then increased in larvae (7.9%), pupae (10.4%) and adult brains (21.1%). The differential expression of Dscam mRNA splicing variants at different developmental stages can account for the differences we detected in the abundance of distinct endodomains at the protein level. These results suggest that the dynamic expression of distinct Dscam endodomains is likely controlled through regulation of alternative splicing.

Together, these observations not only indicate that distinct Dscam endodomain isoforms do exist in vivo, but also demonstrate that the miRNA-based RNAi reagents work effectively to antagonize one while minimally affecting the alternative. Induction of RNAi specifically silencing one Dscam endodomain isoform (e.g., Dscam+19 or Dscam+23) reveals the protein distribution of the other Dscam endodomain isoform (e.g., Dscam−19 or Dscam−23) in vivo. Examining the expression of Dscam+23 and Dscam−23 uncover two distinct sets of Dscam-positive structures that potentially use different Dscam endodomains for supporting different morphogenetic processes.

Wiring of the adult fly neural circuitry involves Dscam transcripts lacking exons 19 and 23

Based on the above analyses of endogenous expression patterns of Dscam endodomain variants, Dscam variants lacking exons 19 and 23 may support diverse aspects of neuronal morphogenesis during larval and pupal stages. This raises the possibility that wiring of the fly neural circuitry primarily involves Dscam variants lacking exons 19 and 23. To test this hypothesis, we examined how silencing of Dscam transcripts encoding specific endodomains by targeted RNAi affects morphogenesis of two distinct types of larval-born neurons where Dscam loss-of-function phenotypes are known. We found in both MB neurons (Fig. 4A–C) and the ellipsoid body neurons (data not shown) that induction of UAS-18/20RNAi or UAS-22/24RNAi, but not UAS-19RNAi or UAS-23RNAi, effectively elicited the phenotypes characteristic of loss of Dscam function.

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Figure 4.

Role of Dscam endodomain variants in embryonic CNS and larval MB axonal morphogenesis. A–C, Quantitative analysis of wandering larval MB axon lobe (A, arrows) phenotype, following GAL4-OK107-dependent induction of various UAS-Dscam RNAis plus UAS-mCD8::GFP. Skinny lobe phenotype (B, arrows) was observed when Dscam−19 or Dscam−23 was eliminated. D–F, Quantitative analysis of embryonic CNS commissureless phenotype, following tubP-GAL4-dependent induction of various UAS-Dscam miRNAs. No phenotype (D, arrows) was observed when all Dscam proteins were knocked down alone or in abl mutant embryos. In contrast, commissureless phenotype (E, arrows) was observed when all Dscam isoforms or Dscam+19 were eliminated in the abl mutant embryos. The embryonic CNS longitudinal fascicles and commissure pathways were visualized by immunostaining with monoclonal antibody BP102.

In MB neurons, Dscam governs axon bifurcation by preventing sister growth cones from migrating along the same path (Wang et al., 2002). It ensures formation of paired MB lobes that normally lie perpendicularly with each other and are comparable in bundle thickness (Fig. 4A). In contrast, when Dscam is absent, MB axons undergo excessive bifurcation and their sister branches are no longer consistently segregated. These abnormalities underlie overgrowth of one MB lobe at the expense of its paired one (Fig. 4B). To determine the roles of Dscam+19, Dscam−19, Dscam+23 or Dscam−23 in MB morphogenesis, we examined which of the above anti-Dscam miRNA transgenes could be induced to elicit these loss-of-Dscam MB phenotypes. We first demonstrated that GAL4-OK107-driven induction of UAS-18RNAi caused defects in MB lobe morphology (Fig. 4B). Instead of having two comparable axon lobes, 88% of the MBs acquired one huge and one skinny lobe (Fig. 4C). This anomaly replicated Dscam mutant MB phenotypes (Wang et al., 2002). Since silencing Dscam with UAS-18RNAi eliminates all Dscam isoforms, this result has not only confirmed the efficiency of knocking down endogenous Dscam by transgenic RNAi, but also given us an opportunity for learning what Dscam “null” phenotypes could be obtained with similar manipulations.

We subsequently examined the effects of UAS-19RNAi, UAS-18/20RNAi, UAS-23RNAi or UAS-22/24RNAi on MB morphology (Fig. 4C). While induction of UAS-19RNAi or UAS-23RNAi to deplete Dscam+19 or Dscam+23 failed to affect MB morphogenesis, 68 and 77% of the MBs acquired asymmetric axon lobes following silencing of Dscam−19 or Dscam−23 using UAS-18/20RNAi or UAS-22/24RNAi (Fig. 4C). This suggests that Dscam−19 and Dscam−23 are indispensable for gross morphogenesis of the MBs. However, the penetrance of UAS-18/20RNAi-induced MB phenotypes increased from 68 to 89% following coinduction with UAS-19RNAi. This implies a lesser role for Dscam+19 in mediating the neuronal morphogenetic process which is mainly governed by Dscam lacking exons 19 and 23. In contrast, coinduction of UAS-22/24RNAi and UAS-23RNAi minimally enhanced the MB phenotype, suggesting again that Dscam+23 is dispensable to the neuronal morphogenesis. Together, these results indicate that the Dscam-dependent MB lobe formation primarily involves Dscam variants lacking exons 19 and 23, which is consistent with Dscam−19−23 being predominantly expressed through postembryonic CNS development.

Dscam+19 is involved in embryonic neuronal morphogenesis

In contrast with Dscam+23 whose usage remains low through different developmental stages, both Dscam+19 and Dscam−19 are abundantly expressed in embryonic CNS. To reveal the role of Dscam exon 19 variants in neural development, we examined whether eliminating Dscam+19 or Dscam−19 affects embryonic neuronal morphogenesis. We characterized neurite tracts in the embryonic ventral ganglion that typically consists of bilaterally symmetric longitudinal fascicles connected across the midline via repeated pairs of commissure bundles. Although aberrant neurite tracts in Dscam mutant embryos have been reported (Schmucker et al., 2000), ubiquitous induction of UAS-18RNAi by tubP-GAL4 failed to disrupt the ladder-like neuronal tracts in otherwise wild-type embryos (Fig. 4F). Kidd's group recently showed that Dscam loss-of-function phenotypes in embryos can be significantly enhanced in tyrosine kinase abelson (abl) mutant backgrounds (Andrews et al., 2008). Prompted by this observation, we examined how silencing Dscam by RNAi might affect neural tract development in abl mutant embryos (Fig. 4D–E). As in the Dscam-depleted embryos caused by tubP-GAL4-dependent induction of UAS-18RNAi, the ladder-like neurite tracts were grossly normal in abl²/abl ⁴ mutant embryos (Fig. 4F). In contrast, the ubiquitous induction of UAS-18RNAi in abl²/abl ⁴ mutant embryos effectively blocked midline crossing of neurites and caused commissureless phenotypes in 19% of the ventral ganglion segments (Fig. 4E,F). These observations set the stage for determining the roles of various Dscams in embryonic neuronal morphogenesis by RNAi using distinct Dscam-targeted miRNAs.

To tell the respective contributions made by Dscam+19 and Dscam−19, we silenced Dscam transcripts containing or lacking exon 19 in the abl²/abl⁴ mutant background. Following analogous induction with tubP-GAL4, UAS-19RNAi and UAS-18/20RNAi elicited commissureless phenotypes in 9 and 1% of the ventral ganglion segments, respectively (Fig. 4F). Together, most embryonic Dscams carry exon 19 (Fig. 3G) and depleting exon 19-containing Dscam potently disrupts wiring of embryonic CNS. These results demonstrate that Dscam+19 plays a more important role than Dscam−19 in the development of the Drosophila embryonic CNS.

Differential rescue of axon branching phenotypes by transgenic Dscam+19 or Dscam−19

To investigate further the functions of Dscams carrying distinct endodomains, we examined whether ectopic expression of Dscams with different endodomains may act differentially in vivo. Although MB morphogenesis involves Dscam variants lacking exons 19 and 23, past studies have shown that the Dscam+19 +23 transgene, no matter which ectodomain it carries and whether it is fused with GFP at the C terminus or not, can effectively prevent sister branches from extending into the same axon bundle in single-cell clones of Dscam mutant neurons (Wang et al., 2004; Zhan et al., 2004; Soba et al., 2007). We wondered whether Dscam transgenes that encode Dscams with distinct endodomains may vary in their ability to restore the normal pattern of axon bifurcation and segregation in Dscam mutant MB neurons.

UAS-Dscam::GFP transgenes that encode the same ectodomain connected to one of the four possible endodomains through the exon 17.2-defined transmembrane domain were obtained. Transgenic lines with a comparable level of induction were identified (Fig. 5A). We then examined how induction of such Dscam transgenes in Dscam mutant single-cell MARCM clones affects their axon bifurcation and segregation at the terminus of MB peduncle. We analyzed MB α/β neurons, whose axons extend through the peduncle, undergo bifurcation at the peduncle end, and then innervate the MB α and β lobes with only one primary branch from each neuron (Fig. 5C). Single-cell MARCM clones of MB α/β neurons were derived following mitotic recombination induced during mid-pupal development. Loss of the GAL4 repressor, GAL80, in the clones led to derepression of GAL4-OK107, which in turn drove expression of UAS-mCD8::GFP for marking the clones uniquely. In the rescue experiments, the derepressed GAL4-OK107 also activated one of the UAS-Dscam::GFPs specifically in the clones. Given that these Dscam transgenes vary only in the presence or absence of exon 19 or 23, any differences in the phenotypes of Dscam mutant α/β neurons that have expressed different UAS-Dscam::GFPs can be fully ascribed to the differences in Dscam protein distribution and/or function due to the involvement of different endodomains.

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Figure 5.

Rescue of MB α/β multibranch phenotype by transgenic Dscam with distinct endodomains. A, Dscam endodomain variants with a similar expression level were chosen to rescue the multibranch Dscam mutant MB α/β phenotype. Heat shock-GAL4 was used to drive the expression of different Dscam endodomain variants by brief heat shock at 37°C. The expression levels were determined by Western blot. B–G, Quantitative analysis of different MB α/β neuronal phenotypes in Dscam mutant clones expressing distinct Dscam endodomain variants (B). Wild-type axons consistently bifurcate and send one primary branch into each of the MB α/β lobes (C). D–F, In contrast, Dscam mutant single-cell clones often extend multiple branches into one MB lobe. G, Note an unusual single-branch phenotype with no axon bifurcation at the peduncle end in some “rescued” single-cell clones. Anti-1D4 staining (magenta) reveals two strong α/β lobes and a weak γ robe in adult MB. Anti-mCD8 antibody (green) was used to reveal mCD8::GFP in MARCM single-cell clones.

As reported previously, single-cell MARCM clones of Dscam mutant MB α/β neurons could not effectively segregate their sister branches to prevent multiple neurites of the same cellular origin from extending into the same MB lobe (Wang et al., 2002). Although Dscam mutant neurons might acquire supernumerary branches, this segregation problem was evident even in the mutant axons that only bifurcated once at the peduncle end. Failure in the divergent segregation of sister branches appears to be the primary defect (Fig. 5D–F). Ninety-seven percent of Dscam mutant single-cell MARCM clones of MB α/β neurons sent two or more neurites into one MB lobe, exhibiting the multibranches/lobe phenotype (Fig. 5B–F). By this measure, single-isoform Dscam−19 transgenes were comparable with single-isoform Dscam+19 transgenes in the ability to correct the Dscam mutant multibranches/lobe phenotype (Fig. 5B). Failure in divergent segregation (multibranches/lobe) was observed in 22, 16, 25, and 22% of Dscam mutant MB α/β neurons (as opposed to 97% in the absence of any rescuing construct) that were rescued by the transgenes of Dscam+19+23, Dscam−19+23, Dscam+19–23 and Dscam-19–23, respectively (Fig. 5B). In addition, among the rescued single-cell MARCM clones (without the multibranches/lobe phenotype), we detected a single-branch/neuron phenotype in which the axon did not bifurcate at the peduncle end and stochastically projected into the α or β lobe. Intriguingly, many more rescued single-cell MARCM clones exhibited the single-branch/neuron phenotype (no bifurcation despite presence of both α and β lobes), when the mutant clones were supplemented with Dscam−19 transgenes (Fig. 5G). Rescue with Dscam−19+23 and Dscam−19–23 caused the single-branch/neuron phenotype in 11 and 13% of Dscam mutant single-cell clones (Fig. 5B). In contrast, with Dscam+19+23 or Dscam+19–23, only 5% of mutant axons failed to bifurcate at the peduncle terminus (Fig. 5B). In addition, transgenic Dscam−19+23 hardly affected wild-type single-cell clones; axon bifurcation at the peduncle terminus was suppressed in only 3% of Dscam−19–23-expressing wild-type clones (Fig. 5B). Together, no matter whether exon 23 is present or not, Dscam−19 transgenes more potently modulate the axon bifurcation and segregation patterns than Dscam+19 transgenes. These observations suggest some exon 19-dependent modification of protein distribution or function that is relevant to where or how Dscam may signal to govern MB axonal morphogenesis.

Differential subcellular localization of Dscam containing or lacking exon 19

To understand the logic behind the exon 19-dependent functional differences, we visualized the protein distribution of Dscam+19::GFP and Dscam−19::GFP transgenes in vivo. Previously, we have reported an assay to visualize Dscam protein distribution in the larval MBs by inducing UAS-Dscam::GFP transgenes in mature larval MB neurons using GAL4–201Y (Wang et al., 2004). Notably, the Dscam+19::GFP and Dscam−19::GFP were found to be enriched in cell bodies and neurites of the larval MBs, respectively (data not shown). This raises the possibility that Dscam protein distribution may vary depending on the presence or absence of exon 19.

However, GAL4–201Y drove expression of Dscam::GFPs in excessive amounts and in mature MB neurons, which is in contrast with the endogenous Dscam being expressed at a much lower level and typically in newly derived postmitotic neurons (Zhan et al., 2004). To ascertain the role of exon 19 in Dscam protein distribution, we examined whether Dscam+19::GFP and Dscam−19::GFP remain differentially localized upon weak induction in young MB neurons. Transient binary induction of UAS-transgene is possible with the TARGET system, in which GAL4 activity is suppressed until inactivation of temperature-sensitive GAL80 at a restrictive temperature (McGuire et al., 2003). Derepression of GAL4-OK107 following a heat shock to inactivate GAL80^ts allowed one to weakly express UAS-transgene in young MB neurons as judged by the central localization of neurites in the age-dependent concentrically organized axon bundles (Kurusu et al., 2002). Transient induction of UAS-Dscam::GFPs revealed that Dscam−19::GFP was selectively enriched in the neurites, while barely detectable in cell bodies (Fig. 6B,D). In contrast, Dscam+19::GFP accumulated in cell bodies as well as neurites (Fig. 6A,C). A similar pattern of differential subcellular localization of Dscam+19 and Dscam−19 was also observed when Dscam+19 and Dscam−19 transgenes were induced in projection neurons (PNs) of the antennal lobe (AL). GAL4-Mz19 (Jefferis et al., 2004) labels a small subset of PNs that connect three AL glomeruli (DA1, VA1d, and DC3) to the lateral horn (LH) and MB calyx (Fig. 7A,B). Upon induction with GAL4-Mz19, Dscam-19::GFPs with either transmembrane domain choice (17.1 or 17.2) were enriched in PN neurites (Fig. 7D,F). Dscam+17.1–19::GFP further accumulated in the glomeruli where PN dendrites elaborate (Fig. 7D). In contrast, Dscam+19::GFPs with either exon 17.1 or 17.2 preferentially localized in PN cell bodies (Fig. 7C,E). These results suggest that Dscam−19 is better targeted to neuronal processes, possibly explaining why transgenic Dscam−19 is more potent than the Dscam+19 in modulating postembryonic neuronal morphogenesis.

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Figure 6.

Exon 19-dependent differential distribution of transgenic Dscam in MB neurons. A–D, TARGET-mediated acute induction of Dscam+19 versus Dscam−19 in larval MB neurons. Transgenic Dscam−19::GFP was primarily targeted to the MB axon bundles and the dendritic calyx (B, D). In contrast, Dscam+19::GFP accumulated in cell bodies as well as neurites (A, C). Antibody 1D4 (magenta) was used to reveal larval MB lobes. Anti-GFP antibody (green) was used to reveal Dscam::GFP.

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Figure 7.

Differential distribution of Dscam variants in AL PNs. A, B, Schematic drawing (A) shows GAL4-Mz19-positive PNs that connect thee AL glomeruli (DA1, VA1d, and DC3) to the lateral horn (LH) ad MB calyx, as revealed by labeling of entire neuronal morphology with UAS-mCD8::GFP in B. DA1 and VA1d are two prominent glomeruli and can be easily visualized in B, D, and F. The position of DC3 is posterior to DA1 and VA1d and the outline of DC3 is depicted in dashed line (A). C–F, Analogous induction of various UAS-Dscam::GFPs exhibits different protein distribution patterns. Transgenic Dscam+19::GFPs primarily localize in cell bodies (C, E), while Dscam−19::GFPs are enriched in neurites (D, F). Furthermore, as reported previously, Dscam−19::GFP with the 17.1 transmembrane domain can be selectively targeted to dendrites (D). DA1 and VA1d are outlined with two circles in C and E.