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Previous Article | Next Article
The Journal of Neuroscience, April 15, 2002, 22(8):3117-3129
Ectopic Expression in the Giant Fiber System of Drosophila Reveals Distinct Roles for Roundabout (Robo), Robo2, and Robo3 in Dendritic Guidance and Synaptic Connectivity
Tanja A. Godenschwege1, Julie H. Simpson2, Xiaoliang Shan1, Greg J. Bashaw2, Corey S. Goodman2, and Rodney K. Murphey1 1 Department of Biology, Morrill Science Center, University of Massachusetts, Amherst, Massachusetts 01003, and 2 Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
The Roundabout (Robo) receptors have been intensively studied for their role in regulating axon guidance in the embryonic nervous system, whereas a role in dendritic guidance has not been explored. In the adult giant fiber system of Drosophila, we have revealed that ectopic Robo expression can regulate the growth and guidance of specific motor neuron dendrites, whereas Robo2 and Robo3 have no effect. We also show that the effect of Robo on dendritic guidance can be suppressed by Commissureless coexpression. Although we confirmed a role for all three Robo receptors in giant fiber axon guidance, the strong axon guidance alterations caused by overexpression of Robo2 or Robo3 have no effect on synaptic connectivity. In contrast, Robo overexpression in the giant fiber seems to directly interfere with synaptic function. We conclude that axon guidance, dendritic guidance, and synaptogenesis are separable processes and that the different Robo family members affect them distinctly.
Key words: axon; dendrites; guidance; giant fiber; Drosophila; roundabout; robo; slit; synapse; commissureless
INTRODUCTION
In the past decade, an array of receptors and ligands have been identified that control growth cone pathfinding and determine axon trajectory (Tessier-Lavigne and Goodman, 1996
). The changes in axon trajectory induced by mutations in pathfinding receptors would be expected to have consequences for synaptic connectivity by simply diverting axons toward or away from their targets. However, whether these receptors have guidance-independent roles in synaptogenesis and the formation of functional synaptic circuitry are not clear. This is attributable in part to technical reasons: it is difficult to record electrophysiologically in the embryo, and it is difficult to separate synaptic effects that are secondary consequences of pathfinding errors from synaptic defects that are attributable to a independent use of receptor-ligand molecules in synaptogenesis. A role for pathfinding receptors in synaptogenesis may also have been previously overlooked because of the focus on their role in axon guidance.
One of the pathfinding receptor families that has been extensively characterized is known as Roundabout (Robo). In Drosophila, the Robo receptors (Robo, Robo2, and Robo3) and their ligand Slit were first identified and characterized for their role in regulating whether axons cross the midline (Seeger et al., 1993
To assess the role of the Robo receptors in regulating circuit formation in the CNS, we used a system amenable to functional studies. The Drosophila giant fiber (GF) system is responsible for a jump-and-flight response to visual stimuli (Tanouye and Wyman, 1980
MATERIALS AND METHODS
Drosophila stocks. All stocks were grown at 22-25°C on standard medium. Two P[GAL]4 lines expressed in the GF system were used. P[GAL4] A307 (Phelan et al., 1996
The shakB(lethal)-Gal4 line, hereafter referred to as shakB-Gal4, was used to drive expression postsynaptically in the giant fiber. shakB(lethal)-Gal4 drives expression in the PSI, the TTMn, and the dorsal longitudinal motor neuron but not in the GF (Jacobs et al., 2000
CC2+
Immunocytochemistry. CNSs of adults and pupas were dissected in 100 mM phosphate buffer (PB) and immediately fixed in 4% paraformaldehyde in PB for at least 30 min at room temperature. Preparations were washed twice in PB, treated with 2N HCl in PBT for 30 min, and further washed four times (10 min each) to remove the acid. After blocking for 2 hr in PAT (100 mM PB, 1% bovine serum albumin, and 0.1% Triton X-100), the tissue was incubated overnight with a rabbit polyclonal anti
-galactosidase (
Physiology and retrograde staining of the TTMn. Intracellular recordings from muscles were obtained from adult flies in a method similar to that described by Tanouye and Wyman (1980)
were driven through the cuticle into the muscle fibers, and intracellular recordings were amplified using a Getting 5A amplifier (Getting Instruments, Iowa City, IA).
Each animal was subjected to two standard tests: response latency and following frequency. For latencies, each fly was given 10 single pulses. Measurements were taken from the beginning of the stimulation artifact to the beginning of the EPSP. For following frequency, each animal was given 10 pulses from a Grass S48 stimulator at 100 Hz. The signals were amplified using a Getting 5A microelectrode amplifier and stored on a personal computer with pClamp software and a DMA interface board (Axon Instruments, Foster City, CA). Analysis was performed on a personal computer using pClamp and Excel 97 software (Microsoft, Redmond, WA).
The recording electrode contained 0.5% neurobiotin, and we injected the TTM muscle iontophoretically at 8-10 sites for 30 min in each specimen. Flies were incubated in a moist chamber for 10 min at room temperature, and the CNS was fixed overnight in 4% paraformaldehyde in PB at 4°C. The motor neuron took up the dye and could be revealed by staining the neurobiotin with the DAB reaction. On many occasions, the TTMn was dye-coupled to the GF axon, and both presynaptic and postsynaptic cells were revealed by staining.
Image capturing and processing. Images in several focal planes were captured from whole-mount CNS preparations using a SPOT digital camera (Diagnostic Instruments Inc., Sterling Heights, MI) and imported into Adobe Photoshop 5.0 software (Adobe Systems Inc., San Jose, CA) on an Apple (Cupertino, CA) Macintosh G3 computer. Montages were then constructed using the "rubber stamp tool" showing axonal projections that cross several planes of focus in the whole-mounted specimen in a single image.
RESULTS
Wild-type expression of Slit and Robo in pupas and adults
The GF and the TTMn are thought to be born during the embryonic wave of neurogenesis (Allen et al., 1998
To determine whether Robo and Slit could be influencing the normal guidance and synaptogenesis of the GF, we used antibodies to determine Slit and Robo expression patterns during various pupal and adult stages during and after GF guidance (Fig. 1). Specific Slit labeling occurs in the midline of the suboesophageal neuromere and in all of the thoracic and abdominal neuromeres presumably on the midline glia (Fig. 1C). Expression of Slit was strongest in early pupas (0-50% of pupal development) and was not detectable in late pupas (after 75%) or in adults. Antibodies to Robo strongly labeled the CNS in a complementary manner; the entire neuropil was labeled with the exception of the midline at all pupal stages (Fig. 1D) and was not detected in adult flies. No specific staining using Robo2 and Robo3 antibodies could be seen in the CNS in pupae or adults, suggesting that Robo2 and Robo3 are expressed weakly or not at all at these stages (data not shown). However, it should be noted that the antibody to Robo2 is very buffer-sensitive and may not work well in the conditions needed to fix pupal tissues.
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Figure 1. Wild-type expression pattern of Robo and Slit in pupae. A, Schematic of the morphology of the GF within the fly CNS. Boxes indicate the regions of the brain and the thoracic ganglion depicted in B-D. B, Control adult (UAS-lacZ/+;A307/+) CNS whole-mount preparation stained for
Overexpression of Commissureless and a Robo dominant negative reveals an endogenous role for Robos in the GF
In the embryonic CNS, Commissureless (Comm) functions to downregulate Robo receptors (Tear et al., 1996
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Figure 2. Coexpression of wild-type Comm rescues Robo2-induced lateral displacement. A, Expression of dominant negative UAS-roboc
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Table 1. Summary of anatomical and physiological phenotypes induced by Robo, Robo2, and Robo3 expression in the GF Ectopic expression of Robo, Robo2, and Robo3 in the GF alters axon trajectory
Overexpression of Robo receptors in the GF reveals that axon trajectory is affected both by the gene being expressed and by the dosage of that particular gene. We used two different p[Gal4] enhancer trap lines, c17 (Trimarchi et al., 1999
When expressed at high levels with the A307 driver, each of the constructs, UAS-robo, UAS-robo2, and UAS-robo3, deflected the GF axon trajectory laterally with 100% penetrance (Fig. 3, Table 1). Expression of Robo causes the giant axons to deflect mildly in the posterior half of first thoracic neuromere (T1) and in the target area in T2 (Fig. 3B). Robo2 expression produces an intermediate phenotype. The axons are strongly deflected in the posterior regions of T1 and T2 (Fig. 3C). Finally, Robo3 is able to deflect the GF axons to the extreme lateral edge of the connective in ~15% of the specimens, and these axons are deflected even further laterally as they approach the target area (Fig. 3D). The shift in trajectory to the lateral edge of the connective was never observed with UAS-robo or UAS-robo2 constructs at any dosage (Fig. 3D, asterisk).
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Figure 3. Overexpression of Robo, Robo2, and Robo3 causes lateral displacement of the GF. A, Control adult (UAS-lacZ/+;A307/+) CNS whole-mount preparation stained for
We measured the lateral position of each GF axon with respect to the midline at a single position just anterior to the axon bend. For this analysis, we divided the CNS into 13 intervals, defining a scale from 0 at the midline to 13 at the lateral edge of the CNS (Fig. 3A, white bar). Control axons grew at a position between 0 and 1 just lateral to the midline (Fig. 3F). To eliminate the possibility that the postsynaptic expression of the A307 driver was affecting the GF trajectory and to clarify the gene dosage effects, we expressed the various constructs with the weaker Gal4 driver c17 and quantified the lateral shift of the GF axons. The penetrance with the c17 driver was less complete than with the A307 driver, but the effect of dosage was clarified (Table 1). For example, two copies of UAS-robo resulted in a greater lateral displacement than one copy of UAS-robo, demonstrating the dosage dependence of the system for Robo.
In contrast, the maximum repulsive output of each receptor is not dependent on dosage. Comparisons between genes confirmed the interactions among dosage, the gene being driven, and the degree of lateralization and eliminated differences in dosage that may exist because of expression levels of each UAS construct. For example, when A307 drives one copy of UAS-robo2, it has a greater repulsive output than two copies of UAS-robo, but when the c17 driver was used with the same UAS constructs, the strength of the repulsive output was reversed (1×UAS-robo2 in comparison with 2×UAS-robo). Our conclusion, based on the work using both GF enhancers, is that axon trajectory is sensitive to both dosage and the gene being expressed. The various qualitative and quantitative data are consistent in ranking the strength of the repulsive output for axons in the context of the GF: Robo < Robo2 < Robo3.
The lateral shifts induced by overexpression of Robo and Robo2 in the GF can be suppressed by co-overexpression of Comm. When UAS-comm was coexpressed with UAS-robo or UAS-robo2, the lateral displacement of the GF axons was rescued in 100% of the cases (Fig. 2C; data only shown for Robo2). In some of these preparations, the GF axons cross the midline in the target area, suggesting that Comm expression can completely block the Robo-induced response to Slit (Fig. 2C, arrow). The specificity of this effect was tested by coexpressing a truncated UAS-comm
In contrast to the axonal aberrations induced by expression of additional Robo, Robo2, and Robo3, the dendritic structure of the GF in the brain appeared normal (data not shown). This finding may not be surprising, because the GF dendrites are located in the tritocerebrum, where no slit expression was found (Fig. 1B,C). As will be seen below, this result for the GF is in direct contrast to the results for the motor neuron.
Induction of the bendless-like phenotype
One of the GF anatomical phenotypes seen with expression of UAS-robo but not UAS-robo2 or UAS-robo3 was a "bendless-like" phenotype observed in approximately one-fourth of the GFs using A307 (Fig. 3E1, Table 1) and was only seen in close approximation of the target area but not in the brain or connective. The phenotype varies, because the GF in individual specimens may have a tapered ending similar to the original bendless (ben) mutant phenotype (Muralidhar and Thomas, 1993
Drosophila Robo has four conserved cytoplasmic motifs that it shares with its homologs in other species (CC0-CC3). The CC2 and CC3 cytoplasmic motifs that bind to Enabled and Abelson are shared by Robos throughout different species but are not present in Robo2 and Robo3 (Bashaw et al., 2000
Presynaptic overexpression of Robo affects synaptic function, but Robo2 and Robo3 do not
We used standard electrophysiological methods (Tanouye and Wyman, 1980
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Figure 4. Physiology of the GF circuit. A, Schematic depiction of the two methods of stimulation as well as the method for recording from the TTM muscle. Brain stimulation was used to activate the GF; thoracic stimulation was used to excite the TTMn directly. B, Schematic of the GF and the TTMn with approximate conduction times. The estimated response latency is shown for brain (0.8 msec) as well as thoracic stimulation (0.6 msec). C, Responses of control (C1) and Robo gain-of-function (C2-C5) flies to brain and thoracic stimulation. Note that c17/+;UAS-robo/+ and A307/+;UAS-robo/+ flies have an increased response latency (1.4 and 1.9 msec) and are not able to follow repetitive stimulation at 100 Hz (C2, C3). Some Robo gain-of-function flies (A307/+;UAS-robo/+) show no response to brain stimulation (C4, asterisk), but thoracic stimulation reveals that the neuromuscular junction of the TTMn is normal (C5).
Overexpression of Robo has a large impact on synaptic connectivity. When UAS-robo constructs are driven by A307, approximately one-fourth of the GFs were completely disconnected from the TTMn, and another one-fourth to one-half of the flies show an increased response latency (Fig. 4, Table 1) and are not able to follow stimuli given with a frequency of 100 Hz (Fig. 4C3, Table 1). The specimens that exhibited no response when the GF was stimulated showed a normal response when the TTMn was stimulated directly, demonstrating that the locus of the defect is the GF
TTMn synapse, not the neuromuscular junction (Fig. 4C4,C5). In contrast, when Robo2 or Robo3 was expressed in the GF, the latencies were normal, and only very subtle defects in following frequency were detected (Table 1). Even with two copies of UAS-robo2, we could detect no physiological effect. Apparently the axon trajectory alterations caused by ectopic expression of Robo2 and Robo3 do not affect synaptic connectivity.
There is a strong correlation between the probability of an absent connection and the ben-like anatomy suggesting that these two are related (Table 1). A single copy of UAS-robo driven by A307 caused 23% functional disconnection and 27% ben-like anatomy. We confirmed this correlation by examining the anatomy in the same specimens that were disconnected physiologically. In >75% of the disconnected specimens, the GF was anatomically ben-like, demonstrating a strong cause and effect between the bendless anatomy and the disconnected physiology. Even the relatively weak expression produced by the c17 enhancer trap line disrupted synaptic transmission and weakened the synapse in approximately one-third of the tested animals. The average response latency was increased, and the synapse was not able to follow stimuli given with a frequency of 100 Hz (Table 1, Fig. 4C2). The extreme low probability of a complete disconnection with c17 can be explained by the low probability of the event and the fact that both GFs are normally connected to both TTMns (Phelan et al., 1996
The response latency of specimens overexpressing UAS-robo
We wondered whether the weak synaptic responses seen in specimens expressing Robo or Robo
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Figure 5. Weak or laterally displaced GF
Robo disrupts the dendrites of the TTMn motorneuron, but Robo2 and Robo3 do not
To investigate the role of the Robo receptors on the postsynaptic neurons, we expressed the various constructs exclusively postsynaptically. We used the shakB-Gal4 line to target expression to the TTMn but not to the GF (Jacobs et al., 2000
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Figure 6. Robo mediates dendritic repulsion of the TTMn, but Robo2 and Robo3 do not. Whole-mount preparations of the thoracic ganglion of late pupas (50-75%) were stained for
Ectopic expression of Robo in the TTMn results in stunted dendrites with a penetrance of 100% (Table 2, Fig. 6B). The medial dendrites do not reach the midline and appear stalled and distorted 20-30 µm lateral to the midline (Fig. 6B, arrows). The lateral dendrite is often absent or abnormal in these animals as well (Fig. 6B, asterisk). Consistent with the distorted anatomy of the medial dendrite, the physiology of the GF
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Table 2. Summary of anatomical and physiological phenotypes induced by Robo, Robo2, and Robo3 expression in the TTMn Expression of UAS-comm or UAS-robo
Because expression of a single copy of UAS-robo in the GF using A307 caused 23% of the flies to show a disconnection phenotype, and A307 is known to express in the motor neurons, the phenotype in these animals may also be attributable to motor neuron expression (Table 1). However, no disconnection was found when a single copy of UAS-robo was expressed strongly and exclusively in the TTMn, suggesting that the weak postsynaptic expression by the A307 driver is not responsible for the disconnection phenotype (Table 2).
The CC2 and CC3 motifs are not responsible for the effect of Robo on dendrites
To test the possibility that the CC2 and CC3 motifs give Robo the ability to affect TTMn dendrite guidance, we expressed UAS-robo
Simultaneous presynaptic and postsynaptic overexpression of Robo and Robo2
To further assess the role of axon pathfinding in the choice of synaptic partners, we examined the synaptic connections made when Robo or Robo2 was expressed on both sides of the synapse, in the TTMn and the GF. We combined a GF enhancer (A307 or c17) and the motor neuron enhancer (shakB-Gal4) to visualize the GF and the TTMn simultaneously (examples are shown only for c17/shakB-Gal4). We dissected pupae in the middle of pupal development (50-75%) when both drivers are active. Despite the fact that the presynaptic and postsynaptic cells are stained the same color in these experiments, the points of contact are unique sites and are readily identified (Fig. 7A,B). These unique contact regions are often enlarged (Fig. 7A, right GF) just as in wild type. In those cases in which we know the contact is completely functional electrophysiologically, such as with robo2 (Table 3, data for A307/shakB-Gal4), these unique contact sites must include the synaptic apparatus. A similar argument applies to the weakened contacts, seen with robo expression; they are unique sites of overlap, although seldom as large as wild type, and functionally they are weaker synapses. If we see no contact, we conclude there is no monosynaptic connection.
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Figure 7. Ectopic GF
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Table 3. Summary of anatomical and physiological phenotypes induced by Robo, Robo2, and Robo3 presynaptic and postsynaptic expression in the GF and the TTMn An unusually informative specimen is shown in Figure 7A. The right side is representative of most specimens, whereas the left side shows one of the numerous variations that occurred at low penetrance. The GF on the right veers laterally as it approaches the target region and contacts the TTMn dendrite in a more lateral position than normal. The region of anatomical contact and overlap between the GF and the TTMn is enlarged, as it would be in wild-type specimens (Fig. 7A, arrow on right). The TTMn dendrite can be seen to extend beyond the contact region and reach the midline (Fig. 7A, arrowhead on right). A remarkable potential of the GF to compensate for misguidance and eventually to find its target was seen on the left side of the same specimen. The left GF first grew laterally (Fig. 7A, white arrows), contacted the TTMn neurite (Fig. 7A, white arrowhead), and grew along it to finally contact the medial dendrite and grow along it from lateral to medial (Fig. 7A, left black arrow).
We could detect two competing effects when Robo was overexpressed presynaptically and postsynaptically: an increase in the number of completely wild-type connections and an increase in the number of completely disconnected synapses. When Robo was expressed only in the motor neuron, not a single fly with a wild-type synaptic connection was found, but coexpression both presynaptically and postsynaptically resulted in 22% wild-type flies (Table 3). This improvement of synaptic connectivity is probably attributable to the lateral deflection of the GF toward the repelled medial TTMn dendrite.
When robo was expressed in the TTMn, we demonstrated that the dendrite never came closer than 20 µm from the midline (Fig. 6B), and yet the GF is usually functionally connected to TTMn, suggesting that the GF must project away from the midline to contact its target. We demonstrated this directly by coexpression of robo both presynaptically and postsynaptically. In one specimen, both GFs extend laterally to reach the displaced dendrite (Fig. 7B). In another specimen, the right GF extends laterally to contact the dendrite, whereas the left GF terminates near the midline and does not contact the TTMn (Fig. 7C, right arrow). When the GF makes contact in these cases, the contacts are considerably smaller than those of controls, consistent with the weaker physiological connection seen when robo was expressed.
Simultaneous presynaptic and postsynaptic overexpression of Robo also disconnected the GF
Distribution of the ectopically expressed Robo-myc and Robo2-myc proteins
To determine whether the differences observed between the UAS-robo-myc and UAS-robo2-myc transgenes were attributable to differential protein distribution, we stained with anti-myc antibody for the ectopically expressed proteins. Three variables influenced the distribution of the ectopic protein: the gene, the neuron examined, and the process (axon or dendrite) examined. The GF axons were stained similarly when the UAS-robo-myc and UAS-robo2-myc were driven by the A307 line. The axons were uniformly labeled, with a slight increase of staining intensity near the synaptic terminals (Fig. 8A2,B2, arrowheads). The GF dendrites were stained weakly (Fig. 8A1, arrow) if at all (Fig. 8B1). Increasing the dosage of Robo2-myc to match the GF somata staining seen with Robo-myc still failed to stain the GF dendrites (Fig. 8B1), suggesting that the Robo2 receptor is being excluded from the GF dendrites. A307 is an enhancer trap line that drives expression also in some unidentified neurons outside the GF, resulting in a high "background" for the Robo-myc but not for Robo2-myc, and unidentified dendrites are clearly stained in the brain with anti-Robo-myc but not with anti-Robo2-myc (Fig. 8A1,B1).
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Figure 8. Protein distribution of Robo and Robo2 in the GF and the TTMn. A1, Localization of anti-myc staining when Robo-myc was expressed under the control of A307 (A307/+;UAS-robo-myc/+). Note the staining in the somata (arrowheads) but only very weakly in dendrites (arrow). A2, Robo-myc localization in the axons of the same specimen as in A1. Note the staining in the presynaptic terminal (arrowheads). A3, Robo-myc localization in the motor neurons at ~50% of pupal development (the genotype is shakB-Gal4/+;UAS-robo-myc/+). Note the very dense label in the dendrites (arrows) and the fact that the medial dendrites never reach the midline (asterisk). The axons (arrowhead) and somata are more weakly labeled. A4, Example of Robo-myc localization in motor neurons at ~70% of development (shakB-Gal4/+;UAS-robo-myc/+). B1, B2, Localization of anti-myc staining when Robo2-myc was expressed by A307 (B1, A307/+;UAS-robo2-myc/+; B2, A307/A307;UAS-robo2-myc/+). The somata (B1, arrowheads) and axons are labeled weakly, and the presynaptic terminal is labeled slightly more strongly (B2, arrowheads), but the dendrites could not be detected. Note the increased background and processes of unknown neurons in A1, A2 in comparison with B1, B2. B3, Localization of Robo2-myc in the TTMn. Note the strongest labeling in the dendrites (arrows) and the lowest in the axons (arrowhead). Although the label is much weaker, the same differential distribution appears to occur for Robo-myc as for Robo2-myc. The genotype is shakB-Gal4/+;UAS-robo2-myc/+. B4, An increase of Robo2-myc protein in the shakB-Gal4/shakB-Gal4;UAS-robo2-myc/UAS-robo2- myc specimen exhibits the same pattern of expression and, although stronger, still does not deflect the lateral TTMn dendrite from the midline. Scale bars, 20 µm.
In the TTMn, the pattern of protein distribution contrasts sharply with the GF, because the dendrites are intensely stained. The overall pattern of protein distribution in the TTMn was similar for Robo-myc and Robo2-myc, but the dendrites consistently stained more intensely than axons or cell bodies (Fig. 8A3,A4,B3,B4). The level of expression in the GF for Robo-myc staining was similar to that for Robo2-myc (Fig. 8A3,B3). Despite the relatively high levels of Robo2-myc protein in TTMn, the dendrites were not repelled from the midline, whereas less Robo2-myc protein was sufficient to deflect the GF axons from the midline (Fig. 8, compare B2, B4). Because the dendritic guidance effects were so different for the two constructs, we wondered whether this was merely a dosage effect. However, an increase of Robo2-myc dosage did not alter the dendritic structure of the TTMn (Fig. 8B3,B4). It is worth noting that it was difficult to increase the strength of staining of Robo2-myc even with the highest possible dosage. Most of the specimens with multiple copies of the UAS-robo-2-myc transgene (shakB-Gal4/shak-Gal4;UAS-robo2-myc/+ or shakB-Gal4/+;UAS-robo2-myc/UAS-robo2-myc) show only slight increases in the strength of staining. This suggests that some other variables, such as degradation rates, are controlling protein expression levels, and we have not achieved control of this unknown factor. In summary, these results support the idea that it is not expression dosage that is responsible for the functional difference between Robo and Robo2 in the regulation of dendritic growth but rather an intrinsic difference between the receptors.
DISCUSSION
The GF system has allowed us to characterize the differences and similarities between the function of the three Robo receptors in Drosophila and to assess the consequences of their expression on the assembly of an identified synapse. The presynaptic GF axon and the postsynaptic TTMn dendrite respond differently to overexpression of the various Robos. In the GF, as in the embryonic CNS (Rajagopalan et al., 2000a
Endogenous function of Robo Receptors in the GF
Considerable evidence suggests that Robo has an endogenous function in guiding the GF axon. First, during pupal development, we could detect the Robo protein throughout the neuropil, and Slit is expressed at the midline. Second, the GF is capable of Slit-mediated repulsion, because overexpression of Robo, Robo2, and Robo3 leads to lateral displacement of the GF. The results are consistent with work in the embryonic CNS in which Robo2 and Robo3 induce a different "final" lateral position than Robo (Rajagopalan et al., 2000a
Role of Robo receptors in dendritic guidance
A dramatic difference between Robo and Robo2 or Robo3 was revealed when each was expressed in the jump motor neuron (TTMn). Robo had a very powerful effect on the TTMn dendrites, repelling them from the midline, whereas Robo2 and Robo3 had no influence whatsoever on the dendritic projection. There is complementary evidence from loss-of-function experiments in the embryonic nervous system that Robo has a function in determining the dendritic projection of some motor neurons. In wild-type specimens, the dendrites of the raw prawn 2 (RP2) neuron do not cross the midline, but in the robo loss-of-function mutant, the dendrites do cross the midline (Wolf and Chiba, 2000
We considered a number of possible explanations for the functional differences among Robo, Robo2, and Robo3 in dendritic guidance. We were able to show that it is not attributable to differential receptor targeting within the neurons, because no difference in the relative distribution between Robo-myc and Robo2-myc was found. In addition, the functional difference cannot be explained by an obvious difference in their cytoplasmic domains; the CC2 and CC3 motifs are present in Robo but not in Robo2 or Robo3, but their removal in the Robo receptor had no affect on dendritic guidance, suggesting that other motifs in the Robo receptors are responsible for the functional difference. Robo2 and Robo3 may be regulated separately from the regulation of Robo by Comm, and two other comm-like genes have been identified in Drosophila (Rajagopalan et al., 2000a
Although Robo apparently does not function normally in the TTMn, we were able to rescue the Robo-induced misguidance of the TTMn dendrite by Comm coexpression. This demonstrates that the ectopic Robo-Comm machinery can function in dendrites and supports the idea that Robo-Comm interaction may be used to guide dendrites in a manner similar to that seen for axons.
Impact of Robo receptors on synaptic connectivity
Our results reveal two relatively independent roles for the Robo receptor during synaptogenesis: (1) an indirect regulation of synapse formation by the influence of Robo receptors on anatomical overlap of the axons and dendrites of the two cells; and (2) a direct disruptive effect by weakening the synapse.
There is a powerful effect of the Robos on synaptic connectivity through their regulation of presynaptic and postsynaptic anatomy. When Robo was expressed exclusively postsynaptically, the synapse was weakened in all specimens. This was correlated with the fact that the TTMn dendrites were always pushed laterally, and the GF connections never appeared anatomically normal. However, simultaneous presynaptic and postsynaptic expression could improve the connection so that 22% of these flies had normal connections. Presumably by pushing the TTMn dendrite and the GF axon laterally, the chances for overlap and strengthening the connection are improved. By regulating the overlap of the axonal and dendritic processes, the Robos control whether the cells are within synaptic grasp of one another, and this provides the outlines of the circuit diagram that will emerge. This may be considered an indirect, although critical, role of the Robo receptors on synaptogenesis.
In addition, Robo appears to have a direct disruptive effect on the GF
Simultaneous presynaptic and postsynaptic expression enhanced the penetrance of the ben-like phenotype and the disconnection phenotype, synergistically demonstrating the involvement of the postsynaptic cell in the expression of this phenotype (Table 3). These findings suggest that the presynaptic and postsynaptic partners have found one another, and pathfinding is complete before the emergence of this severe synaptic defect. Furthermore, because simultaneous presynaptic and postsynaptic overexpression is supposed to compensate for the pathfinding errors, because both GF and its TTMn target are shifted laterally, the increase in the number of totally disconnected neurons is likely to be attributable to a synaptic effect rather than the secondary consequence of a guidance defect.
Our interpretation is that the Robo expression on either side of the synapse interferes with synapse formation, but the presence of Robo on both sides synergistically enhances the disruptive effect of Robo on synapse maturation. These results suggest that possibly the Robo receptor needs to be removed from both growth cones and dendrites for synaptogenesis to proceed normally. A similar idea has been proposed by Wolf et al. (1998)
FOOTNOTES Received Sept. 19, 2001; revised Dec. 7, 2001; accepted Jan. 23, 2002.
This work was supported by National Science Foundation Grant IBN9904957 to R.K.M., National Institutes of Health Grant NS18366, and Christopher Reeve Paralysis Foundation Grant GBC1-9801-2 to C.S.G. J.H.S. is a predoctoral fellow and C.S.G. is an investigator with the Howard Hughes Medical Institute. G.J.B. is a recipient of a Burroughs Wellcome Fund career award in the biomedical sciences. We thank Dr. A. Chiba for providing all UAS-comm fly lines, the Spyros Artavanis-Tsakonas laboratory for providing the anti-Slit antibody, and the J. Bacon and J. Davies laboratories for the shakB(lethal)-Gal4 line.
Correspondence should be addressed to Dr. Rodney K. Murphey, Department of Biology, Morrill Science Center, University of Massachusetts, Amherst, MA 01003. E-mail: rmurphey{at}bio.umass.edu.
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