A Combinatorial Semaphorin Code Instructs The Initial Steps Of Sensory Circuit Assembly In The Drosophila CNS

doi:10.1016/j.neuron.2011.02.050

Neuron. Author manuscript; available in PMC 2011 October 28.

Published in final edited form as:

Neuron. 2011 April 28; 70(2): 281–298.

doi: 10.1016/j.neuron.2011.02.050

PMCID: PMC3095019

NIHMSID: NIHMS293142

A Combinatorial Semaphorin Code Instructs The Initial Steps Of Sensory Circuit Assembly In The Drosophila CNS

Zhuhao Wu,¹ Lora B. Sweeney,² Joseph C. Ayoob,¹^* Kayam Chak,¹ Benjamin J. Andreone,¹ Tomoko Ohyama,³ Rex Kerr,³ Liqun Luo,² Marta Zlatic,³ and Alex L. Kolodkin^1,⁴

¹The Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, Howard Hughes Medical Institute, Baltimore, MD 21205, USA

²Department of Biology and Neurosciences Program, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA

³Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA

⁴ Correspondence to: Email: kolodkin/at/jhmi.edu

^*Current address: Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15207, USA

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SUMMARY

Longitudinal axon fascicles within the Drosophila embryonic CNS provide connections between body segments and are required for coordinated neural signaling along the anterior-posterior axis. We show here that establishment of select CNS longitudinal tracts and formation of precise mechanosensory afferent innervation to the same CNS region are coordinately regulated by the secreted semaphorins Sema-2a and Sema-2b. Both Sema-2a and Sema-2b utilize the same neuronal receptor, plexin B (PlexB), but serve distinct guidance functions. Localized Sema-2b attraction promotes the initial assembly of a subset of CNS longitudinal projections and subsequent targeting of chordotonal sensory afferent axons to these same longitudinal connectives, while broader Sema-2a repulsion serves to prevent aberrant innervation. In the absence of Sema-2b or PlexB, chordotonal afferent connectivity within the CNS is severely disrupted, resulting in specific larval behavioral deficits. These results reveal that distinct semaphorin-mediated guidance functions converge at PlexB and are critical for functional neural circuit assembly.

INTRODUCTION

Neurons extend processes over long distances during development, establishing complex yet precise connections to achieve mature neuronal functions. During this process growing neuronal processes recognize and interpret numerous cues as they navigate to their appropriate targets (Raper and Mason, 2010; Tessier-Lavigne and Goodman, 1996). In both vertebrates and invertebrates, longitudinal neural tracts extending along the anterior-posterior axis within the nerve cord serve to exchange and integrate information between different body segments and the brain. To establish these tracts, developing neurites must extend across segmental boundaries, often fasciculating with related neurites from a myriad of possible partners in adjacent segments. In addition, longitudinal pathways often receive neural input from sensory afferents and other local interneurons critical for processing specific sensory information and modulating appropriate motor responses. These two aspects of longitudinal tract assembly could be intrinsically linked in order to better achieve select targeting of neuronal projections that belong to the same circuit.

Cellular experiments in both invertebrates and vertebrates demonstrate the importance of contact with pioneer neurons for the establishment of continuous rostral-caudal neuronal pathways (Goodman et al., 1984; Kuwada, 1986; Wolman et al., 2008). Genetic analyses in the Drosophila embryonic CNS reveal molecular mechanisms governing important aspects of longitudinal pathway organization within the nerve cord. The initial projections of some CNS axons that extend longitudinally are first guided by selectively localized netrin on the surface of adjacent axons (Hiramoto et al., 2000). Subsequently, these axons lose their responsiveness to netrin, continue projecting longitudinally, and cross segmental boundaries through the action of Slit/Robo signaling (Hiramoto and Hiromi, 2006). Slit-mediated repulsion specifies three lateral positions (medial, intermediate, and lateral) for distinct longitudinal axon tracts based on differential expression of Robo receptors (Evans and Bashaw, 2010; Rajagopalan et al., 2000; Simpson et al., 2000; Spitzweck et al., 2010). Related functions of Slit-Robo signaling for CNS longitudinal tract formation have also been observed in vertebrates (Farmer et al., 2008; Long et al., 2004; Lopez-Bendito et al., 2007; Mastick et al., 2010). Interestingly, sensory afferent input to the Drosophila embryonic CNS utilizes this same Slit-Robo code to regulate the projection of different sensory axon classes to distinct CNS lateral positions (Zlatic et al., 2003), restricting both the pre- and postsynaptic components of this first synapse for sensory circuits to a limited region. It remains to be determined how neuronal projections within these specific regions selectively fasciculate with one another.

Several homophilic cell adhesion molecules, including FasII, L1, and Tag1, have been observed to promote the fasciculation of CNS longitudinal projections (Harrelson and Goodman, 1988; Lin et al., 1994; Wolman et al., 2007; Wolman et al., 2008). In the grasshopper and in Drosophila, anti-FasII monoclonal antibodies (MAbs) specifically label several longitudinal fascicles on each side of the CNS, and in Drosophila (utilizing the 1D4 MAb) these appear as three discrete longitudinal axon tracts when viewed from a dorsal aspect (Bastiani et al., 1987; Grenningloh et al., 1991; Landgraf et al., 2003). However, the 1D4-positive (1D4⁺) tracts in the Drosophila embryonic CNS represent only a small subset of the total CNS longitudinal pathways within each lateral region specified by the Slit-Robo code, and they are closely associated with other longitudinal projections that are 1D4-negative (Bastiani et al., 1987; Lin et al., 1994; Rajagopalan et al., 2000; Simpson et al., 2000). Chordotonal (ch) sensory afferent inputs to the CNS, which specifically exhibit axonal branching and elongation along the intermediate 1D4⁺ longitudinal tract (Zlatic et al., 2003), are also 1D4-negative. Taken together, these observations suggest that additional factors govern these specific fasciculation events within each CNS region.

Repulsive semaphorin guidance cues signaling through their cognate plexin receptors have been implicated in longitudinal tract formation and in the restriction of sensory afferent projections to distinct CNS targets in both Drosophila and mouse (Pecho-Vrieseling et al., 2009; Yoshida et al., 2006; Zlatic et al., 2009). In Drosophila, Plexin A (PlexA) and Plexin B (PlexB) are the only plexin receptors, and both play important roles during CNS longitudinal tract development. PlexA is the receptor for transmembrane semaphorin-1a (Sema-1a) and is required for CNS longitudinal tract formation, however only in the most lateral region of the nerve cord (Winberg et al., 1998b). The PlexB receptor, in contrast, is specifically required for the organization of CNS longitudinal tract only in the intermediate region (Ayoob et al., 2006), however the identity of the PlexB ligand(s) required for this function is still unclear. There are two secreted semaphorins in Drosophila, semaphorin-2a (Sema-2a) and semaphorin-2b (Sema-2b). Sema-2a signals repulsion and contributes in part to PlexB-mediated sensory afferent targeting within the CNS, however CNS longitudinal projections appear to be less affected in Sema-2a mutants as compared to PlexB mutants (Zlatic et al., 2009).

Here, we show that both Sema-2a and Sema-2b are PlexB ligands during embryonic CNS development and mediate distinct functions. The PlexB receptor integrates both Sema-2a repulsion and Sema-2b attraction to coordinately regulate the assembly of specific CNS longitudinal projections and select sensory afferent innervation within that same CNS region. Perturbation of PlexB-mediated signaling during the establishment of sensory afferent connectivity within the CNS results in larval sensory-dependent behavioral deficits. These results suggest that a combination of semaphorin cues, acting in concert with the longer-range Slit gradient in the embryonic Drosophila CNS, ensures the fidelity of both CNS interneuron projection organization and sensory afferent targeting, both of which are critical for the establishment of a functional neural circuit.

RESULTS

PlexB is required for appropriate projection of both CNS interneurons and ch sensory afferents within the intermediate domain of the embryonic Drosophila CNS

In the absence of PlexB, interneuron projections that form a group of longitudinal connectives in the developing Drosophila embryonic CNS are disorganized (Ayoob et al., 2006). Interestingly, the targeting of ch sensory afferent projections to the CNS occurs within this same intermediate CNS region, as determined by intracellular labeling of individual ch neurons (Merritt and Whitington, 1995; Zlatic et al., 2003). By genetically labeling ch neurons with GFP using the iav-GAL4 driver (Kwon et al., 2010) and visualizing CNS longitudinal tracts with 1D4 immunohistochemistry (Figures 1A–D), we asked whether or not sensory afferent targeting to the CNS also requires PlexB. As previously reported (Ayoob et al., 2006), in PlexB^−/− null mutant (PlexB^KG00878) embryos the intermediate 1D4⁺ longitudinal tract (1D4-i) is severely disorganized (including defasciculation, disorganization, and wandering of axon bundles within this intermediate position); however, the medial and lateral 1D4⁺ tracts (1D4-m and 1D4-l) appear normal (Figures 1E and 1F). In PlexB^−/− mutants, ch axons extend into the CNS but fail to form regular terminal branches in the region of the disrupted 1D4-m tract, instead remaining stalled with expanded, splayed out, morphologies over the region where they normally would elaborate their synaptic contacts (Figures 1G and 1H). The other plexin receptor in Drosophila, PlexA, is required for the formation of the 1D4-l tract but not the 1D4-i tract, and in PlexA^−/− mutants ch afferent projections target to the medial region of the CNS in a relatively normal fashion (Figures S1A–D). These results show that PlexB-mediated signaling is required for appropriate projection of both CNS interneurons and ch sensory afferents to the same intermediate region within the developing CNS.

Figure 1

PlexB is required for the organization of select CNS longitudinal connectives and chordotonal sensory afferent innervation within the embryonic Drosophila CNS

Both Sema-2a and Sema-2b are PlexB ligands required for appropriate projection of CNS interneurons within the intermediate longitudinal region

During Drosophila embryonic neural development, the 1D4-i tract is established prior to ch sensory afferent targeting and elongation along this tract (Figures S1E–J). Therefore, we first addressed how PlexB regulates the formation of the 1D4-i tract within the CNS by defining the ligands required for this function. The semaphorin protein Sema-2a is thought to be a PlexB ligand in both the PNS and CNS (Ayoob et al., 2006; Bates and Whitington, 2007; Zlatic et al., 2009). However, previous analyses using different P element-derived Sema-2a^−/− mutant alleles show no, or very weak, CNS 1D4⁺ longitudinal tract phenotypes (Winberg et al., 1998a; Zlatic et al., 2009). To address the involvement of Sema-2a in CNS development, we made a Sema-2a null allele by generating an FRT-derived genomic deletion called Sema-2a^B65 (see Figure S2A for details). Sema-2a^B65 null mutant embryos do show pronounced CNS 1D4-i tract defasciculation defects (Figure 2C; see Figure 4F for rescue), however the phenotypes are less severe than those observed in PlexB^−/− mutants (Figures 2B and 2I). Therefore, there must be at least one additional PlexB ligand that functions to organize CNS longitudinal projections.

Figure 2

Secreted semaphorins Sema-2a and Sema-2b are both PlexB ligands and are required for select CNS longitudinal tract formation

Figure 4

Sema-2a is broadly distributed in the embryonic CNS and, unlike Sema2b, functions as an axonal repellent to position longitudinal tracts

Of the four other Drosophila semaphorins, the secreted semaphorin Sema-2b is the best candidate PlexB ligand. Sema-5c is not expressed in the CNS during the embryonic development (Khare et al., 2000), and Sema-1a and Sema-1b bind to PlexA but not to PlexB (Ayoob et al., 2006; Winberg et al., 1998b). The Sema-2b protein is most closely related to Sema-2a, exhibiting 68% amino acid sequence identity. To analyze Sema-2b function, we made a Sema-2b null mutant by generating an FRT-derived genomic deletion called Sema-2b^C4 (see Figure S2A for details). Sema-2b^C4 null mutant embryos show pronounced disorganization and defasciculation defects in the 1D4-i tract (Figure 2D; see Figure 3M for rescue), suggesting that Sema-2b also serves as a PlexB ligand during the embryonic CNS development. However, the CNS phenotypes observed in Sema-2b^C4 null mutants are also not as severe as those observed in the PlexB^−/− mutant (Figures 2B and 2I). To ask whether both Sema-2a and Sema-2b are required for PlexB-mediated functions, we generated a Sema-2a, Sema-2b double null allele called Sema-2ab^A15 (see Figure S2A for details). Sema-2ab^A15 homozygous mutant embryos fully recapitulate the phenotypes observed in PlexB^−/− mutants regarding defects in the 1D4-i tract (Figures 2F and 2I). We also analyzed Sema-2b ^C4; PlexB double null mutant embryos (Figure 2G) and Sema-2ab ^A15; PlexB double null mutant embryos (which are null for Sema2a, Sema2b, and PlexB) (Figure 2H); both genotypes exhibit 1D4-i defects identical to those observed in PlexB^−/− single mutants and Sema-2ab ^A15 homozygous mutants with equal penetrance (Figure 2I), indicating that both Sema-2a and Sema-2b function in the same genetic pathway as PlexB. Interestingly, Sema-2a ^B65/+,Sema-2b ^C4/+ trans-heterozygous mutant embryos exhibit a much lower penetrance of CNS longitudinal connective defects than embryos of either single mutant (Figures 2E and 2I), suggesting that Sema-2a and Sema-2b functions are distinct and contribute to different aspects of intermediate longitudinal connection formation.

Figure 3

Sema-2b protein is expressed along specific CNS longitudinal connectives and functions to promote the assembly of select longitudinal connectives

To complement our genetic analyses we next performed alkaline phosphatase (AP)-tagged ligand binding assays on live dissected embryos (Fox and Zinn, 2005). We first confirmed that AP alone does not bind to the CNS of dissected Drosophila embryos in our assay (data not shown). We then observed that Sema-2a–AP and Sema-2b–AP both bound to endogenous CNS receptors in dissected wild-type embryos (Figures 2J and 2L), but not to endogenous CNS receptors in PlexB^−/− mutants (Figures 2K and 2M). Compared to Sema-2a–AP, Sema-2b–AP bound more robustly to endogenous CNS receptors (Figure 2N). We also expressed PlexB in a Drosophila S2R+ cell line and observed that Sema-2b–AP bound strongly to these cells but not to PlexA-expressing S2R+ cells (Figures S2G and S2D), as observed previously for Sema-2a (Ayoob et al., 2006) (Figures S2B–F). These ligand-receptor binding specificities correlate well with the functions of these proteins in CNS longitudinal track formation. PlexB^−/− and PlexA^−/− mutant embryos exhibit distinct CNS longitudinal tract defects (Ayoob et al., 2006; Winberg et al., 1998b), and Sema-1a^−/− mutants have defects similar to those observed in PlexA^−/−, but not PlexB^−/−, mutants (Yu et al., 1998) (Figures S2H and S2I). In addition, we observed that Sema-1a, Sema-2b double null mutants and Sema-1a; PlexB double null mutants both show disorganization of the 1D4-l and 1D4-m tracts (Figures S2J and S2K), further supporting the idea that Sema-1a–PlexA and Sema-2b–PlexB signaling direct distinct aspects of embryonic longitudinal tract formation. Taken together, these results show that Sema-2a and Sema-2b signaling through the PlexB receptor accounts for most, if not all, PlexB functions in embryonic CNS intermediate longitudinal tract formation.

Sema-2b protein is expressed along specific CNS longitudinal connectives and functions to promote select longitudinal connective assembly

We next assessed Sema-2b protein distribution in Drosophila embryos using a polyclonal antibody specific for Sema-2b (Sweeney et al., manuscript in preparation). Sema-2b is weakly expressed on CNS commissures and more robustly on two longitudinal pathways (Figure 3B). Sema-2b expression is strong along the 1D4-i tract, and also is localized on a more medial pathway that lies directly adjacent to the 1D4-m tract (Figure 3C). Sema-2b protein is completely absent in Sema-2b^C4 null mutant embryos (Figure 3D).

To better define Sema-2b CNS expression, we labeled Sema-2b–expressing neurons and their processes using a genomic fragment containing ~35Kb of DNA upstream of the Sema-2b protein coding region to construct a Sema-2b reporter (2b^L-τGFP; Figure 3A). The 2b^L-τGFP reporter labels two distinct longitudinal axon tracts, recapitulating the staining pattern for endogenous Sema-2b expression, and the outer of these two GFP⁺ tracts occupies the same lateral position as the 1D4-i connectives (Figures 3E and 3F). Sema-2b, a secreted protein, is most likely released from these 2b^L-τGFP pathways. Therefore, the correct formation of these 2b^L-τGFP longitudinal pathways is likely to be required for normal Sema-2b expression and, perhaps, subsequent fasciculation and organization of the 1D4-i axons. To determine if these Sema-2b–expressing pathways themselves require Sema-2b for their assembly, we first examined the 2b^L-τGFP pathways in the Sema-2b^C4 null mutant. We found that the outer 2b^L-τGFP pathway appeared disorganized in the absence of Sema-2b, while the medial 2b^L-τGFP pathway appeared to remain largely intact (Figure 3G), suggesting that Sema-2b functions in a cell-type autonomous manner to promote the fasciculation of Sema-2b–expressing longitudinal axons in the intermediate region. However, given the difficulty in discerning the integrity of these Sema-2b–expressing 2b^L-τGFP pathways, we used the more selective Sema2b-τMyc (2b-τMyc) reporter. This reporter labels only a subset of the Sema-2b–expressing neurons in the CNS (Rajagopalan et al., 2000), and we observed that these neurons normally express very high levels of Sema-2b (Figures S3A–C). In wild-type embryos, neurons labeled by the 2b-τMyc reporter line extend their axons across the midline along the anterior commissure and then turn anteriorly, subsequently fasciculating with 2b-τMyc axons in the next anterior segment and thereby forming a continuous longitudinal connective (Figure 3H). During neural development, the 2b-τMyc–labeled longitudinal tract is formed prior to the 1D4-i fascicle, which subsequently forms directly adjacent to it (Figure 3I; Figure S3D–L). In Sema-2b^C4 null mutants, the number and cell body position of 2b-τMyc neurons remains unchanged and their axons project normally across the CNS midline, turning anteriorly in their normal lateral position. However, they then often wander off their correct path and fail to fasciculate with 2b-τMyc axons in the next anterior segment, resulting in an aberrant 2b-τMyc longitudinal axon tract (Figure 3J). Some Sema-2b^C4 mutant axons (~0.73 per embryo) exhibit shifting of their anterior projections to a more medial position (“medial detour”), however a greater fraction of misdirected Sema-2b^C4 axons (~2.54 per embryo) shift laterally (“lateral detour”). Some 2b-τMyc axons (~0.61 per embryo) turn and exit laterally from the anterior pathway in Sema-2b^C4 mutant (“lateral exit”) (Figure 3J; see quantification in Figure 5H). These phenotypes are never observed in wild-type embryos. These results suggest that that Sema-2b is required cell-type autonomously for 2b-τMyc longitudinal pathway formation. Importantly, the formation of 2b-τMyc pathway does not depend upon Sema-1a or PlexA (Figures S3M and S3N).

Figure 5

Neuronal PlexB mediates the organization of both CNS longitudinal connectives and ch sensory afferent CNS projections

We next restored Sema-2b expression in the Sema-2b^C4 null mutant using a BAC transgene that covers only the Sema-2b genomic region (however, with the Menl-1/2 genes removed; see Figure S2 for details). This ~60Kb BAC transgene (BAC:Sema-2b) fully rescues the Sema-2b^C4 longitudinal connective defects, including those in both the 2b-τMyc⁺ pathway and the 1D4-i tract (Figures 3K and 3M; quantification in Figure 5H). To assess how secreted Sema-2b promotes the fasciculation and organization of Sema-2b–expressing longitudinal axons and also the 1D4-i tract, we conducted a similar rescue experiment using a modified BAC transgene (BAC:Sema-2b™) that expresses a membrane-tethered Sema-2b otherwise identical to BAC:Sema-2b. The BAC:Sema-2b™ transgene also rescues most of the Sema-2b^C4 null mutant phenotypes seen in both the 2b-τMyc⁺ and the 1D4-i tracts (Figures 3L and 3N; quantification in Figure 5H). We find that a small fraction (~1 axon per embryo) of the 2b-τMyc axons are still diverted laterally in this BAC:Sema-2b™ rescue, however unlike in Sema-2b^C4 null mutant, these pathways often re-join 2b-τMyc axons in the next anterior segment (Figure 3L, empty arrowhead). Therefore, expression of secreted Sema-2b serves to facilitate 2b-τMyc axon fasciculation, and since a transmembrane Sema-2b also can function in this capacity, these results strongly suggest that Sema-2b functions at short-range as an axonally delivered guidance cue, mediating axon-axon recognition and fasciculation during Drosophila embryonic CNS development.

Sema-2a is broadly distributed in the embryonic CNS and unlike Sema2b functions as a repellent to position longitudinal tracts

Using MAb 19C2, which specifically recognizes Sema-2a (Bates and Whitington, 2007), we found that Sema-2a is concentrated along ventral midline structures and commissures during neural development, exhibits lower expression levels toward the lateral regions of the CNS and is diffusely distributed along the region of the CNS longitudinal tracts (Figure 4A). 19C2 staining is absent in Sema-2a^B65 null mutant embryos (Figure 4B). The 2b-τMyc axons cross the CNS midline along the anterior boundary of the anterior commissure and then form their longitudinal connective in a lateral CNS region where relatively lower levels of Sema-2a are found (Figure 4C). In Sema-2a^B65 null mutant embryos, the 2b-τMyc⁺ axons still remain tightly fasciculated with one another and form their characteristic continuous longitudinal pathway. However, some 2b-τMyc axons (~0.67 per embryo) detour medially, sometimes extending to the CNS midline and crossing over to the contralateral side (~0.3 per embryo): phenotypes never observed in wild-type embryos (Figure 4D; quantification in Figure 5H). These inappropriate projections are located in regions where Sema-2a is normally highly expressed in wild-type embryos. We restored Sema-2a expression in the Sema-2a^B65 null mutant using a ~36Kb BAC transgene covering the entire Sema-2a genomic region (BAC:Sema-2a), resulting in full rescue of all CNS defects observed in both the 2b-τMyc pathway and 1D4⁺ tracts (Figures 4E and 4F). Consistent with previous studies (Zlatic et al., 2009), our results suggest that Sema-2a serves as a repulsive cue to constrain axons within select regions of the CNS.

Sema-2a and Sema-2b proteins share 68% amino-acid identity, however our results suggest that they mediate distinct functions. To directly assess differences in how these closely related ligands guide axons, we performed two gain-of-function (GOF) experiments. We first asked whether Sema-2a and Sema-2b mediate distinct functions in CNS longitudinal tract formation. We engineered two different BAC constructs using the same portion of the Sema-2b promoter (Figure 3A), and we expressed either Sema-2a (BAC:2b^L-Sema-2a) or Sema-2b (BAC:2b^L-Sema-2b) in the Sema-2b^C4 genetic background. The BAC:2b^L-Sema2b transgene fully rescued the Sema-2b^C4 mutant phenotypes, (Figures 4G and 4H). In contrast, the BAC:2b^L-Sema2a transgene failed to rescue the guidance defects observed in the Sema-2b^C4 mutants. Interestingly, the BAC:2b^L-Sema2a transgene did result in the appearance of more severe defects in 2b-τMyc pathway. These mostly include individual defasciculated 2b-τMyc⁺ axons that project laterally toward the margins of the CNS (Figure 4I; quantification in Figure 5H). The 1D4-i tract was also severely disrupted and multiple ectopic crossings of the midline by 1D4⁺ axons were observed in ~90% of the segments following Sema-2a expression in the Sema-2b expressing neurons (Figure 4J; 45 out of 50 segments scored), a phenotype never observed in Sema-2b^C4 mutants or the corresponding BAC:2b^L-Sema2b rescue experiments. These results show that Sema-2a and Sema-2b can mediate distinct guidance functions in the same neuronal pathways.

We next asked whether or not Sema-2a and Sema-2b can also mediate distinct guidance functions in other parts of the nervous system. Drosophila embryonic motor pathways labeled by 1D4 show stereotypic projection patterns and innervate distinct peripheral muscles (Figures S4A, S4B, and S4E), (Bate and Broadie, 1995), providing a simple yet robust system to study guidance cue functions (Vactor et al., 1993). Using the 5053A-GAL4 line (Ritzenthaler et al., 2000), transmembrane versions of Sema-2a or Sema-2b were ectopically expressed solely on peripheral muscle-12 in developing embryos (Figure S4C). Sema-2a™ GOF in muscle-12 led to a loss of normal ISNb RP5 innervation in ~50% of hemisegments examined (Figure 4K, empty arrowheads; Figures S4D and S4E), consistent with the known repulsive effects of Sema-2a on embryonic motor neurons (Ayoob et al., 2006; Carrillo et al., 2010; Matthes et al., 1995; Winberg et al., 1998a), while Sema-2b™ GOF in muscle-12 had no affect on ISNb RP5 formation (Figure 4L, arrowheads; Figure S4D). However, Sema-2b™ over-expression in peripheral muscle-12 did have a pronounced effect on the lateral branches of the SNa pathway, which were observed to retain ectopic contact with muscle-12 in ~30% of hemisegments, a phenotype never observed in wild-type embryos (Figure 4L, arrows; Figures S4D and S4E). Over-expression of Sema-2a™ from the same muscle had no effect on SNa motor axons (Figure 4K, arrows; Figure S4D), further demonstrating that Sema-2a and Sema-2b mediate distinct guidance functions.

Taken together, these GOF experiments demonstrate that Sema-2a and Sema-2b function differently in both CNS longitudinal connectives and motor axons: Sema-2b functions to promote axonal attraction, whereas Sema-2a functions as a repellent.

Neuronal cell-type autonomous requirements for PlexB in longitudinal tract and sensory afferent targeting in the CNS

To understand how PlexB mediates secreted semaphorin signaling during CNS development, we first examined its requirement for 2b-τMyc pathway formation. In PlexB^−/− mutant embryos the 2b-τMyc pathway formation is severely disrupted; 2b-τMyc longitudinally projecting axons are often defasciculated, and individual axons are diverted both medially and laterally (Figures 5A and 5H). This phenotype is a combination of both the Sema-2a^−/− and Sema-2b^−/− mutant phenotypes (Figures 5G and 5H). Using the elav-GAL4 driver to express PlexB in all neurons in the PlexB^−/− mutant, we observed full rescue of the 2b-τMyc pathway (Figure 5B) and also full rescue, as previously reported (Ayoob et al., 2006), of the adjacent 1D4-i tract (Figure 5C). These data further support PlexB functioning to integrate both Sema-2a-mediated repulsion and Sema-2b–mediated attraction, resulting in proper organization of select CNS longitudinal tracts.

PlexB is enriched in the intermediate and lateral regions of the CNS scaffold (Figures S5A-C). To determine in which neurons PlexB functions, we next assessed the requirement for PlexB in distinct neuronal populations. In a wild-type background, pan-neuronal overexpression, using the elav-GAL4 driver of a modified PlexB receptor lacking its cytoplasmic domain (PlexB^EcTM) leads to the disorganization of both the 2b-τMyc pathway and the 1D4-i tract (Figure 5D), phenocopying the PlexB^−/− null mutant and showing that PlexB^EcTM functions as a dominant-negative receptor. The MP1 neurons, which can be genetically labeled by the sim-GAL4 driver (Hulsmeier et al., 2007), serve as pioneer axons for the 1D4-i tract (Figures S5D–F) (Hidalgo and Brand, 1997). The MP1 longitudinal pathway resides in the same intermediate region as the 2b-τMyc pathway and lies directly adjacent to it (Figures S5G–S5I). Expressing PlexB^EcTM selectively in these neurons disrupts 1D4-i tract formation; however, the 2b-τMyc pathway remains intact (Figures (Figures5E5E and S5J–L). A similar neuronal cell-type autonomous requirement for PlexB was apparent following PlexB^EcTM over-expression solely in ch neurons under control of the iav-Gal4 driver. We observed disruption of ch axon targeting within the CNS similar to what we observe in PlexB^−/− mutants (Figure 5F) even though CNS longitudinal pathways in iav-GAL4, UAS: PlexB^EcTM embryos remain intact. Importantly, overexpression of full length PlexB using the same iav-GAL4 driver leads to no such phenotype in ch afferent targeting (data not shown). These results indicate that PlexB function is autonomously required in both central and peripheral neurons for correct patterning of their projections within the intermediate domain of the neuropile, presumably through recognition and integration of both Sema-2b attraction and Sema-2a repulsion. By directing the projections of both sensory afferents and CNS interneurons to the same narrow region of the neuropile, PlexB allows for correct synaptic connections and circuit formation between ch axons and their CNS postsynaptic partners.

Ch sensory afferent targeting within the CNS requires secreted semaphorins

To determine how Sema-2a and Sema-2b directly regulate PlexB-mediated CNS targeting of ch sensory afferents, we analyzed ch CNS targeting in Sema-2a^−/−, Sema-2b^−/−, and Sema-2a^−/−, Sema-2b^−/− double null mutant embryos. In Sema-2a^B65 null mutant embryos, ch axon terminals within the CNS still exhibit longitudinally continuous branches along the lateral extent of the 1D4-i tract; in addition, some ch axons display ectopic projections medially (Figures 6A–C, 6J, and 6K; quantification in Figures S6A–F). In the Sema-2b^C4 null mutants, however, ch axons fail to elaborate their characteristic morphology within the CNS, most often terminating in a position that is lateral to the location where the 1D4-i connective normally forms and failing to form a continuous longitudinal branch between segments (Figures 6D–F, 6J, and 6K; quantification in Figures S6A–F). In Sema-2ab^A15 double null mutants, ch axons project within the CNS in a zone that includes the intermediate longitudinal region, however terminal braches are completely disorganized (Figures 6G–K; quantification in Figures S6A–F), exhibiting both ectopic lateral and medial projections as they do in PlexB^−/− mutants (Figures (Figures1H1H and and6J).6J). These results support PlexB–Sema-2b signaling acting to attract extending axons to the intermediate longitudinal region of the neuropile, whereas Sema-2a acts as a repellent; both ligands utilize the same receptor and act in concert to ensure the accurate assembly of sensory afferents with correct CNS connectives (Figure 6L).

Figure 6

Sema-2a and Sema-2b cooperate to ensure correct targeting of ch sensory afferent projections within the CNS

Normal larval responses to vibration require precise ch sensory afferent targeting within the CNS

Our genetic analyses show that PlexB–Sema-2b signaling is critical for correct ch afferent innervation and CNS interneuron projections within the same intermediate region of the embryonic CNS. Termination of sensory afferents and their putative postsynaptic partners within the same narrow region of the neuropile may be necessary for proper synaptic connection and circuit assembly. Therefore, we next asked whether functional consequences result from the neuronal wiring defects we observe following disruption of Sema-2b–PlexB signaling.

Ch neurons in the Drosophila adult have been implicated as mechanosensory transducers for acoustic signals (Eberl, 1999), and also are presumed to be involved in larval propriosensation and mechanosensation (Caldwell et al., 2003). To assess larval ch sensory neuron functions in a high throughput manner, we developed a novel assay for larval vibration sensation. Approximately one hundred larvae were placed on a large agar-filled dish located above a loud speaker. We used the novel Multi-Worm Tracker (MWT) software (sourceforge.net; Swierczek et al., submitted) to automatically deliver vibration stimuli with the speaker while tracking the entire larval population on the dish. Prior to the onset of vibration larvae engage in normal foraging behavior, mostly crawling straight and occasionally making turns. We found that vibration induces a stopping response, followed by head turning (Figures 7A and 7A’; Supplementary Material Movies S1 and S2). Larval head turning in response to vibration is highly reproducible and readily quantifiable using the MWT software (Figures 7B and 7E). This “startle” reaction to mechanical stimuli may allow the larva to sample its environment and change crawling direction following detection of potentially harmful stimuli.

Figure 7

Normal larval responses to vibration require precise ch sensory afferent targeting within the CNS

We found that atonal (ato¹) mutant larvae, which lack ch neurons (Jarman et al., 1993), do not exhibit a normal response to vibration. Upon stimulation, they show a small decrease in crawling speed (data not shown) with no head turning (Figures 7C and 7E). We inhibited synaptic transmission in ch neurons by combining the iav-GAL4 with UAS-TNT (tetanus toxin) and found that iav-TNT larvae, which have inactivated ch neurons, do not show significant increases in head turning in response to vibration as compared to control larvae that express GFP (iav-GFP) in ch neurons (Figures S7A, S7B, and S7D). Therefore, ch neurons are a major class of larval sensory neurons involved in sensing vibration, and their proper synaptic input to the CNS is required for inducing normal head turning behavior in response to vibration.

In Sema-2b^C4 mutant larvae we also observed an abnormal response to vibration. Sema-2b^C4 mutant larvae do reduce their speed significantly in response to vibration (data not shown), however they show no head turning (Figures 7D and 7E), similar to the vibration responses observed in ato¹ mutant larvae. These results suggest that defective larval vibration responses observed in the absence of Sema-2b result from ch neurons being unable to establish appropriate sensory afferent connectivity within the CNS (Figure 6F).

The behavioral deficits observed in Sema-2b^C4 null mutant larvae could also be contributed by requirements for Sema-2b–PlexB signaling in other neuronal populations that are part of the circuitry essential for vibration responses. Therefore, we genetically manipulated PlexB signaling solely in ch neurons. As shown above (Figure 5F), expressing a dominant-negative PlexB receptor selectively in ch neurons (iav-PlexB^EcTM) severely disrupts CNS targeting of ch sensory afferents. We found that the response to vibration in iav-PlexB^EcTM larvae is severely compromised as compared to control larvae that express GFP (iav-GFP) in ch neurons (Figure 7E, see trace in Figure S7C), similar to the head-turning deficit we observe in ato¹, iav-TNT, and Sema-2b^C4 mutant larvae. This indicates that the proper targeting of ch afferent innervation in CNS is indeed important for normal larval vibration behavior. Therefore, PlexB-mediated signaling regulates normal targeting and elaboration both of ch afferent synaptic input and interneuron connective assembly in the same target area, thereby ensuring correct assembly of circuitry involved in processing ch sensory information and generating appropriate responses to vibration.

DISCUSSION

A Semaphorin-plexin code functions together with the Slit-Robo code to assemble CNS neural circuits

The establishment of CNS longitudinal tracts in Drosophila occurs sequentially, from medial to lateral, through a series of distinct guidance events. These include extension of processes that pioneer these trajectories, and subsequent fasciculation and defasciculation events that allow additional processes to join these pathways, cross segment boundaries, and establish connectives that span the rostrocaudal axis of the embryonic CNS (Hidalgo and Booth, 2000). During this process, a repulsive Slit gradient acts over a long range to establish three distinct lateral regions for longitudinally projecting axons, the choice of which is determined by differential expression of Robo receptors (Dickson and Zou, 2010). Once they settle within an appropriate lateral region, individual axons that are part of the same bundle must then adhere to one another and remain fasciculated. We find that Sema-2b signals through PlexB to accomplish this task for longitudinal connectives in the intermediate region.

Interestingly, this Sema-2b–PlexB-mediated organization is inherently connected to Silt-Robo-mediated patterning. The lateral position of intermediate longitudinal processes, including the 2b-τMyc pathway, is initially determined by Robo3-mediated signaling (Evans and Bashaw, 2010; Rajagopalan et al., 2000; Simpson et al., 2000; Spitzweck et al., 2010). Therefore where Sema-2b is expressed reflects lateral positional information derived from the Robo code. Then, this lateral information is further conveyed by the continuous Sema-2b expression over the entire anterior/posterior axis, mediating local organization of both CNS interneurons and sensory afferent projections through the PlexB receptor. When PlexB signaling is disrupted, 2b-τMyc axons still project across the CNS midline and turn rostrally at the appropriate medial-to-lateral position; however, they subsequently wander both medially and laterally, often crossing the medio-lateral regional boundaries set by the Robo code (Figure 5G). Therefore, PlexB-mediated Sema-2b signaling solidifies specific projection positioning originally established by the Robo code. Together, these two distinct Robo and plexin guidance cue signaling modules function in a sequential and complementary fashion to specify both long range medial-to-lateral positioning (Robo) and short-range local fasciculation (PlexB). PlexA, the other Drosophila plexin receptor, and its ligand Sema-1a are specifically required for the proper formation of the 1D4-l pathway (Winberg et al., 1998b; Yu et al., 1998). However, Sema-1a does not show restricted expression within the medio-lateral axis of the nerve cord analogous to that observed for Sema-2b (Yu et al., 1998), suggesting a different mechanism may underlie Sema-1a–PlexA regulation of fasciculation in the most lateral CNS longitudinal region.

Following medio-lateral specification by Slit-Robo signaling and general organization of longitudinal regions by Sema-plexin signaling, additional cues are likely to mediate local interactions among neural processes already restricted to defined regions in the neuropile. Several cell surface proteins may serve such functions; for example, the cell adhesion molecule (CAM) connectin, like Sema-2b, shows exquisitely restricted expression along a subset of longitudinal projections (Nose et al., 1992). More widely expressed CAMs also play important roles in maintaining the fasciculated state of longitudinally projecting processes that are part of the same connective; indeed, in the absence of the Drosophila Ig super family member FasII, axons that contribute to the MP1 pathway show reduced association when examined at high resolution (Lin et al., 1994). Therefore, an ensemble of short-range cues expressed in distinct subsets of longitudinally projecting neurons allows for individual pathways to be established following more global restriction to appropriate locations, and as we demonstrate here, this process is critical for the neural circuit function. It seems likely that similar mechanisms underlie the segregation of complex trajectories, the establishment of laminar organization, and the formation of discrete neural maps in other regions of invertebrate and vertebrate nervous systems (Matsuoka et al., 2011; Sanes and Yamagata, 2009).

Plexin B mediates both Sema-2b attraction and Sema-2a repulsion

Our analyses allow for a comparison between the effects of the secreted semaphorins Sema-2a and Sema-2b on both CNS interneuron trajectories and sensory afferent targeting within the CNS. We observed in both LOF and GOF genetic paradigms that Sema-2a acts as a repellent, consistent with previous observations (Ayoob et al., 2006; Bates and Whitington, 2007; Carrillo et al., 2010; Matthes et al., 1995; Winberg et al., 1998a; Zlatic et al., 2009). Sema-2b, in contrast, serves an opposite guidance function and promotes neurite fasciculation. The highly restricted expression of Sema-2b within the intermediate domain of the nerve cord serves to assemble select longitudinal tracts and ch sensory afferents in this region, strongly suggesting that Sema-2b functions as a local attractive cue to define a specific CNS sub-region and influence the organization of specific circuits.

Though both Sema-2b and Sema-2a signal through the same receptor, PlexB, they appear to do so independently. In the absence of Sema-2a, Sema-2b is still required for fasciculation and organization of the 2b-τMyc and 1D4-i tracks, and also for correct ch afferent innervation in the intermediate region of the nerve cord. In the absence of Sema-2b, Sema-2a expression alone results in potent repellent effects within the CNS for both the 2b-τMyc pathway and ch sensory afferent targeting. The distinct attractive and repulsive functions of Sema-2b and Sema-2a, respectively, are further revealed by the different phenotypes observed in GOF experiments. In the CNS of Sema-2b^−/− mutant embryos, expression of Sema-2a under the control of the Sema-2b promoter results in both 2b-τMyc and 1D4⁺ tract defasciculation much more severe than what is observed in the Sema-2b mutant alone; similar expression of Sema-2b fully rescues the discontinuous and disorganized Sema-2b^−/− longitudinal connective phenotypes. Moreover, membrane-tethered Sema-2b is similarly capable of rescuing the Sema-2b^−/− mutant phenotype, further supporting the idea that Sema-2b is a short-range attractant. In the periphery, misexpression of transmembrane versions of both Sema-2b and Sema-2a in a single body wall muscle demonstrates that Sema-2b™ overexpression results in motor neuron attraction, whereas Sema-2a™ in this same misexpression paradigm functions as a motor axon repellent.

We also show that PlexB is the receptor that mediates both Sema-2a and Sema-2b functions in the intermediate region of the developing nerve cord. Only Sema-2a^−/−, Sema-2b^−/− double null mutants, and not either single mutant, fully recapitulates the PlexB^−/− mutant phenotype, and ligand binding experiments demonstrate that PlexB is the endogenous receptor for both Sema-2a and Sema-2b in the embryonic nerve cord. However, both ligands exert opposing guidance functions despite sharing over 68% amino acid identity and also very similar protein structures (R. Robinson, Z. Wu, A. Kolodkin, and Y. Jones; data not shown). In vertebrates, distinct plexin co-receptors often bias the sign of semaphorin-mediated guidance events (Bellon et al., 2010; reviewed by Mann et al., 2007). We find that the Drosophila ortholog of Off-Track, a transmembrane protein implicated in modulation of vertebrate and invertebrate plexin signaling (Toyofuku et al., 2008; Winberg et al., 2001), apparently does not function in the Drosophila PlexB-mediated guidance events investigated here (data not shown). It will be important to define the relevant differences between the Sema-2a and Sema-2b proteins that are critical for affecting divergent PlexB signaling, and whether or not unique ligand-receptor protein-protein interactions result in differential PlexB activation of signaling cascades with diametrically opposed effects on cytoskeletal components (Hu et al., 2001).

Semaphorin-mediated sensory afferent targeting within the CNS

We find here that Sema-plexin signaling critical for specifying a subset of intermediate longitudinal pathways is also utilized to generate precise mapping of ch sensory input onto CNS neurons. In Drosophila, different classes of sensory axons target to distinct regions of the nerve cord neuropile (Merritt and Whitington, 1995), and the same Robo code essential for positioning CNS axons also regulates the medio-lateral positioning of sensory axons within the CNS (Zlatic et al., 2003; Zlatic et al., 2009). In addition to slit-mediated repulsive effects on sensory afferent targeting, Sema-1a and Sema-2a also restrict the ventrally and medially projecting afferents of the pain sensing Class IV neurons within the most ventral and most medial portions of the nerve cord neuropile (Zlatic et al., 2009). This is reminiscent of recent observations in the mammalian spinal cord showing that a localized source of secreted Sema3e directs proprioceptive sensory input through plexin D1 signaling, ensuring the specificity of sensory-motor circuitry in the spinal cord through repellent signaling (Pecho-Vrieseling et al., 2009). In addition, the transmembrane semaphorins Sema-6C and 6D provide repulsive signals in the dorsal spinal cord that direct appropriate proprioceptive sensory afferent central projections (Yoshida et al., 2006). However, little is known about the identity of cues that serve to promote selective association between sensory afferents and their appropriate central targets in vertebrates or invertebrates. We find that PlexB signaling guides ch sensory terminals to their target region in the CNS through Sema-2b–mediated attraction. Selective disruption of PlexB function in ch neurons severely abolishes normal ch afferent projection in the CNS. Using a novel high-throughput assay for quantifying larval behavioral responses to vibration, we confirm a role for ch sensory neurons in larval mechanosensation (Caldwell et al., 2003). Using this assay we are also able to show that precise ch afferent targeting is required for central processing of vibration sensation and subsequent initiation of appropriate behavioral output. At present we do not know the precise postsynaptic target of ch axons, though our analysis suggests the Sema-2b⁺ neurons are good candidates. Combining vibration response assays with visualization of activated constituents of the ch vibration sensation circuit will allow for a comprehensive determination of input and output following proprioceptive sensation.

Common molecular mechanisms underlying sequential organization of interneuron and sensory afferent CNS connectivity

The formation of a functional circuit relies on the precise assembly of a series of pre- and postsynaptic components. Robo3-mediated signaling is required both for the targeting of ch axons and a subset of the longitudinally projecting interneurons to the same broad intermediate domain of the neuropile (Zlatic et al., 2003). We show here that PlexB-mediated signaling is important for both the assembly of distinct longitudinal projections and also the targeting of ch sensory axon terminal arborizations within the same restricted sub-region of the Robo3-defined intermediate domain of the Drosophila embryonic nerve cord. We find that the secreted semaphorin Sema-2b is a PlexB ligand that plays a central role in both of these guidance events during Drosophila neural development. Sema-2b–PlexB signaling promotes selective fasciculation of the small population of longitudinally projecting axons that express Sema-2b and also immediately adjacent longitudinal projections in the intermediate medio-lateral region of the development CNS. Sema-2b also facilitates targeting of ch afferent terminals that subsequently arrive and establish synaptic contacts in this intermediate region of the developing nerve cord. Sema-2b–PlexB signaling ensures the correct assembly of the circuit that processes ch sensory information, and in its absence larval vibration responses are dramatically compromised. Interestingly, the other PlexB ligand within the CNS, Sema-2a, plays an opposing role to Sema-2b by preventing aberrant targeting through repulsion; together, these two secreted semaphorin ligands act in concert to assure precise neural projection in the developing CNS. Therefore, a combinatorial guidance code utilizes both repulsive and attractive semaphorin cues to mediate the accurate connection of distinct CNS structures and, ultimately, to ensure functional neural circuit assembly.

EXPERIMENTAL PROCEDURES

Generation of transgenes

From large Drosophila BAC clones (RPCI-98: BACPAC resource at CHORI, see clone coordinates in Figure S2A), genomic fragments containing Sema-2b (CG33960, FlyBase) or Sema-2a (CG4700) were retrieved by gap-repair into the attB-P[acman]-ApR vector; constructs were then integrated into engineered attP landing sites in Drosophila (Venken et al., 2006). For Sema-2b promoter (2b^L) BAC transgenes, a ~35Kb genomic fragment upstream from the Sema-2b protein coding sequence was used to drive the expression of τGFP, Sema-2a, or Sema-2b. The Sema-2b coding sequence was subcloned from a cDNA construct (a gift from B. Dickson, Institute of Molecular Biotechnology of the Austrian Academy of Sciences) corresponding to M₄₉-V₇₈₄ in the ACL83134 (GenBank) protein sequence. For membrane-tethered modification of Sema-2a and Sema-2b in pUASt constructs or BAC constructs, the TM-GFP region of the mCD8-GFP protein (Lee and Luo, 1999) was cloned in frame to the C-terminus of these secreted semaphorins. To generate PlexB^EcTM, the entire extracellular and transmembrane regions (1468aa in total) of the Myc-PlexB protein (Ayoob et al., 2006), followed by a stop codon, were subcloned into the pUASt vector.

Genetic analyses

To generate Sema-2a and Sema-2b null alleles, genomic deletions were made using Flippase Recognition Target (FRT) sites (Ryder et al., 2004) to remove Sema-2a (FDD-000938: Sema2a^B65), Sema-2b (FDD-0012943: Sema-2b^C4), or Sema-2a and Sema-2b (FDD-0012939: Sema-2ab^A15) (see deleted regions, Figure S2A). All other mutant stocks have been previously described: plexB^KG00878 (Ayoob et al., 2006), Sema-1a^P1 (Yu et al., 1998), and plexA^Df(4)C3 (Winberg et al., 1998b). Specific GAL4 drivers were used to label and manipulate particular subsets of neurons and their projections, including: iav-GAL4 (gift of C. Montell, Johns Hopkins University) for chordotonal sensory neurons; and sim-GAL4 (Hulsmeier et al., 2007) for MP1 neurons. Other GAL4 drivers used were elav-GAL4 (Yao and White, 1994) and 5053A-GAL4 (Swan et al., 2004). The 2b-τMyc pathway was labeled with the Sema2b-τMyc marker (Rajagopalan et al., 2000). For overexpression studies, the following UAS transgenes were used: UAS:Sema-2a-TM-GFP, UAS:Sema-2b-TM-GFP, and UAS:myc-plexB^EcTM (this work); UAS:myc-plexB (Ayoob et al., 2006), UAS:syt-GFP (Bloomington Stock Center #6926).

Immunohistochemical analyses

Embryo collections and stainings were performed as described (Ayoob et al., 2006; Yu et al., 1998) using the following primary antibodies: anti-Fas II MAb 1D4, (1:4; Vactor et al., 1993), anti-Sema-2a MAb 19C2 (1:4; Winberg et al., 1998a), rabbit polyclonal anti-Sema-2b (1:1000, Sweeney et al., manuscript in preparation), rabbit anti-GFP (1:1000, Molecular Probes), anti-Myc MAb 9E10 (1:1000, Sigma), anti-Myc MAb 71D10 (1:1000, Cell Signaling), and rabbit anti-Tau (1:200, AnaApec). Rabbit anti-PlexB antibody was generated by New England Peptide according to the peptide sequence CRYKNEYDRKKRRADFGD in the extracellular domain of the PlexB protein, custom affinity purified and used at 1:200. HRP-conjugated goat anti-mouse and anti-rabbit IgG/M (1:500, Jackson Immunoresearch), Alexa488 or Alexa546-conjugated goat anti-mouse IgG, and Alexa647-conjugated goat anti-rabbit IgG (1:500, Molecular Probes) were used as secondary antibodies. Embryos at select developmental stages were dissected to reveal the CNS from the dorsal side, and images were acquired as described (Ayoob et al., 2006) or using a Zeiss LSM 510 confocal microscope.

Quantification of the 1D4-i tract defects

To quantify 1D4-i tract defects, the CNS region of dissected embryos was observed from the dorsal side at 40× under bright-field. T2, T3, and A1-8 segments were included for analysis from each embryo. The measure of 1D4-i trajectory disorganization was whether or not two or more 1D4⁺ bundles in the intermediate region of the longitudinal connectives were observed to have a separation of more than one wild type 1D4⁺ bundle width; if so, the hemisegment was scored as disorganized. This determination was made halfway between adjacent ISN nerve roots for each segment scored.

Alkaline phosphatase-binding assays

The binding of alkaline phosphatase (AP)-tagged ligands to Drosophila S2R+ cells, or to dissected embryonic ventral nerve cords, was assessed as described (Ayoob et al, 2006; (Fox and Zinn, 2005). To select live plexB^−/− mutant embryos, the plexB ^KG00878 allele was placed over a fourth chromosome GFP marker (a gift from B. McCabe, Columbia University). Homozygous mutant embryos were then identified by their lack of GFP fluorescence using a Zeiss LUMAR.V12 fluorescent stereoscope. To produce AP-ligands, Sema-2a or Sema-2b cDNAs were cloned into the APtag-5 vector (GenHunter) using NheI and BglII sites. The entire DNA fragment expressing secreted Sema-2a–AP, or Sema-2b–AP fusion protein ligand, was subcloned to the pUASt vector using NotI and XbaI sites. The pUASt constructs were co-transfected with an Act-GAL4 plasmid into S2R+ cells cultured in a serum-free Schneider’s Drosophila medium (1x, Gibco). Four days after transfection, the cell culture supernatants were collected and concentrated. Freshly prepared ligands were used each time, and ligand quality was assessed using western blot. Ligand concentrations were measured by quantifying AP activity, and a concentration of 6nM was used for ligands in all analyses.

Quantification of the ch afferent terminal distribution in the embryonic CNS

To quantify ch afferent distribution within the embryonic CNS, stage 16.5 embryos were stained with 1D4 (for reference coordinates) and anti-GFP to visualize ch terminals expressing UAS:syt-GFP under the iav-GAL4 driver. High-resolution Z-stack pictures were taken using a Zeiss LSM 510 confocal microscope from a dorsal view to generate a series of optical cross-sections. Only hemisegments A2–A4 were scored for quantification (from 4 embryos/genotype for a total of 24 hemisegments/genotype; ~60.5μm optical sections/hemisegment for a total of ~1500 sections/genotype). We avoided the region ~3μm to either side of the ch afferent entry point into the VNC to eliminate excessive background signals from the entering ch nerve bundles and their initial branching within the CNS. At each anterior-posterior position, we used the plot profile function from NIH ImageJ software (Rasband WS, ImageJ, U.S. National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/, 1997–2009) to determine both 1D4 and anti-GFP fluorescent signal distributions along the medial-to-lateral axis in the cross-section. For each optical section, the peak position of the 1D4-m tract signal was used as a reference point (lateral position defined as = 0μm). Then, the lateral GFP signal distributions from all optical cross-sections were averaged to generate a normalized distribution for further analysis. Peak position of the normalized GFP distribution was defined as the lateral position of the highest GFP value in relation to the 1D4-m peak; peak width was measured at half peak height in the plotted distribution curve.

Behavioral tests

Drosophila stocks were constructed using balancers with Tubby or GFP markers to allow selection of live larvae with desired genotypes. The Sema-2b^C4 mutant was prepared in three genetic backgrounds: an isogenic line in w1118 background; an isogenic line with the wild-type white⁺ gene on the X-chromosome in the same w1118 background (W⁺/w1118); and an isogenic line through 10 backcrosses to Canton-S line. In all three genetic backgrounds we observed similar behavioral deficits in vibration responses in mutant larvae as compared to the wild-type. We used the same W⁺/w1118 genetic background for all stocks analyzed in our behavioral paradigms. For vibration response tests, third instar larvae (before the wandering stage) were placed on a flat agar plate surface that permits free movement. Using the novel Multi-Worm Tracker (MWT) and Choreography softwares (sourceforge.net; Swierczek et al. submitted), behavior of the entire larval population on the dish was tracked and analyzed. Vibration stimuli were delivered automatically. A dish with larvae was placed directly above a speaker and eight short (1 sec) pulses and a longer (30 sec) pulse of 1000 Hz, 1V vibration stimuli were applied at close range. The larval head turning response (“kink”) was measured in Choreography, the analysis software that accompanies the MWT, using the absolute angle between the head (20% of skeleton) and the main body axis (remaining 80% of skeleton). This kink angle was quantified and compared between wild-type and mutant larvae to evaluate startle responses upon vibration-stimulation.

Supplementary Material

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ACKNOWLEDGMENTS

We are very grateful to K. Venken and H. Bellen for expert support with BAC transgenic techniques, B. Dickson for the Sema2b-τMyc marker line and Sema-2b cDNA construct, C. Montell for the iav-GAL4 stock, B. McCabe for the fourth chromosome GFP marker, M. Pucak and the NINDS Multi-photon Core Facility at JHMI for confocal imaging, and D. McClellan for her helpful comments on the manuscript. We also thank J. Cho for mapping the UAS:PlexB^EcTM stock, C. Nacopoulos for assistance with fly genetics, and members of the Kolodkin, Luo, and Zlatic laboratories for their helpful discussions throughout the course of this project. We are grateful to N. Swierczek for writing the MWT software, D. Hoffmann for building the behavioral rigs and D. Olbris, R. Svirskas and E. Trautman for their help with behavior data analysis. We also thank the Bloomington Stock Center and the Drosophila Genome Research Center for fly stocks. This work was supported by NIH R01 NS35165 to A.L.K., R01 DC005982 to L.L., and by Janelia Farm HHMI funding to M.Z. and R.K.. R.K. and M.Z. are Fellows at Janelia Farm Howard Hughes Medical Institute; A.L.K. and L.L. are Investigators of the Howard Hughes Medical Institute.

Footnotes

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REFERENCES

Ayoob JC, Terman JR, Kolodkin AL. Drosophila Plexin B is a Sema-2a receptor required for axon guidance. Development. 2006;133:2125–2135. [PubMed]
Bastiani MJ, Harrelson AL, Snow PM, Goodman CS. Expression of fasciclin I and II glycoproteins on subsets of axon pathways during neuronal development in the grasshopper. Cell. 1987;48:745–755. [PubMed]
Bate M, Broadie K. Wiring by fly: the neuromuscular system of the Drosophila embryo. Neuron. 1995;15:513–525. [PubMed]
Bates KE, Whitington PM. Semaphorin 2a secreted by oenocytes signals through plexin B and plexin A to guide sensory axons in the Drosophila embryo. Dev Biol. 2007;302:522–535. [PubMed]
Bellon A, Luchino J, Haigh K, Rougon G, Haigh J, Chauvet S, Mann F. VEGFR2 (KDR/Flk1) signaling mediates axon growth in response to semaphorin 3E in the developing brain. Neuron. 2010;66:205–219. [PubMed]
Caldwell JC, Miller MM, Wing S, Soll DR, Eberl DF. Dynamic analysis of larval locomotion in Drosophila chordotonal organ mutants. Proc Natl Acad Sci U S A. 2003;100:16053–16058. [PubMed]
Carrillo RA, Olsen DP, Yoon KS, Keshishian H. Presynaptic activity and CaMKII modulate retrograde semaphorin signaling and synaptic refinement. Neuron. 2010;68:32–44. [PMC free article] [PubMed]
Dickson BJ, Zou Y. Navigating intermediate targets: the nervous system midline. Cold Spring Harb Perspect Biol. 2010;2:a002055. [PMC free article] [PubMed]
Eberl DF. Feeling the vibes: chordotonal mechanisms in insect hearing. Curr Opin Neurobiol. 1999;9:389–393. [PubMed]
Evans TA, Bashaw GJ. Functional diversity of Robo receptor immunoglobulin domains promotes distinct axon guidance decisions. Curr Biol. 2010;20:567–572. [PubMed]
Farmer WT, Altick AL, Nural HF, Dugan JP, Kidd T, Charron F, Mastick GS. Pioneer longitudinal axons navigate using floor plate and Slit/Robo signals. Development. 2008;135:3643–3653. [PMC free article] [PubMed]
Fox AN, Zinn K. The heparan sulfate proteoglycan syndecan is an in vivo ligand for the Drosophila LAR receptor tyrosine phosphatase. Curr Biol. 2005;15:1701–1711. [PubMed]
Goodman CS, Bastiani MJ, Doe CQ, du Lac S, Helfand SL, Kuwada JY, Thomas JB. Cell recognition during neuronal development. Science. 1984;225:1271–1279. [PubMed]
Grenningloh G, Rehm EJ, Goodman CS. Genetic analysis of growth cone guidance in Drosophila: fasciclin II functions as a neuronal recognition molecule. Cell. 1991;67:45–57. [PubMed]
Harrelson AL, Goodman CS. Growth cone guidance in insects: fasciclin II is a member of the immunoglobulin superfamily. Science. 1988;242:700–708. [PubMed]
Hidalgo A, Booth GE. Glia dictate pioneer axon trajectories in the Drosophila embryonic CNS. Development. 2000;127:393–402. [PubMed]
Hidalgo A, Brand AH. Targeted neuronal ablation: the role of pioneer neurons in guidance and fasciculation in the CNS of Drosophila. Development. 1997;124:3253–3262. [PubMed]
Hiramoto M, Hiromi Y. ROBO directs axon crossing of segmental boundaries by suppressing responsiveness to relocalized Netrin. Nat Neurosci. 2006;9:58–66. [PubMed]
Hiramoto M, Hiromi Y, Giniger E, Hotta Y. The Drosophila Netrin receptor Frazzled guides axons by controlling Netrin distribution. Nature. 2000;406:886–889. [PubMed]
Hu H, Marton TF, Goodman CS. Plexin B mediates axon guidance in Drosophila by simultaneously inhibiting active Rac and enhancing RhoA signaling. Neuron. 2001;32:39–51. [PubMed]
Hulsmeier J, Pielage J, Rickert C, Technau GM, Klambt C, Stork T. Distinct functions of alpha-Spectrin and beta-Spectrin during axonal pathfinding. Development. 2007;134:713–722. [PubMed]
Jarman AP, Grau Y, Jan LY, Jan YN. atonal is a proneural gene that directs chordotonal organ formation in the Drosophila peripheral nervous system. Cell. 1993;73:1307–1321. [PubMed]
Khare N, Fascetti N, DaRocha S, Chiquet-Ehrismann R, Baumgartner S. Expression patterns of two new members of the Semaphorin family in Drosophila suggest early functions during embryogenesis. Mech Dev. 2000;91:393–397. [PubMed]
Kuwada JY. Cell recognition by neuronal growth cones in a simple vertebrate embryo. Science. 1986;233:740–746. [PubMed]
Kwon Y, Shen WL, Shim HS, Montell C. Fine thermotactic discrimination between the optimal and slightly cooler temperatures via a TRPV channel in chordotonal neurons. J Neurosci. 2010;30:10465–10471. [PMC free article] [PubMed]
Landgraf M, Sanchez-Soriano N, Technau GM, Urban J, Prokop A. Charting the Drosophila neuropile: a strategy for the standardised characterisation of genetically amenable neurites. Dev Biol. 2003;260:207–225. [PubMed]
Lee T, Luo L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron. 1999;22:451–461. [PubMed]
Lin DM, Fetter RD, Kopczynski C, Grenningloh G, Goodman CS. Genetic analysis of Fasciclin II in Drosophila: defasciculation, refasciculation, and altered fasciculation. Neuron. 1994;13:1055–1069. [PubMed]
Long H, Sabatier C, Ma L, Plump A, Yuan W, Ornitz DM, Tamada A, Murakami F, Goodman CS, Tessier-Lavigne M. Conserved roles for Slit and Robo proteins in midline commissural axon guidance. Neuron. 2004;42:213–223. [PubMed]
Lopez-Bendito G, Flames N, Ma L, Fouquet C, Di Meglio T, Chedotal A, Tessier-Lavigne M, Marin O. Robo1 and Robo2 cooperate to control the guidance of major axonal tracts in the mammalian forebrain. J Neurosci. 2007;27:3395–3407. [PubMed]
Mann F, Chauvet S, Rougon G. Semaphorins in development and adult brain: Implication for neurological diseases. Prog Neurobiol. 2007;82:57–79. [PubMed]
Mastick GS, Farmer WT, Altick AL, Nural HF, Dugan JP, Kidd T, Charron F. Longitudinal axons are guided by Slit/Robo signals from the floor plate. Cell Adh Migr. 2010;4:337–341. [PMC free article] [PubMed]
Matsuoka RL, Nguyen-Ba-Charvet KT, Parray A, Badea TC, Chedotal A, Kolodkin AL. Transmembrane semaphorin signalling controls laminar stratification in the mammalian retina. Nature. 2011 Doi:10.1038/nature09675. [PMC free article] [PubMed]
Matthes DJ, Sink H, Kolodkin AL, Goodman CS. Semaphorin II can function as a selective inhibitor of specific synaptic arborizations. Cell. 1995;81:631–639. [PubMed]
Merritt DJ, Whitington PM. Central projections of sensory neurons in the Drosophila embryo correlate with sensory modality, soma position, and proneural gene function. J Neurosci. 1995;15:1755–1767. [PubMed]
Nose A, Mahajan VB, Goodman CS. Connectin: a homophilic cell adhesion molecule expressed on a subset of muscles and the motoneurons that innervate them in Drosophila. Cell. 1992;70:553–567. [PubMed]
Pecho-Vrieseling E, Sigrist M, Yoshida Y, Jessell TM, Arber S. Specificity of sensory-motor connections encoded by Sema3e-Plxnd1 recognition. Nature. 2009;459:842–846. [PMC free article] [PubMed]
Rajagopalan S, Vivancos V, Nicolas E, Dickson BJ. Selecting a longitudinal pathway: Robo receptors specify the lateral position of axons in the Drosophila CNS. Cell. 2000;103:1033–1045. [PubMed]
Raper J, Mason C. Cellular strategies of axonal pathfinding. Cold Spring Harb Perspect Biol. 2010;2:a001933. [PMC free article] [PubMed]
Ritzenthaler S, Suzuki E, Chiba A. Postsynaptic filopodia in muscle cells interact with innervating motoneuron axons. Nat Neurosci. 2000;3:1012–1017. [PubMed]
Sanes JR, Yamagata M. Many paths to synaptic specificity. Annu Rev Cell Dev Biol. 2009;25:161–195. [PubMed]
Simpson JH, Bland KS, Fetter RD, Goodman CS. Short-range and long-range guidance by Slit and its Robo receptors: a combinatorial code of Robo receptors controls lateral position. Cell. 2000;103:1019–1032. [PubMed]
Spitzweck B, Brankatschk M, Dickson BJ. Distinct protein domains and expression patterns confer divergent axon guidance functions for Drosophila Robo receptors. Cell. 2010;140:409–420. [PubMed]
Swan LE, Wichmann C, Prange U, Schmid A, Schmidt M, Schwarz T, Ponimaskin E, Madeo F, Vorbruggen G, Sigrist SJ. A glutamate receptor-interacting protein homolog organizes muscle guidance in Drosophila. Genes Dev. 2004;18:223–237. [PubMed]
Tessier-Lavigne M, Goodman CS. The molecular biology of axon guidance. Science. 1996;274:1123–1133. [PubMed]
Toyofuku T, Yoshida J, Sugimoto T, Yamamoto M, Makino N, Takamatsu H, Takegahara N, Suto F, Hori M, Fujisawa H, et al. Repulsive and attractive semaphorins cooperate to direct the navigation of cardiac neural crest cells. Dev Biol. 2008;321:251–262. [PubMed]
Vactor DV, Sink H, Fambrough D, Tsoo R, Goodman CS. Genes that control neuromuscular specificity in Drosophila. Cell. 1993;73:1137–1153. [PubMed]
Venken KJ, He Y, Hoskins RA, Bellen HJ. P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science. 2006;314:1747–1751. [PubMed]
Winberg ML, Mitchell KJ, Goodman CS. Genetic analysis of the mechanisms controlling target selection: complementary and combinatorial functions of netrins, semaphorins, and IgCAMs. Cell. 1998a;93:581–591. [PubMed]
Winberg ML, Noordermeer JN, Tamagnone L, Comoglio PM, Spriggs MK, Tessier-Lavigne M, Goodman CS. Plexin A is a neuronal semaphorin receptor that controls axon guidance. Cell. 1998b;95:903–916. [PubMed]
Winberg ML, Tamagnone L, Bai J, Comoglio PM, Montell D, Goodman CS. The transmembrane protein Off-track associates with Plexins and functions downstream of Semaphorin signaling during axon guidance. Neuron. 2001;32:53–62. [PubMed]
Wolman MA, Regnery AM, Becker T, Becker CG, Halloran MC. Semaphorin3D regulates axon axon interactions by modulating levels of L1 cell adhesion molecule. J Neurosci. 2007;27:9653–9663. [PubMed]
Wolman MA, Sittaramane VK, Essner JJ, Yost HJ, Chandrasekhar A, Halloran MC. Transient axonal glycoprotein-1 (TAG-1) and laminin-alpha1 regulate dynamic growth cone behaviors and initial axon direction in vivo. Neural Dev. 2008;3:6. [PMC free article] [PubMed]
Yao KM, White K. Neural specificity of elav expression: defining a Drosophila promoter for directing expression to the nervous system. J Neurochem. 1994;63:41–51. [PubMed]
Yoshida Y, Han B, Mendelsohn M, Jessell TM. PlexinA1 signaling directs the segregation of proprioceptive sensory axons in the developing spinal cord. Neuron. 2006;52:775–788. [PMC free article] [PubMed]
Yu HH, Araj HH, Ralls SA, Kolodkin AL. The transmembrane Semaphorin Sema I is required in Drosophila for embryonic motor and CNS axon guidance. Neuron. 1998;20:207–220. [PubMed]
Zlatic M, Landgraf M, Bate M. Genetic specification of axonal arbors: atonal regulates robo3 to position terminal branches in the Drosophila nervous system. Neuron. 2003;37:41–51. [PubMed]
Zlatic M, Li F, Strigini M, Grueber W, Bate M. Positional cues in the Drosophila nerve cord: semaphorins pattern the dorso-ventral axis. PLoS Biol. 2009;7:e1000135. [PMC free article] [PubMed]