In this study, we demonstrate that Dscam endodomain variants are dynamically and differentially expressed in the developing Drosophila CNS. This conclusion derives from: (1) the analysis of Dscam transcript compositions by RT-PCR, (2) the localization of specific Dscam endodomains by depleting the alternatives via RNAi against exon 19, exon 23, or the unique exon-exon junctions derived from skipping of exon 19 or exon 23 (), and (3) the direct visualization of Dscam+19 using Ab19 as opposed to labeling all the Dscam isoforms with Ab18 (). Post-embryonic neuronal morphogenesis utilizes Dscam variants lacking exons 19 and 23 (), while Dscam+19 plays a more important role in the wiring of embryonic neural tracts (). Skipping exon 19 prevents accumulation of Dscams in neuronal cell bodies, implicating a mechanism for regulating Dscam protein targeting by the alternative splicing of exon 19 ( and ). In addition, exon 23 is dispensable for most Dscam-dependent neuronal morphogenetic processes but present in probably all the Dscam molecules in certain neural structures, suggesting an unidentified Dscam function in the developing Drosophila CNS ().
Four different Dscam endodomain variants arise from independent alternative splicing by skipping exon 19 or exon 23. Possible approaches to study the role of these Dscam endodomain variants in neuronal morphogenesis include: (1) manipulating Dscam
at the genomic level by gene targeting (Gong and Golic, 2003
), or (2) silencing different Dscam
endodomain transcripts by RNAi (Chen et al., 2007
; Shi et al., 2007
). Deleting genomic sequence to manipulate alternative exon choice might be problematic, since elimination of specific Dscam
endodomain variants inevitably leads to expression of other isoforms in much broader patterns and/or at higher levels than the normal unperturbed conditions. In contrast, knock-down of Dscam endodomain variants at the translational level by RNAi should deplete the isoforms of interest while minimally affecting the expression of others. Further, by targeting the junction spanning different exons (), the miRNA-based silencing approach allowed us to selectively deplete isoforms that lack any unique exon shared by the isoform subset. It is also worth noting that transgenic miRNA could eliminate endogenous Dscam expression at the embryonic stage, while double-strand RNA transgenes perform poorly at this stage (; Yu and Lee, unpublished observation).
Using miRNA-based knockdown and immunostaining by isoform-specific antibodies, we discovered that Dscam+19 and Dscam−19 redundantly govern neuronal morphogenesis but are preferentially utilized at different developmental stages (–). Dscam+19 is primarily used during embryogenesis, whereas Dscam−19 abundantly exists in the post-embryonic nervous system (). Despite the dynamic changes in the relative abundance, Dscam+19 and Dscam−19 exhibit similar spatial expression patterns (). Consistent with these expression profiles, endogenous Dscam−19 plays an essential role in post-embryonic neuronal morphogenesis ( and ), while Dscam+19 plays a role in the formation of embryonic CNS ( and ).
In contrast to the dynamic usage of exon 19, Dscam+23 and Dscam−23 are enriched in different neural structures at the same developmental stages (). It appears that the high-expression domains exclusively consist of Dscam−23 and depleting Dscam−23 transcripts is sufficient to recapitulate the loss-of-Dscam phenotype (). Notably, Dscam+23 is selectively expressed in some midline cells of the embryonic CNS (). Further investigation is needed to determine the identity of these midline cells (e.g. unpaired midline neurons or glia).
Skipping exon 19 produces the Dscam without a proline-rich motif and an ITAM-like (). An ITAM is defined by a motif containing two tyrosine residues within the consensus sequence of YxxI/Lx(6–12)
YxxI/L (Fodor et al., 2006
). Unlike most ITAM motifs which carry Ile/Leu adjacent to the second tyrosine residue, Ala is present in that position of Dscam (). The utilization of YxxA in ITAM is not unprecedented since the same variation occurs in RhoH, a hematopoietic-specific GTPase-deficient member of Rho GTPase family (Gu et al., 2006
). ITAM-mediated signals control a variety of cellular responses, ranging from phagocytosis, cell migration, proliferation, differentiation to gene induction (Fodor et al., 2006
). Whether the ITAM-like in Dscam can mediate the canonical ITAM signal transduction to govern similar cellular responses awaits investigation. Intriguingly, transgenic Dscams are enriched in different subcellular compartments depending on the presence or absence of exon 19 (). In the MBs, transgenic Dscam−19 is preferentially targeted to neurites while transgenic Dscam+19 is distributed throughout the neurons. Induction of transgenic Dscams
with different endodomains in AL PNs revealed similar patterns of differential protein distribution (). These correlate with the phenomena that transgenic Dscam−19 acted more potently than Dscam+19 in altering neurite projection patterns in both MB neurons and PNs (; Yu and Lee, unpublished observation). It remains to be determined whether the proline-rich motif and/or the ITAM-like within exon 19 help regulate protein distribution of Dscam.
The notable difference between Dscam+23 and Dcam−23 is the latter variant lacking exon 23 and thus losing a PDZ-binding motif (). However, transgenic Dscams behaved indiscriminately in the presence or absence of exon 23, yielding no insight into why Dscam−23 is utilized in known Dscam-dependent neuronal morphogenetic processes (). In addition, Dscam+19+23::GFP
transgene that abolishes the potential PDZ-binding motif located at the carboxyl terminus of Dscam remains as potent as unmodified Dscam in preventing sister branches from extending into the same axon bundle in single-cell clones of Dscam
mutant neurons (Soba et al., 2007
). To unravel the function of the PDZ-binding motif in Dscam+23 may require more sensitive assays or studies in different model systems.
Transgenic Dscam−19 not only effectively prevents multiple self-branches from extending into the same MB lobe in Dscam
mutant single-cell MARCM clones, but it also blocks axon bifurcation which results in single-branch/neuron phenotype in a significant number of the rescued single-cell MARCM clones (). The single-branch/neuron phenotype is apparently elicited by a cell-autonomous mechanism, and cannot be readily explained based on our current model about the roles of Dscam in controlling axon arborization (). Two known mechanisms may suppress MB bifurcation at the peduncle end. First, the competition among self-branches for the available separate fascicles probably underlies the phenomenon that neurons can reliably make the correct numbers of branches based on the numbers of fascicles that project off the branch point. This may explain why MB α/β axons do not bifurcate when the α or β lobe is missing (Wang et al., 2002
). Second, promiscuous competition from non-self branches is thought to occur and, as a consequence, stop most axons from extending beyond the bifurcation point, when the endogenous Dscam
gene loses its ectodomain diversity or a single-isoform Dscam
transgene is ubiquitously expressed (Hattori et al., 2007
; Wang et al., 2004
; Zhan et al., 2004
). Both pathological conditions are caused by environmental factors through non-autonomous mechanisms. By contrast, in the clone-specific rescue experiments, the α and β lobes were both normal, and the Dscam transgene was only expressed in the neuron that exhibited the single-branch/neuron phenotype (). These results clearly suggest a novel cell-autonomous mechanism for Dscam in governing neurite arborization. Further, it is unlikely to be due to excessive Dscam expression, since the UAS-transgenes
are expressed at low levels in newborn single-cell MARCM clones, owing to the perdurance of GAL80 inherited from the heterozygous ganglion mother cells. In addition, overexpressing UAS-Dscam−19+23
in wild-type MB α/β MARCM clones should increase the overall Dscam amount, but did not cause obvious single-branch/neuron phenotype (). Therefore, the suppression of axon bifurcation is likely to result from loss of Dscam ectodomain diversity in a single neuron. This suggests that the huge molecular diversity in the Dscam ectodomain is not only essential for self-recognition among numerous migrating growth cones but also critical to the behavior of an isolated growth cone. It is possible that the complexity of the expressed Dscam ectodomains in a given neuron at a given time may determine the strength of Dscam-Dscam homophilic signaling between the nascent split growth cones and, thus, help govern how divergently the sister growth cones should migrate away without compromising each other.
Taken together, we substantiate the presence of four possible Dscam endodomains and demonstrate that Dscams with specific endodomains support specific neural developmental processes. The identification and characterization of Dscam endodomains are essential for further elucidation of the roles of Dscam and its immense molecular diversity in neural development as well as the innate immunity of insects (Watson et al., 2005
). It also shed new light on how the diversity in the Dscam ectodomain may cell-autonomously govern neurite arborization in the development of Drosophila