Alternative splicing in Drosophila neuronal development.

J. Neurogenetics, 28(3–4): 199–215 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 0167-7063 print/1563-5260 online DOI: 10.3109/01677063.2014.936437

Review

Alternative splicing in Drosophila neuronal development Carmen Mohr1,2,3 and Britta Hartmann1,3

J Neurogenet Downloaded from informahealthcare.com by Dicle Univ. on 11/17/14 For personal use only.

1

Institute of Human Genetics, University Medical Center Freiburg, Freiburg, Germany 2 Research Training Program GRK 1104, Freiburg, Germany 3 BIOSS - Centre for Biological Signaling Studies, Freiburg, Germany

Abstract: Post-transcriptional pre-mRNA splicing has emerged as a critical step in the gene expression cascade greatly influencing diversification and spatiotemporal control of the proteome in many developmental processes. The percentage of genes targeted by alternative splicing (AS) is shown to be over 95% in humans and 60% in Drosophila. Therefore, it is evident that deregulation of this process underlies many genetic diseases. Among all tissues, the brain shows the highest transcriptome diversity, which is not surprising in view of the complex inter- and intracellular networks underlying the development of this organ. Reports of isoforms known to function at different steps during Drosophila nervous system development are rapidly increasing as well as knowledge on their regulation and function, highlighting the role of AS during neuronal development in Drosophila. Keywords: Alternative splicing, Drosophila, neurogenesis, post-transcriptional regulation

Introduction Post-transcriptional events have the potential to greatly expand the transcriptome and proteome of a cell and to control the deployment of proteins in both spatial and temporal dimensions. One central aspect is the process of alternative splicing (AS) that allows the modulation of gene information in a most efficient manner by alteration of exon usage (Figure 1A). Splicing occurs in the nucleus and can be coupled to transcription or other key steps of the gene expression cascade (reviewed in Han et al., 2011). Transcription of a gene generates a direct copy of the genomic template, the precursor messenger RNA (pre-mRNA), which still contains exonic and intronic sequences. The introns are spliced out of the pre-mRNA to produce the messenger RNA (mRNA) which is then exported and translated (Blencowe & Graveley, 2007 and references within). Splicing is achieved by a multiprotein complex, the spliceosome, consisting of appr. 300 proteins and 5 RNAs binding to the core-splicing motifs (splicesites, polypyrimidine tract, and branch point) (Figure 1B) (Wahl et al., 2009). AS results in the complete or partial exclusion or inclusion of exons and/or introns from the message thus generating different mRNAs from a single

pre-mRNA template. This is achieved by regulatory transacting factors, the splicing factors, which in analogy to DNA transcription factors bind to cis-regulatory elements that are located within exonic and intronic pre-mRNA sequences (Figure 1B) (Wang & Burge, 2008). AS has the potential to modulate the information encoded in the mRNA thereby affecting translation efficiency, protein structure, mRNA localization and stability, and microRNA binding sites. Hence, AS is largely responsible for the enormous transcriptome and proteome diversity in higher eukaryotes despite the relative low number of encoded genes. Recent reports describe that over 95% of human genes and 60% of Drosophila genes are subject to AS (Graveley et al., 2011; Pan et al., 2008). The role of all these different transcripts is largely unknown and it is still in debate how much AS is simply due to the noise of the system (i.e., without functional relevance). However, large-scale studies have shown that numerous AS events are tissue specific or developmentally regulated supporting the idea of functional relevance for these transcripts (Sultan et al., 2008; Wang et al., 2008). RNA-Seq and microarray studies comparing different tissues in humans and mice have shown that the brain is among the organs with the highest transcriptome

Received 16 January 2014; accepted 15 June 2014 Address correspondence to Britta Hartmann, University Medical Center Freiburg, Institute of Human Genetics, 79102 Freiburg, Germany. Tel:  49 761 27070270. Fax:  49 761 27070410. E-mail: [email protected] 199

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Figure 1. (A) Schematic representation of different mechanisms to generate transcriptome diversity. AS can affect whole exons (cassette or mutually exclusive exons), parts of exons (alternative 5’ or 3’splice-site usage) or introns can be retained. Additionally, alternative polyadenylation or alternative transcription initiation can affect mRNA composition. (B) Regulation of AS through cis- and trans-acting factors. The core-spliceosomal elements include the 5’- and 3’splice-site (ss), branch point, and a polypyrimidine tract (Py-tract) just upstream of the 3’splice-site which are bound by the core-spliceosomal components (including the hnRNPs U1, U2, U4,U5, U6, U2AF, and SF1). Additional regulation of AS is achieved by splicing factors that bind to intronic and/or exonic reguatory motifs.

diversity (Wang et al., 2008). In line with this observation, a number of neurological diseases are caused by defects of the splicing machinery or mis-spliced transcripts (e.g., Licatalosi & Darnell, 2006) including the locus of Frontotemporal Dementia with Parkinsonism Linked to Chromosome 17 (FTDP-17) and an autosomal dominant form of retinitis pigmentosa (RP). This emphasizes the importance of the splicing process itself and the necessity of its tight, spatial, and temporal regulation in neuronal tissues.

Neurogenesis of Drosophila starts in the embryo with the formation of neuronal stem cells, the primary neuroblasts (NB) that undergo two phases of proliferation to generate the primary and secondary neuronal lineages. In the first phase, each NB divides asymmetrically, with one cell maintaining NB characteristics and the other becoming a ganglion mother cell (GMC). Each GMC divides further to produce a pair of primary differentiating neurons, building the larval neuronal system. During a second round of NB divisions, secondary neurons and glial


Alternative splicing in Drosophila neuronal development

cells are produced, which differentiate during metamorphosis to form the adult brain together with restructured primary neurons (e.g., Boyan & Reichert, 2011; CamposOrtega, 1995; Hartenstein et al., 2008; Hartenstein & Wodarz, 2013; Knoblich, 2010; Li et al., 2013; Lin & Lee, 2012; Urbach & Technau, 2004). The mature adult brain is composed of an outer layer formed by the cell bodies of neurons and glia and an inner neuropile that consists of highly branched axons and dendrites. During nervous system development, large numbers of neurons have to be generated which ultimately adopt different morphologies, connect with specific synaptic targets, and express distinct ion channels, receptor molecules, and neurotransmitters. In this review we summarize the current knowledge on AS during Drosophila neuronal development. While there are many splicing factors known to be expressed in the nervous system and many splice-isoforms have been observed, for most of them their target pre-mRNAs are unknown and the molecular mechanisms by which these isoforms are generated as well as their biological function are unclear. However, some prominent examples of neuron-specific isoforms demonstrate the fundamental role of AS and illustrate its potential to shape developmental processes. AS in neuronal development There are only a few well-characterized tissue-specific splicing factors and one of the best known is the neuronal splicing factor Elav (embryonic lethal abnormal vision). Elav was the first identified member of the ELAV/Hu gene family characterized by embryonic lethality as well as eye and optic lobe defects indicating that the gene is important during embryogenesis and the development of the visual system (Campos et al., 1985). Members of the highly conserved ELAV/Hu gene family encode for RNA-binding proteins (RBPs) characterized by three RNA recognition motives (RRMs), the second and the third being separated by a hinge region (Birney et al., 1993). Interestingly, the first two RRMs of ELAV family members show a high homology to Sex-lethal (Sxl) protein, a splicing factor that regulates AS in the sex determination cascade (Birney et al., 1993; Lisbin et al., 2000; Robinow et al., 1988). In general, the sequences of the three Drosophila and four human ELAV/Hu family members show a high sequence similarity of 52–82% (Okano & Darnell, 1997; Pascale et al., 2008; Samson, 2008). Elav functions in neuronal AS and regulates translation levels during neuronal development including translation of its own message (Borgeson & Samson, 2005; Hilgers et al., 2012; Samson, 1998). Transcripts from the elav gene are expressed in most cells of the central and peripheral nervous system during all stages of neural development (Robinow & White,

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1991). In neurons, Elav protein is located in the nucleus, whereas in glial cells it is predominantly detected in the cytoplasm (Berger et al., 2007). Genetic analyses of elav in flies have shown that it is required for proper differentiation of neurons during neurogenesis and maintenance of mature neurons (Antic & Keene, 1997; Campos et al., 1985; Good, 1995; Robinow et al., 1988; Robinow & White, 1991; Yao & Samson, 1993). Furthermore, Elav was shown to be involved in the regulation of axonal midline crossing by positively controlling commissureless (comm) mRNA expression levels in commissural neurons (Simionato et al., 2007). Comm directs midline crossing by sorting the Roundabout (Robo) receptor into vesicles which are bound for degradation. High levels of Comm result in low levels of Robo receptor, a prerequisite for axonal midline crossing (Keleman et al., 2002, 2005; Kidd et al., 1998). So far, neuronal AS regulated by Elav has been shown for three pre-mRNAs: erect wing (ewg), neuroglian (nrg) and armadillo (arm) (Koushika et al., 1996, 2000; Lisbin et al., 2001; Loureiro & Peifer, 1998; Soller & White, 2003). For all three genes, Elav promotes the generation of a neuron-specific isoform (Haussmann et al., 2008; Koushika et al., 2000; Lisbin et al., 2001). Direct binding of Elav to pre-mRNAs has been shown for ewg and nrg. In the case of ewg pre-mRNA, Elav binds and forms a multimeric complex in intron 6 just distal to the polyA (pA) site, thereby promoting intron 6 splicing and inhibiting 3′ end processing. This results in an increase of Ewg protein in the nervous system. In non-neuronal tissues, in the absence of Elav, ewg transcripts terminate in intron 6 and become polyadenylated using intronic pA sites. The neuron-specific ewg isoform was shown to be sufficient for complete rescue of viability and neuronal function of ewg mutant flies (Koushika et al., 1996, 2000; Soller & White, 2005). Ewg acts mainly through increasing the levels of mRNA of genes that are involved in transcriptional and posttranscriptional regulation of gene expression. It genetically interacts with components of the Wingless, Notch, TGF-ß, and AP1 signaling pathways to restrict synaptic growth (Haussmann et al., 2008). Using the R8 photoreceptor neuron subtype of the Drosophila retina as an example, it was recently shown that ewg is also important for the down-regulation of Hippo pathway activity, and therefore the determination of R8 neuronal subtype fate (Hsiao et al., 2013). In a similar way as described for ewg, Elav promotes the usage of a more distal neuronspecific 3¢ splice site in nrg pre-mRNA, resulting in an isoform with a distinct cytoplasmic domain. In a RNA interference-(RNAi) based screen for cell adhesion molecules that play a role in the maintenance of synaptic connections, Nrg was identified to be a central coordinator of synaptic growth, function, and stability. Nrg was shown to bind to the intercellular interaction motif of Ankyrin2 in vivo linking Nrg to the cytoskeleton. This interaction


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has been suggested to be important for directly controlling the balance between synapse formation and stability (Enneking et al., 2013). In the case of arm (the Drosophila homolog of ß-catenin), Elav facilitates exclusion of exon 6 of the ubiquitously expressed arm pre-mRNA, creating a protein with a truncated carboxy-terminus (Koushika et al., 2000). Arm plays an important role in the Wingless (Wg) signaling pathway and cadherin-based cell-cell adhesions in epithelial and neuronal tissue (Valenta et al., 2012). The neuronal-specific isoform of arm accumulates in differentiating neurons and functions in Wg signal transduction to determine NB fate early in CNS development. Furthermore, arm functions in the construction of the axonal scaffold later during neuronal development (Loureiro & Peifer, 1998). For all three targets of Elav, little information is available on the role of their neuronal isoforms. Further investigations are required to elucidate the exact mechanisms of generation and functions of neuron-specific isoforms of arm, nrg and ewg. Despite the demonstration that Elav is not responsible for regulating AS of all neuronal genes (Koushika et al., 2000), it will be of interest to investigate whether more pre-mRNAs are subject to regulation by this splicing factor. RNA-binding protein 9 (rbp9) and found in neurons (fne) also encode RNA-binding proteins of the Drosophila ELAV family. Rbp9 is detected from pupal stages onward in the cortex of the CNS and in the ovarian cystocytes (Kim & Baker, 1993). Rbp9-null mutant flies show no apparent developmental defects; however, with proceeding age-reduced locomotor activity, and a markedly shorter lifespan have been observed (Kim et al., 2010; Kim-ha et al., 1999). Work performed by the group of Kim-ha suggests that Rbp9 might regulate the splicing of cell adhesion molecules, which are necessary for the formation and maintenance of the blood–brain barrier, but to date no direct target was identified (Kim et al., 2010). Contrary to the nuclear localization pattern of Elav and Rbp9, the third member of the family, Fne, is present in the cytoplasm of neuronal cells throughout development suggesting that it rather functions in translational control than in AS. Fne null mutant flies are viable but show discrete fusion of the ß-lobes of the mushroom bodies and male flies exhibit short courtship indices (Zanini et al., 2012). Over-expression of fne in neurons leads to developmental arrest during larval stages and a decrease of stable transcripts from the elav and fne locus that underscores its potential role in translational control (Samson & Chalvet, 2003). In summary, the three members of the ELAV protein family, Elav, Rbp9, and Fne, show different subcellular localization patterns in neuronal tissue and exhibit distinct functions in the regulatory pathways to maintain the functions of differentiated neurons. Additionally, Elav plays a critical role


during neuronal differentiation as a neuron-specific splicing factor. Similar to the Drosophila ELAV-family, human ELAV (hELAV) proteins were shown to directly regulate AS besides their well-studied involvement in the regulation of mRNA target stability, translatability, and export to the cytoplasm (Ince-Dunn et al., 2012; Lebedeva et al., 2011; Mukherjee et al., 2011). In addition, hELAV has been shown to couple transcription and splicing processes by direct interaction with RNA polymerase II, Cyclindependent kinase 9, and Class I histone deacetylase enzyme 2 (HDAC2; Zhou et al., 2011). AS in glial cell development AS processes have also been reported in various aspects of glial cell development. Among them, the best characterized is the interplay between Held out wings (How) and Crooked neck (Crn) that regulates AS of neurexinIV (nrxIV) (Edenfeld et al., 2006; Rodrigues et al., 2012). This presents a beautiful example of AS regulation depending on controlled subcellular localization and integration of signaling cues (Figure 2A). Glial cells have many distinct roles during development and are essential for the functionality of the nervous system (Parker & Auld, 2006). They provide growth cues for axons, promote neuronal survival, and prune axons during metamorphosis. Another function of glia is to protect the fly neurons from the high ionic levels in the hemolymph throughout life, serving as a blood–brain barrier (BBB). The BBB is built by perineurial glia and the subperineurial glial cells which arise from mesodermal and ectodermal lineages, respectively (Edwards et al., 1993; Fredieu & Mahowald, 1989). Important for the integrity of the BBB are the pleated septate junctions (pSJ) connecting the perineurial and subperineurial glial cells. One of the proteins found in the pSJ and essential for BBB function is NrxIV (Baumgartner et al., 1996). Due to mutually exclusive AS of either exon 3 or 4, nrxIV has two functionally different isoforms that provide distinct adhesive properties (Stork et al., 2009). NrxIVexon4 is found in neurons and interacts with Wrapper in midline glial cells, a member of the Ig-superfamily (Stork et al., 2009), The isoform NrxIVexon3 is expressed in cells that form septate junctions (Edenfeld et al., 2006; Rodrigues et al., 2012). In a genetic screen, the gene crn encoding for a splicing factor was identified to be important for glial cell migration and differentiation (Edenfeld et al., 2006). Glial cells of crn mutants do not fully migrate along the axons, fail to differentiate, and do not form septate junctions (Edenfeld et al., 2006). Crn is ubiquitously expressed during embryonic development (BDGP, Tomancak et al., 2002, 2007) and at low to moderate levels in diverse tissues during


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Figure 2. (A) Model of How/Crn function. How-S and Crn interact in the cytoplasm and translocate to the nucleus. Because timing of nrxIV AS during glial cell development is important, at least one, most likely two signals have to be readout by How/Crn. Cdk12 is one potential kinase to phosphorylate the C-terminal domain (CTD) of Polymerase II (Pol II). Phosphorylation of the CTD results in the recruitment of Prp40 and the spliceosome. Crn can interact with Prp40 and thus favors the binding of How to its site at the nrxIVpre-mRNA, resulting in more neuron-specific isoform. (B) Schematic representation of the Dscam1 genomic organization, AS pattern, and protein structure. Exon-clusters 4 (12 exons), 6 (48 exons), 9 (33 exons) and 17 (2 exons) generate the large transcript diversity affecting the amino acid composition of the Ig-domains (Ig domains 2, 3, and 7) and the transmembrane domain ™. Ig: immunoglobulin domain; TM: transmembrane domain; FN: Fibronectin typeIII domain.

later stages (Gelbart & Emmert, 2013). In glial cells, Crn protein can be detected in the nucleus as well as in the cytoplasm (Edenfeld et al., 2006). It contains 16 tetratricopeptide repeats and bears over 40% similarity to the yeastsplicing factor Clf1, which has been shown to interact with Prp40, a component of the U1-spliceosomal subunit.

Crn itself cannot directly bind RNA (Chung et al., 1999). It interacts molecularly with How, which belongs to the signal-transduction and activation of RNA (STAR) family of RNA-binding proteins and is a homolog of human QUAKING (QKI). STAR proteins have been implicated in many developmental aspects ranging from germline


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development and wing patterning to epithelial-to-mesenchymal transition (Volk & Artzt, 2010). They are composed of one large K homology (KH) domain that mediates RNA binding. The activity of STAR proteins can be regulated through post-translational modifications by signaling pathways or other regulatory cues (reviewed in Volk & Artzt, 2010). Moreover, how and other members of the STAR family are alternatively spliced themselves, resulting in multiple isoforms with diverse functions. During glial cell development, How is expressed predominantly in subperineurial glial cells (Rodrigues et al., 2012). Three protein isoforms (How-L, -M, and -S) differing in their N-termini and with different cellular localization can be generated from the how locus. In subperineurial glial cells, How-L is localized in the nucleus and How-S in the cytoplasm where it directly interacts with Crn. The complex of How-S and Crn translocates to the nucleus, directly binds nrxIV pre-mRNA, and regulates its AS. Nuclear How-L also shows some splicing activity, but to a less extent. Because the pSJs of the BBB are formed at late stages of glia development, timing of nrxIV AS has to be tightly regulated. So far, regulatory mechanisms of this event have not been resolved, but the kinase Cdk12 was identified to be implicated in nrxIV AS, possibly mediating a temporal cue (Rodrigues et al., 2012). The current model proposes that How-S/Crn complex translocation to the nucleus is triggered by an external signal. During transcriptional elongation, the carboxyterminal domain (CTD) of RNA polymerase II is phosphorylated, possibly by Cdk12, thus recruiting Prp40 to the CTD. Interaction of this complex with nuclear How/ Crn directs efficient AS of nrxIV (Figure 2A) (Rodrigues et al., 2012). It will be exciting to uncover the signaling pathways that control the timing of nrxIV AS and to find out whether How and Crn fulfill similar functions in other developmental processes possibly responding to different signaling cues. Interestingly, QUAKING, the mouse homolog of How, was also shown to be important for glial cell ensheathment of axons. QUAKING binds to similar motifs in premRNAs as How and generates three major protein isoforms (QKI-5, QKI-6, QKI-7) that show different subcellular localization, indicating that the molecular mechanism of splicing regulation by the two factors is conserved (Chen & Richard, 1998; Cox et al., 1999; Ebersole et al., 1996; Sidman et al., 1964; Wu et al., 2002). AS in axon growth and synapse formation There are several examples of alternatively spliced transcripts and splicing factors with a role in axon guidance and synapse morphology. The best studied case is the Down syndrome cell adhesion mol-


ecule 1, Dscam1 (Hattori et al., 2007; Schmucker et al., 2000; Wojtowicz et al., 2004; Zhan et al., 2004; Zipursky & Grueber, 2013). Many aspects of Dscam1 AS regulation have been analyzed in detail including the underlying molecular mechanisms as well as the role of specific isoforms in vivo. Dscam1 was identified in the year 2000 due to its interaction with the SH2 and SH3 domains of Dreadlocks (Dock) (Schmucker et al., 2000). Dscam1 expression was detected in the neuropile and homozygous mutants are lethal in early larval stages. Mutant embryos exhibit mild to severe disorganization of axon pathways in the ventral nerve cord. Further analysis using the larval optic nerve (Bolwig’s nerve) as a model showed that Dscam1 acts together with Dock and the serine/threonine kinase Pak to direct axon growth in the embryonic nervous system (Schmucker et al., 2000). The ability of dendrites and axons to discriminate between self and non-self is a key mechanism for correct neuronal wiring ensuring that branches from the same neuron do not overlap resulting in the typically fasciculated morphology of neurons (Kramer & Kuwada, 1983; Kramer & Stent, 1985). Several studies demonstrated that Dscam1 provides the molecular basis for self-avoidance in Drosophila (reviewed in Zipursky & Grueber, 2013). An intriguing feature of Dscam1 is the extraordinary complexity of its splicing behavior as discussed in the following. Dscam1 encodes a transmembrane protein composed of an ectodomain consisting of 10 Ig-domains and six fibronectin type III repeats, a transmembrane domain, and a C-terminal intracellular domain. Theoretically, over 38000 isoforms can be generated from the Dscam1 locus of which 18496 isoforms have been experimentally detected during Drosophila development (Schmucker et al., 2000; Sun et al., 2013). Isoform diversity is generated by four clusters containing one of four mutually exclusive exons (exon 4, 6, 9, and 17) (Figure 2B). Each of these clusters contains 12, 48, or 33 exons, respectively, encoding different Ig-domains (Ig-domain 2, 3 and 7) and two exons encoding the transmembrane domain (Schmucker et al., 2000). The necessity of Dscam1 diversity was first demonstrated by the severe neuronal wiring defects of a transgenic fly which expressed only a single Dscam1 ectodomain isoform (Hattori et al., 2007). In the meantime, isoform-specific studies using dendritic arborization (da) neurons, a group of sensory neurons that are highly branched displaying an extensive overlapping arborization pattern, have highlighted the importance of Dscam1 diversity (Hattori et al., 2009). Expression of 1152 distinct Dscam1 isoforms in da neurons was not sufficient to consistently discriminate between self and non-self but 4752 isoforms were (Hattori et al., 2009). In addition to genetic studies, the isoforms and their binding behavior have been analyzed by biochemical means revealing that the isoforms exhibit strong homophilic



binding and do not bind or bind only with low efficiency to different isoforms. Even small differences in amino acid sequence resulted in disruption of binding (Wojtowicz et al., 2004). In this way, the “Dscam-code” of a neuron determines the complex pattern of neurites (Wojtowicz et al., 2004). Interestingly, the mammalian clustered Protocadherins (Pcdh), a large group of transmembrane proteins of the cadherin superfamily, are required for neurite self-avoidance proposing similar molecular concepts for this process (reviewed in Chen & Maniatis, 2013). A challenge in Dscam1 research is to localize the thousands of isoforms and to address their specific functions. New technologies and sophisticated bioinformatic approaches helped mastering the first step toward this direction. For example, two studies used custom-designed microarrays detecting all 93 variable exons of the extracellular domain of Dscam1. These were hybridized to cDNA isolated from either photoreceptor subtypes (R3/R4 and R7) or mushroom body neurons from 3rd instar larvae (Neves et al., 2004; Zhan et al., 2004). One drawback of such a system is that the data from the microarray is a sum of the cDNAs hybridized and therefore single-cell RT-PCR combined with stochastic approaches were applied to estimate the type and numbers of isoforms expressed in single neurons. Both studies concluded that neurons express a set of 10–50 different Dscam1 proteins and that this set makes them sufficiently distinguishable from their neighbors. Another drawback of exon microarrays is that the exon order cannot be predicted and the abundance is estimated in relation (relative abundance) but not quantitatively. A new strategy called CAMseq based on RNASeq by next generation sequencing (NGS) was developed to circumvent this issue. CAMseq uses circularized Dscam1 cDNA and a 2-step PCR protocol to reduce the size of the cDNA to 1 kb, which can then be sequenced on the Illumina platform (2  150 bp) (Sun et al., 2013). With this approach, isoform-specific expression was analyzed in cell culture and multiple tissues at various developmental stages. The major conclusion from this study was that AS of the four different clusters, containing one of the four mutually exclusive exons, is independent of each other but a certain bias of cluster usage is observed in specific cell-types. Moreover, the number of Dscam1 isoforms expressed varies in different cells, raising interesting questions on how much Dscam1 diversity is required to create the complex network of a brain (Sun et al., 2013). How is Dscam1 AS regulated to create such diversity? A number of groups have characterized some of the mechanisms regulating mutually exclusive AS in the different Dscam1 clusters. RNA structures seem to play an important role in regulation of Dscam1 AS as characteristic secondary structures have been described for each cluster (Kreahling

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& Graveley, 2005; May et al., 2011; Yang et al., 2011; Yue et al., 2013). For example, a long, conserved RNA structure, the inclusion stem (iStem) is responsible for inclusion or exclusion of the complete exon 4 cluster, but has no influence on the choice of the variable exons themselves (Kreahling & Graveley, 2005). A combination of sterical hindrance and an RNA structural element masking the splice-sites have been proposed for cluster 17 regulation (Yue et al., 2013). Additionally, RNA-binding proteins have been implicated in Dscam1 AS regulation. For example the splicing repressor hnRNP36 has been shown to be partially responsible for AS of cluster 6 (Olson et al., 2007). An RNAi screen in Drosophila S2 cells to detect splicing regulators of exon 4 and 17 AS identified 8 regulators important for exon 4 choice and 29 factors affecting cluster 17 AS (Park et al., 2004). Interestingly, these 8 factors important for cluster 4 AS also affect exon choice of cluster 17. In summary, AS of each Dscam1 cluster is regulated by distinct secondary RNA structures, possibly in combination with splicing factors that can be shared between clusters. It will be intriguing to see how these mechanisms are utilized in vivo to create such diversity. AS has also been implicated in the regulation of synaptic growth and plasticity. Here we present two examples that are both connected to Fasciclin II (FasII), which is like Dscam1, a member of the immunoglobulin superfamily (IgSF). In 1991, Drosophila FasII, the homolog of human Neural Cell Adhesion Molecule (NCAM), was identified and characterized as a neuronal cell adhesion molecule, that allows growth cones to distinguish between different axon pathways, and additionally being essential for synaptogenesis (Grenningloh et al., 1991; Kristiansen & Hortsch, 2010; Packard et al., 2003). Several FasII isoforms are generated in vivo. Transmembrane isoforms of FasII are found in neurons whereas GPI-linked isoforms are expressed exclusively in glial cells (Wright & Copenhaver, 2000). Recently, the group of McCabe characterized the spliceosomal proteins Beag and Dsmu1 (synonym: Smu1) as potential candidates to regulate FasII AS (Beck et al., 2012). Both factors are important for the development of synapses at the neuromuscular junction (NMJ) in Drosophila. Flies mutant for beag or Dsmu1 showed significantly fewer synaptic boutons at the NMJ and decreased neurotransmitter release compared to wildtype flies. Further experiments placed Dsmu1 upstream of beag. Both proteins co-localize with Elav in the nucleus. At the molecular level, lower amounts of the neuron specific isoform of FasII were observed in beag and Dsmu1 mutants. Over-expression of a neuron-specific transmembrane isoform of FasII was sufficient to rescue the synaptic phenotype of beag mutants suggesting that beag and Dsmu1 co-operate to regulate neuron-specific AS of FasII. However, the neurotransmitter release defect of


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beag mutants was not altered upon FasII overexpression, indicating that the splicing of additional genes might be regulated by Beag and Dsmu1 in addition to FasII (Beck et al., 2012). Future studies addressing the mechanism of AS regulation of FasII and additional target genes of Beag/Dsmu1 and possibly Elav will contribute to our understanding of synapse development. A protein that has been shown to be important for the synaptic localization of FasII in the membrane is Discs large (Dlg), a member of the molecular scaffolding protein family MAGUK (membrane-associated guanylate kinases). Members of this family are expressed in a variety of tissues including the neuronal system (Woods & Bryant, 1991; Woods et al., 1996), and are known to be involved in the organization of neuronal signal transduction cascades, synapse formation, and function (Garner et al., 2000; Oliva et al., 2012). Drosophila Dlg proteins are expressed pre- and postsynaptically in type I synaptic boutons and have been implicated in many different functions in the CNS including the regulation of neurotransmitter release (Budnik et al., 1996; Lahey et al., 1994), localization of Shaker potassium channels (Ruiz-Cañada et al., 2002; Tejedor et al., 1997), synaptic localization of FasII (Thomas et al., 1997; Zito et al., 1997), and regulation of synaptic plasticity (Koh et al., 1999). As expected, flies mutant for dlg show severe defects in neuronal anatomy (Budnik et al., 1996; Lahey et al., 1994; Thomas et al., 1997). Early studies have shown that Dlg interacts via its PDZ-domain with FasII and in dlg mutants FasII is dispersed in a broad region around the boutons (Thomas et al., 1997). Mendoza et al. (2003) showed that dlg premRNA is alternatively spliced generating proteins, differing in their domain structure. Isoforms containing an N-terminal S97N domain are important for proper neuronal differentiation. This S97N domain is homolog to the N-terminus of the mammalian synapse-associated protein 97 (SAP97/hDLG), which is known for its essential functions in the nervous system. Drosophila S97N-isoforms are mainly expressed in neuropile regions of the CNS and at NMJs and knock-down of the proteins via RNAi results in abnormal development of neuronal tissue (Mendoza et al., 2003). So far it has not been clarified which dlg isoform fulfills this function, but it is likely that the S97Ncontaining isoform is involved in the establishment of the neuronal functions of dlg. Sex-specific AS during neuronal development Drosophila female and male flies have characteristic anatomical differences besides the genital organs, such as the sex combs or body color which are established during development and are controlled by a well-characterized sex-determination cascade. Morphological differences


between the sexes are also observed in the central nervous system but it is less clear how these dimorphisms are established at the molecular level. Drosophila melanogaster also exhibits innate sex-specific behaviors such as courtship and aggression which are largely under the control of the sex-determination cascade. The sex in Drosophila is determined by the ratio of X-chromosomes to autosomes ultimately establishing expression of the splicing factor Sex-lethal (Sxl) in female and not in males flies (Figure 3A). Sxl regulates AS of a handful of pre-mRNAs including its own message, male sex-lethal 2 (msl2) which is crucial for dosage compensation, and transformer (tra) encoding for another splicing factor (Bashaw & Baker, 1995; Bell et al., 1991; Inoue et al., 1990; Kelley et al., 1997; Nagoshi et al., 1988). AS regulation by Sxl ans Tra is one of the best characterized splicing cascades and studying its players has revealed fundamental insights in the molecular mechanisms of splicing regulation as well as the impact of post-transcriptional control on cellular and developmental processes. Sxl regulates AS of tra such that only females produce a fully translatable mRNA and thus functional Tra protein (Figure 3A). Tra controls AS of two transcription factors, doublesex (dsx) and fruitless (fru) (Figure 3A) (Baker, 1989; Burtis & Baker, 1989; Ito et al., 1996; Ryner & Baker, 1991; Ryner et al., 1996). Tra-dependent AS of dsx generates a female and male specific protein isoform, DsxF and DsxM, respectively. These isoforms differ in their carboxy-termini due to the usage of an alternative 3’ splice site and display distinct transcriptional activities regulating somatic sex differences (Anand et al., 2001; Baker & Wolfner, 1988; Burtis & Baker, 1989; Christiansen et al., 2002; Lee et al., 2002; Salz, 2011). While sxl and tra are ubiquitously expressed in the CNS, dsx gene products are detectable only from embryonic stage 16 onwards in about 350–450 cells, the majority of which are neurons (Birkholz et al., 2013; Lee et al., 2002). The other target of Tra is fru pre-mRNA (Heinrichs et al., 1998). Already in 1963, a fru mutant was described demonstrating that sex-specific isoforms of fru are required for many gender-specific mating behaviors (Anand et al., 2001; Gill, 1963; Goodwin et al., 2000; Ito et al., 1996; Manoli et al., 2005; Ryner et al., 1996; Villella et al., 1997). Many different transcripts are generated from the fru locus from at least four known promoters (P1–P4) (Figure 3B) (Goodwin et al., 2000). The most distal promoter, P1, is exclusively active in about 2000 neurons which make up 2% of the CNS in both females and in males (Lee et al., 2000; Manoli et al., 2005; Usui-Aoki et al., 2000). Only products transcribed from P1 are alternatively spliced in a sex-specific manner such that only males produce functional Fru protein (FruM) (Figure 3B) (Ito et al., 1996; Ryner et al., 1996). Transcripts encoded by the P1 promoter vary from all



other common, non sex-specifically spliced fru isoforms (FruCOM) in a 101 aa long amino-terminal stretch called the M-sequence. Additionally, AS at the 3′ end of male specific FruM transcripts leads to diversity of the last exons, coding for different zinc finger domains with distinct transcriptional activities (Figure 3C) (Billeter et al., 2006; Dalton et al., 2013, Ryner et al., 1996; Usui-Aoki et al., 2000). Although the M-sequence is the main difference between males and females experimental data clearly demonstrated that it is essential but not sufficient to completely determine femaleness or maleness (Demir & Dickson, 2005; Ferri et al., 2008; Rideout et al., 2007; UsuiAoki et al., 2000). Large scale studies identified about 100 genes potentially regulated by FruM but a more detailed analysis on the effects of this regulation is pending (Dalton et al., 2010; 2013). Furthermore, ectopic expression of FruM in female and male shows different transcriptional activities implying that additional sex-specific factors might influence its activity. One candidate could be hunchback (hb) which was identified as a modifier of FruM function in the formation of male-typical shaping of contralateral axons (Goto et al., 2011) but it still remains unclear how the interaction of FruM and Hb influences the sex-specific morphogenesis of neuronal tissue. Detailed structural differences in the CNS of female and male flies were first described for the number of mushroom body fibers, being significantly higher in females than in males (Technau, 1984). In the meantime, several groups have investigated the gross and fine anatomical differences in the CNS between the sexes (Cachero et al., 2010; Kimura et al., 2005; Technau, 1984). In general, striking volume differences can be observed especially in higher brain centers of female and male flies (Cachero et al., 2010). Several neuronal clusters have been identified that are composed of different number of neurons between the sexes (e.g. the mAL cluster) or are only present in the male CNS such as the P1 cluster (Kimura et al., 2008). Differences in the projection patterns of neurons were identified for taste sensory neurons (Possidente & Murphey, 1989; Taylor, 1989), mAL neurons (Kimura et al., 2005; Koganezawa et al., 2010) and third order olfactory neurons (Cachero et al., 2010; Kohl et al., 2013). This suggests that groups of neurons are differentially connected in the two sexes, building up sex-specific neuronal circuits (reviewed in Yamamoto & Koganezawa, 2013). In addition, different steps of courtship behavior were mapped to the dimorphic regions in the CNS described above (e.g. the P1, mAL and DA1 neurons), implying that morphological differences in these areas might influence the behavior of the fly (Hall, 1977; Hotta & Benzer, 1972, 1976; Yamamoto & Koganezawa, 2013). The mAL neurons, a cluster of interneurons located medially above the antennal lobe (mAL neurons) are

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dimorphic in their number (30 neurons in males, 5 in females) and their axonal projection pattern: e.g. an ipsilateral neurite extending to the suboesophageal ganglion (SOP) is only present in males (Figure 3C) (Kimura et al., 2005). Whether these neurons take a female or male fate depends on the level of FruM and the chromatin state (Datta et al., 2008; Ito et al., 2012; Kimura et al., 2005). Pull down assays showed that FruM forms a complex with the transcriptional cofactor Bonus (Bon), which recruits either of two chromatin remodeling regulators Histonedeacetylase 1 (HDAC1) or Heterochromatin protein 1a (HP1a) (Ito et al., 2012). High levels of FruM and HDAC1 promote male development while low levels of FruM and high levels of HP1a direct the neurons to develop the female specific structure (Ito et al., 2012). However, the transcriptional network and exact chromatin landscape in this developmental switch from female to male development is still unknown. Another example highlighting the principle of fru functioning as a genetic switch gene is the DA1 glomeruli of the antennal lobe which connects with olfactory receptor neurons important for pheromone reception. Female and male third-order olfactory neurons (aSP-f and aSP-m, respectively) build different dendritic connections which genetically depend on FruM (Kohl et al. 2013). Sex-specific FruM dependent wiring of these third-order olfactory neurons activates gender specific behaviors due to differential pheromone processing (Kohl et al., 2013). However, the molecular basis how FruM routes these dendrites is still unknown. Dsx expression studies clarified that besides fru-expressing neurons also dsx neurons (which are smaller in number and partially overlap with the fru clusters) show sex-specific dimorphisms (Cachero et al., 2010; Rideout et al., 2010). Specific dsx positive clusters hold a higher number of neurons in males (900 neurons subdivided into 9 groups) than in females (700 neurons in 5 groups). Besides this dimorphism in number, the topology of neuronal projection is similar between female and male brains. However, the synaptic density and the axonal projections are increased in males compared to females (Rideout et al., 2010). An example to illustrate the importance of dsx sexspecific isoforms for neuronal functionality is the malespecific Posterior 1 (P1) cluster neuron in the dorsal posterior region of the brain. It is believed to be a potent activator of male courtship behavior and expresses both fru and dsx (Demir & Dickson, 2005; Kimura et al., 2008; Stockinger et al., 2005). The morphology of the P1 cluster is not altered in fru mutant males and females expressing fruM still lack the cluster concluding that FruM is not important for the development of male-specific P1 neurons. Further studies showed that the P1 cluster depends on the absence of Tra protein in males supporting a role for the other target of Tra, dsx (Kimura et al., 2008). Indeed, dsx mutant females exhibit the male specific

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Figure 3. (A). The sex-determination cascade in females (left) and males (right). Sxl regulates AS of itself and tra such that functional Sxl and Tra protein are only expressed in female flies. The presence of Tra (in females) and absence (in males) results in AS of dsx and fru pre-mRNA generating either female-specific (F) or male-specific (M) isoforms. The fruF transcripts contains a premature stopcodon so no functional FruF protein can be translated. (B) Schematic representation of the fruitless genomic region. Presented are the experimentally confirmed (P1-P4) and EST-data-predicted promoters (P*) in green. All transcripts include a common set of exons C1C5, coding for a BTB domain. A-E label alternative 3’ exons, encoding different zinc finger domains, S denotes the sex-specifically



P1-cluster whereas depletion of DsxM in males results in a reduced number of P1 neurons proposing a dual role for Dsx being responsible for programmed cell death in females (DsxF) and survival in males (DsxM). This hypothesis was supported by studies using postembryonic lineages of NB which exhibit sex-specific proliferation behavior (Birkholz et al., 2013). The group showed that DsxF induces cell death thus preventing the generation of a postembryonic NB lineage in females. In contrast, DsxM in males is important for NB survival (Birkholz et al., 2013). The molecular mechanisms by which DsxF induces cell death and DsxM promotes NBs survival are not known. Genome-wide studies identified 54 genes expressed in the adult head that are regulated downstream of Dsx (Goldman & Arbeitman, 2007), and 650 additional DSX-binding regions, presenting potential direct targets of DsxF (Luo et al., 2011). Among the candidates is the pro-apoptotic gene reaper (rpr) which might play a role in the mechanism of DsxF induced cell death (Luo et al., 2011). But so far, no experimental data are available that show a direct interaction of DsxF and Rpr or other factors involved in apoptosis. Recently, it was shown that ovulation, a female specific behavior, is controlled by Sxl but independent of tra in a small subset of neurons (tra-insufficient feminization (TIF) branch) (Evans & Cline, 2013). The target on which Sxl acts is unknown, but interestingly, the P1 promoter of fru is partly active in these neurons (Evans & Cline, 2013). Consistently with this observation, a number of studies have shown that sex-specific AS is more widespread than previously thought affecting over hundred genes and provided evidence that a branch besides the Sxl/Tra cascade exists (Chang et al. 2011, Hartmann et al., 2011; McIntyre et al., 2006). Furthermore, RNASeq data revealed that the AS of the well characterized splicing factors of the sex determination cascade, sxl, tra, dsx and fru, is incomplete. Thus, sxl, tra, and fru transcripts coding for the male, non-functional isoform and the female and male dsx isoforms are expressed in both sexes (Graveley et al., 2011; our data, unpublished) demonstrating that sex specific AS is not an all or nothing event although it results in either femaleness or maleness. In summary, many studies clearly demonstrate the importance of sex-specific isoforms of fru and dsx to control differences in number of neurons, axonal branching, and connectivity. However, the underlying molecular

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mechanisms by which sex-specific isoforms of Dsx and Fru confer cellular differences remain unclear. Not all neurons which display sexual dimorphisms express fru and/or dsx, and it will be interesting to see to what extent other sexspecific factors contribute to cellular dimorphism in the Drosophila CNS. Genome-wide approaches Novel technologies facilitate obtaining information on the number of splicing regulators and isoforms that are expressed in the developing nervous system. A combination of bioinformatics and experimental approaches has shown that the Drosophila genome encodes for approximately 445 putative splicing factors or factors involved in RNA metabolism (Gan et al., 2011; Herold et al., 2009). Publically accessible databases such as the global expression analyses from the modEncode project (Graveley et al., 2011) illustrate that 117 splicing factors are expressed at high levels in the larval CNS and up to 353 if we consider low to intermediate levels. These numbers are not surprising, considering that only a handful of tissue or cell specific splicing factors have been characterized and that it has been postulated that rather a cocktail of splicing factors than a single factor determines the splicing pattern (Matlin et al., 2005). In agreement with this, a specific splicing factor is able to function in different developmental processes and several splicing factors can be required for the same developmental process. For example, a recently performed RNAi screen in vivo against known RBPs was performed, searching for RBPs being important for larval dendritic morphology. The screen identified 88 candidates genes which often produced a specific phenotype upon knock-down (Olesnicky et al., 2013). It was shown that different regions of a specific mRNA can be regulated by an overlapping but distinct set of splicing factors. For Dscam1, 29 spliceregulators were identified to be exclusively important for AS regulation of the Dscam1 cluster 4, while 8 factors are involved in the control of the two clusters 4 and 17 (Park et al., 2004). How much AS occurs during Drosophila neuronal development? New technologies such as exon or custom designed splicing-sensitive microarrays and RNA-Seq by NGS now allow not only to quantify the amount of transcripts in a given tissue or developmental stage but

Figure 1. (Continued ) spliced exon of P1 transcripts. Fruitless transcripts. Only transcripts derived from the most-distal promoter P1 are sex specifically spliced due to the usage of an alternative 5’ss in exon S. Male transcripts add 101 aa, the M sequence, to the N terminus of the BTB domain, resulting in functional FruM protein in males, whereas in females no Fru protein is produced from P1 due to a premature stop codon in the S exon. Alternative 3’ exons A C are present in female and male specific transcripts. (C) mAL neurons show a sexual dimorphism in male (left) and female brains (right), differing in their number (30 in males, 5 in females) and projection areas (bilateral with a horsetail-like structure in males, contralateral with Y-shaped branches in females).


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also to gain information about the transcript composition itself. While especially RNA-Seq experiments will be useful to address this question, the bioinformatics required for data analysis are still demanding. For example, different RNA-Seq studies aimed at comparing the transcriptome of adult female and male heads (Chang et al., 2011; Daines et al., 2011; Sturgill et al., 2013; our data not published). While a common result of these independent studies was that there are over hundred genes showing differential AS between the sexes in the adult head, the number of detected isoform-specific transcripts vary between 182 to 1370 depending on the definition of bioinformatics cutoffs and possibly on variation in the biological samples (e.g. age of the flies Samson & Rabinow, 2014). Nevertheless, adapting these technologies to characterize the single-cell transcriptome and splicing factor composition will greatly enhance our understanding of AS in the nervous system. Outlook The generation of mRNA isoforms by means of AS is documented for a number of fundamental processes in Drosophila neuronal development but the underlying mechanisms are often not known in much detail. It will be exciting to unravel the functions exerted by the constantly increasing number of reported neuronal isoforms and to discover which premRNAs are targeted by the splicing factors expressed in the CNS. High throughput analyses focusing on the transcriptome of single cells combined with indepth genetic and molecular studies will help to understand the role of AS underlying neuronal development in Drosophila. Acknowledgments We would like to thank Sarah Ortolf and George Pyrowolakis as well as the anonymous reviewers for their helpful comments on the manuscript. C.M. and B.H. are indebted to the BadenWürttemberg Stiftung (Eliteprogram für Postdocs) for financial support of their research project. C.M. is supported by the GRK 1104. Declaration of interest: The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper. References Anand, A., Villella, A., Ryner, L. C., Carlo, T., Goodwin, S. F., et al. (2001). Molecular genetic dissection of the sex-specific and vital functions of the Drosophila melanogaster sex determination gene fruitless. Genetics, 158, 1569–1595.

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Identifying and characterising key alternative splicing events in Drosophila development.

Histone methylation, alternative splicing and neuronal differentiation.

Cell-specific alternative splicing of Drosophila Dscam2 is crucial for proper neuronal wiring.

Alternative role of HuD splicing variants in neuronal differentiation.

Alternative splicing: role in cancer development and progression.

Genomics of alternative splicing: evolution, development and pathophysiology.

Neuronal cell-type-specific alternative splicing: A mechanism for specifying connections in the brain?

Inositol 1,4,5-trisphosphate receptors: distinct neuronal and nonneuronal forms derived by alternative splicing differ in phosphorylation.

Alternative splicing in plant immunity.

Computational analysis of translational readthrough proteins in Drosophila and yeast reveals parallels to alternative splicing.

NOVA regulates Dcc alternative splicing during neuronal migration and axon guidance in the spinal cord.

LSD1 Neurospecific Alternative Splicing Controls Neuronal Excitability in Mouse Models of Epilepsy.

Arrest is a regulator of fiber-specific alternative splicing in the indirect flight muscles of Drosophila.

Induction of soluble AChE expression via alternative splicing by chemical stress in Drosophila melanogaster.

Alternative mRNA splicing.

Analogous alternative splicing.

RBM10 regulates alternative splicing.

The Interplay of Temperature and Genotype on Patterns of Alternative Splicing in Drosophila melanogaster.

Positive autoregulation of sex-lethal by alternative splicing maintains the female determined state in Drosophila.

Regulation of Shootin1 Gene Expression Involves NGF-induced Alternative Splicing during Neuronal Differentiation of PC12 Cells.

Neuronal cell type-specific alternative splicing is regulated by the KH domain protein SLM1.

H2.

Alternative splicing of Drosophila Nmnat functions as a switch to enhance neuroprotection under stress.

The neurogenetics of alternative splicing.