Dscam1 Forms a Complex with Robo1 and the N-Terminal Fragment of Slit to Promote the Growth of Longitudinal Axons
[['Longitudinal axon guidance is distinguished by long periods of growth independent of intermediate targets .', 'In vertebrates , long distance gradients of Wnt and Shh have been shown to guide longitudinal axons in an anterior-posterior direction [1 , 2] .', 'Longitudinal axons also respond to local cues derived from the central nervous system ( CNS ) midline , notably attractants such as Netrin and repellents such as Slit .', 'The conflicting actions of these cues act to set the dorsal-ventral positions for longitudinal axon pioneers and dopaminergic axons [3–6] .', 'In vitro culture of longitudinal explants with both Netrin and Slit synergistically promotes axon growth [5] , suggesting that the opposing cues not only define accurate lateral positioning but also may promote axon growth .', 'In Drosophila , the highly organized and segmented nature of the nerve cord makes it easy to detect gross defects in longitudinal axon guidance [7] .', 'Mutations in the lola gene specifically disrupt longitudinal formation between segments ( Fig 1F ) .', 'Lola is a transcription factor that regulates the expression of multiple axon guidance genes such as robo1 , slit , and Dscam1 [8 , 9] .', 'Notch and Delta are the only two cell surface genes identified that have highly penetrant longitudinal disruption phenotypes as single mutants [10 , 11] .', 'Notch signaling induces neurons to produce a mesh of projections that link segments , providing a substrate for navigating growth cones [12] .', 'The Netrin receptor Frazzled ( Fra ) is found on this mesh , as well as axons , and traps Netrin diffusing from the CNS midline [13] .', 'Longitudinal pioneer axons grow along the edge of this Netrin-positive region [14] .', 'Netrin therefore has two activities in longitudinal axon guidance as a local contact-dependent cue and as a midline-derived chemoattractant that must be suppressed by other signals to prevent inappropriate midline crossing .', 'The Robo/Slit signaling system plays a key role in defining the lateral position of longitudinal axons in Drosophila , acting as a repellent from the CNS midline [15–18] .', 'The emerging view is that Slit and Netrin signaling balance each other to maintain the lateral position of axons , and this view is supported by mutant analysis in flies and vertebrates [5 , 6 , 14 , 19] .', 'However , in embryos with reduced Robo/Slit signaling in which longitudinal axons inappropriately cross the midline , ectopic slit expression in the longitudinal pathway was sufficient to rescue guidance [14] .', 'Nondirectional slit signals can therefore promote longitudinal trajectories by preventing midline crossing , suggesting that Robo/Slit signaling suppresses the Netrin-induced attraction to the midline [14] .', 'The results of subsequent epistasis experiments examining the behavior of the pCC longitudinal pioneer axon in combinations of fra , robo1 , and slit mutants are consistent with this conclusion [19] .', 'These observations lead to the question of whether this secondary Slit activity is mediated by Robo or alternative receptors .', 'Dscam1 is a high-affinity receptor for Slit with a role in axon branching of adult mechanosensory axons [20] .', 'In the embryo , Dscam1 mutants have strong disruptions to the outermost longitudinal fascicle , and intact fascicles often have a wavy appearance suggesting a role in longitudinal growth and guidance [21] .', 'We find that Dscam1 only binds Slit-N and that binding recruits Robo1 in a complex .', 'A slit mutant that cannot be cleaved ( slit-UC ) [22] retains repulsive activity but displays strong disruption of longitudinal axon guidance , suggesting that Slit-N is required for the progression of longitudinal axons from segment to segment .', 'Several lines of genetic evidence suggest that Slit-N signaling through the Dscam1-Robo1 complex promotes longitudinal axon outgrowth across the segment boundary .'], ['Proteolytic cleavage of Slit does not appear to be required for axon repulsion , as expression of an uncleavable slit transgene rescues slit mutants as effectively as wild-type slit [23] .', 'Subsequent analysis of a slit allele ( slit-UC ) incapable of being cleaved revealed a role for Slit cleavage in muscle migration [22] .', 'We analyzed the effect of the slit-UC allele on CNS formation and found that the axon scaffold superficially resembles that of a robo1 mutant ( Fig 1C and 1D ) .', 'However , while robo1 mutants have a highly regular CNS axon scaffold , slit-UC mutants are variable from segment to segment .', 'The slit-UC longitudinal connectives between segments are often missing or have collapsed into one tract , whereas robo1 mutants almost always have two tracts on either side of the midline [7] .', 'To analyze this phenotype in more detail , we stained with anti-Fasciclin 2 ( Fas2 ) , which labels three parallel longitudinal tracts on either side of the midline .', 'In the slit-UC mutant , all three longitudinal fascicles contribute to form a single thick fascicle that meanders along the midline forming occasional circles of axons ( Fig 1J ) .', 'The outermost fascicle is highly discontinuous and frequently appears to have stalled .', 'This contrasts with the robo1 phenotype in which only the innermost ( pCC/MP1 ) pathway wanders across the midline , while the medial and lateral tracts generally do not , although they can be thinner or show occasional breaks ( Fig 1I ) .', 'The slit-UC allele therefore has a phenotype that is stronger than robo1 mutants alone .', 'The slit-UC Fas2 phenotype resembles strong lola alleles in which the majority of Fas2-positive axons wander back and forth across the midline in a single large fascicle ( Fig 1L ) [8] .', 'As the slit-UC phenotype is more severe than robo1 , we constructed double mutants for both Dscam1 and robo1 to see if any synergy between these Slit receptors was observed .', 'Dscam1 robo1 double mutants have an enhanced robo1 phenotype resembling that of slit-UC because the longitudinal connectives are frequently reduced to one tract ( Fig 1E ) .', 'Unlike slit-UC , which appears variable at all stages of axon outgrowth , Dscam1 robo1 double mutants are relatively regular until stage 16 and then rapidly become more irregular perhaps because the nerve cord often fails to condense ( S1 Fig ) .', 'We stained the longitudinal tracts with anti-Fas2 and found that in Dscam1 robo1 mutants , all the axon tracts are disrupted , with a single thick fascicle wandering across the midline , and the medial and lateral tracts are clearly disrupted , frequently stalled so there is no connection of segments ( Fig 1K ) .', 'This phenotype strongly resembles the slit-UC phenotype .', 'To accurately quantify longitudinal phenotypes , we counted the number of Fas2-positive fascicles at the segment boundaries ( S1 Data ) .', 'We used two different Dscam1 alleles to generate the double mutants , and one combination ( Dscam1P robo4 ) was statistically indistinguishable from slit-UC ( p = 0 . 98 , Tukey honest significant difference [HSD] test , Fig 2 ) .', 'The other combination ( Dscam11 robo4 ) had a weaker phenotype , although it was still significantly stronger than any of the other genotypes tested .', 'This may be due to the nature of the alleles or genetic modifiers on the mutant chromosome .', 'Both Dscam1 and robo1 mutants have breaks in the outermost Fas2-positive fascicle ( Fig 1H and 1I ) , and the double mutant combination had a statistically significant increase in severity of the longitudinal axon guidance phenotype ( p < 0 . 0001 , Tukey HSD test , Fig 2 ) .', 'Mutants in the robo2 and Dscam3 paralogues have disruptions in the longitudinal fascicles ( S2 Fig ) , so we included these genes in the fascicle analysis , finding they make minor contributions ( Fig 2 ) .', 'In summary , Slit cleavage and the Dscam1 and robo1 genes are all required for longitudinal axon guidance , with the high frequency of stalled ( missing ) Fas2-positive tracts suggesting an axon growth defect as well .', 'The longitudinal connectives in the fly embryo are pioneered in part by the pCC interneuron [24 , 25] .', 'To understand what role Slit cleavage might be playing , we examined the behavior of the pCC axon in the slit-UC mutant using anti-Fas2 staining .', 'In early stage 13 slit-UC embryos , the pCC axons can clearly be seen growing at different rates , supporting a role in axon growth ( Fig 3B ) .', 'Cell body positioning in the slit-UC mutant is also irregular , suggesting a role in neuronal migrations .', 'In wild-type embryos , the pCC axon does not cross the midline , projecting ipsilaterally ( Fig 3A ) .', 'In contrast , in robo1 mutants , 96% of pCC axons cross the midline in the first commissure they encounter ( Fig 3C , 3C′ and 3D; S2 Data ) .', 'Surprisingly , given the similarity to robo1 mutants when stained with BP102 , only 30% of pCC axons cross the midline at the first commissure in slit-UC mutants ( Fig 3B′ and 3D; S3 Data ) .', 'This difference is statistically significant ( p < 0 . 0001 , Tukey HSD test ) .', 'The slit-UC allele produces a less stable Slit protein [22] , which may be responsible for the pCC phenotype , but midline repulsion is clearly present .', 'The ascending ( pCC and also vMP2 ) and descending ( MP1 and dMP2 ) pioneers fasciculate to form the first longitudinal tract that spans the segment boundary .', 'In robo1 mutants , the pioneer axons recross the midline on arriving at the next segment ( stage 14 ) , forming the characteristic circular fascicles that give the mutant its name ( roundabout; Fig 3C′′ ) [7] .', 'The same circular fascicles are seen in slit-UC mutants ( Fig 3B′′ ) .', 'We scored the presence of a Fas2-positive midline-crossing fascicle at the posterior of a segment as evidence for pCC crossing the midline and found such a fascicle present in 95% of the segments examined .', 'This frequency is statistically indistinguishable from robo1 mutants ( Fig 3E ) .', 'In summary , in slit-UC mutants most pCC axons do not cross the midline at the first commissure they encounter but do cross at the next commissure ( Fig 3F ) .', 'This phenotype is quite distinct from robo1 mutants ( see the schematic in S3 Fig ) and allows us to experimentally separate midline repulsion from crossing the segment boundary .', 'The phenotype potentially reveals an inability of Slit/Robo signaling to suppress Netrin-based midline attraction , a possibility we test below .', 'Alternative explanations include a decrease in repulsion as the embryo gets older or a need for an increase in growth cone motility to cross the segment boundary as the axon environment grows more complex .', 'As axon guidance receptors can form complexes that alter responses to ligands , we tested whether Dscam1 , Robo1 , and Slit could form a complex in cell culture .', 'The receptors and Slit were expressed in 293 cells , which can proteolytically process Slit to produce Slit-N [26] .', 'Immunoprecipitation of Robo1 can pull down Dscam1 , but only in the presence of Slit , suggesting that Dscam could modify Robo signaling output ( Fig 4A ) .', 'When Robo1 was present alone , Slit-FL was preferentially immunoprecipitated by anti-Robo1 ( Fig 4B ) .', 'However , when plasmids for Robo1 and Dscam1 were present in approximately stoichiometric amounts , Slit-N was preferentially immunoprecipitated , indicating that the Dscam1-Robo1-Slit-N complex forms in preference to Robo1-Slit-FL ( Fig 4B ) .', 'We also tested the ability of Dscam1 to bind Slit isoforms in the absence of Robo1 .', 'We transfected 293 and COS cells with plasmids for full-length Slit and Dscam1 .', 'Immunoprecipitation of Dscam1 pulled down Slit-N , but not Slit-FL from 293 cells ( Fig 4C ) .', 'Our COS cell line cannot proteolytically process Slit , allowing us to test whether Dscam1 will bind to Slit-FL when Slit-N is absent .', 'No evidence for Slit-FL binding was found , strongly suggesting that Dscam1 only binds Slit-N ( Fig 4C ) .', 'Expression of Robo2 increases Slit processing in vivo [27] , but we found no evidence that the presence of Dscam1 influenced cleavage .', 'Because 293 cells process Slit efficiently , the differences in processing may be best visualized in vivo , where processing is more highly regulated .', 'This result provides a molecular explanation for Slit processing and suggests there is an equilibrium between Robo1 homodimers and the Dscam1-Robo1 complexes ( Fig 4I ) .', 'In flies , expression of the Netrin receptor Frazzled lacking a cytoplasmic domain ( FraΔC ) has been used to dissect the function of Fra [28 , 29] .', 'If the role of Dscam1 is simply to inhibit Robo1 , then a Dscam1 dominant-negative construct lacking the cytoplasmic domain ( Dscam1ΔC ) [30] would be expected to mimic expression of a similar robo1 construct ( robo1ΔC ) .', 'Labeling of the ventral nerve cord with anti-Fasciclin 2 ( Fas2 ) reveals three parallel longitudinal tracts on either side of the CNS midline ( Fig 4D ) .', 'Pan-neural expression using the sca-GAL4 driver and a single copy ( 1x ) of Dscam1ΔC is highly disruptive to the longitudinal tracts ( Fig 4E ) .', 'The large number of stalled axons and breaks in the tracts suggests that Dscam1 is playing a role in axon growth .', 'In contrast , a single copy of a robo1ΔC transgene induces mild midline crossing as is seen for weak robo1 mutants , although breaks in the outermost fascicle are also seen ( Fig 4F ) [31] .', 'Surprisingly , when embryos expressing a single copy of Dscam1ΔC ( 1x ) are stained with BP102 to reveal the CNS axon scaffold , it appears more or less like wild type , indicating that the transgenes have a disproportionately strong impact on longitudinal axon guidance .', 'Coexpression of both Dscam1ΔC and robo1ΔC transgenes in single copies did not produce an increase in severity .', 'The contrasting results obtained for robo1ΔC and Dscam1ΔC indicate that Dscam1 is not merely soaking up Slit-N in the embryo ( in which case a robo1-like phenotype should be observed ) but is actively signaling within the longitudinal tracts .', 'Dscam1ΔC is likely inhibiting the activity of other receptors or complexes .', 'Expression of multiple copies of robo1ΔC produces a robo1-like phenotype of increased midline crossing [32] .', 'Increasing the copy number ( 2x ) of Dscam1ΔC and the pan-neural driver results in disruption of both longitudinal and commissural axon guidance , and these connectives are largely missing in affected embryos ( Fig 4H ) .', 'The commissural axon defect confirms our earlier observations that Dscam1 participates in growth towards the CNS midline [33] .', 'We expect Dscam1ΔC to be binding to Slit-N and Robo1 , disrupting the signaling output of the complex .', 'As Robo1 is present in only the longitudinal portions of the CNS axon scaffold [34] , Robo1/Dscam1 complexes are likely participating in longitudinal axon guidance .', 'We previously demonstrated that Slit-N binds with high affinity ( 22 nM ) to the first four immunoglobulin ( Ig ) domains of Dscam1 ( EC4 in Fig 5D ) [20] .', 'We attempted to further map the binding site by constructing an N-terminal deletion series of the Dscam1 gene .', 'In all constructs , the cytoplasmic domain was replaced with a 6xHis tag , and the transmembrane domain was retained ( Fig 5A ) .', 'COS cell expression was confirmed by immunohistochemistry and western blot .', 'Two Slit constructs with an N-terminal myc epitope ( to avoid any steric hindrance at the cleavage site ) , myc-Slit-FL and myc-Slit-N ( Fig 5B ) , produced identical bands of ~123 kD corresponding to Slit-N in National Institutes of Health ( NIH ) 293 cells .', 'In cell overlay studies , Slit-N did not bind to constructs lacking the first five Ig domains or longer ( IgΔ1–5 and IgΔ1–7 constructs , Fig 5C ) , confirming our earlier result that the binding site lies in the N-terminal ectodomain of Dscam1 .', 'Slit-N bound to the full-length Dscam1 ectodomain ( EC-FL ) and to constructs lacking the first ( IgΔ1 ) and first two Ig domains ( IgΔ1–2; Fig 5C ) .', 'This suggested that Slit-N would bind to either Ig3 or Ig4 .', 'However , deletion of either of these domains alone did not prevent binding .', 'We speculated that the binding sites could lie on either side of the variable domains and deleted both Ig1 and Ig4 simultaneously also without disrupting binding .', 'These results strongly suggest that Slit-N has at least two binding sites in the first five Ig domains of Dscam1 .', 'To confirm these results , we utilized a series of constructs in which the Slit ectodomain is deleted from the carboxy-terminal end and the derivatives fused with the Fc region of human IgG ( EC series , Fig 5D ) [35] .', 'We previously demonstrated that EC4 binds to Slit-N fused to alkaline phosphatase and that Slit-N does not bind Fc alone [20] .', 'These constructs are fused to the antibody constant region , allowing immunoprecipitation .', 'Slit proteins were produced using a baculovirus system , and in the insect cell lines used , Slit processing was observed at a low level ( S4 Fig ) .', 'When Slit-FL was tested for binding to the EC constructs , Slit-N was found to bind to all constructs tested , but Slit-FL did not , even though Slit-FL is present in considerable excess ( Fig 5E ) .', 'This implies that there is a Slit binding site in the first three Ig domains of Dscam1 .', 'We confirmed this finding with Slit-N expression alone ( Fig 5F ) .', 'Combined with the results in Fig 3 , this suggests that Dscam1 has a strong preference for binding Slit-N .', 'As a further control , we mapped the binding site of Netrin-B to Ig domains 7 and 8 , as NetB bound to Ec8 but not Ec6 ( Fig 5G ) .', 'This result is consistent with those obtained for vertebrate Dscam and cNetrin-1 , which placed binding in Ig domains 7–9 [36] .', 'In summary , Slit-N binds to at least two sites in the first five Ig domains of Dscam1 , with one site lying in the first three Ig domains ( Fig 5H ) .', 'The preferential binding of Slit-N to Dscam1 led us to identify an assay system in which we could observe differential effects of Slit-FL and Slit-N in vivo .', 'Motor neurons express robo1 [37] and Dscam1 ( S5 Fig ) .', 'Dscam1 mutants show motor neuron innervation defects , suggesting Dscam1 is active in the process .', 'Dscam1 is also seen at a low level at muscle attachment sites ( S5 Fig ) , so Dscam1 may be present on the muscles themselves .', 'Muscles do not normally express slit as they respond to Slit at muscle attachment sites [22 , 38] .', 'Muscle overexpression of UAS-slit-FL in muscles has been previously shown to repel innervating motor neurons [39] .', 'We found that overexpression of UAS-slit-N only repelled 30% of motor neurons innervating the muscle 6/7 cleft , whereas UAS-slit-FL repelled 70% of axons ( Fig 6B and 6C ) .', 'Expression of an uncleavable slit transgene ( UAS-slit-U ) repelled 74% of motor neurons ( Fig 6F ) , the same level as UAS-slit-FL .', 'Removal of Dscam1 activity increased the repulsive effect of UAS-slit-N to a level that is statistically equivalent to that of UAS-slit-FL , but muscle innervation was different from overexpression of UAS-slit-N alone or the failure of innervation seen in Dscam1 homozygotes ( p < 0 . 001 , Tukey HSD test , Fig 6H ) .', 'As muscles do not normally express slit , and Dscam1 may be present on the muscles at a low level , these results must be interpreted with caution .', 'As Dscam1 modulates the response of motor neurons to ectopically expressed Slit-N , it may be acting as a receptor in vivo .', 'However , loss of Dscam1 may allow the expression or activation of an alternative receptor that responds directly to Slit-N .', 'Dscam1-Dscam1 interactions between the motor neuron and muscle could also be mediating a repulsive effect .', 'In the absence of Dscam1 , Slit-N appears to be as repulsive as Slit-FL or Slit-U .', 'Motor neuron growth cones actively integrate both positive and negative cues [40] .', 'Slit-N may have both a repulsive activity and a second activity such as promoting axon outgrowth that may have conflicting effects on motor neurons .', 'Previous genetic analysis of slit suggested that deletion of the epidermal growth factor ( EGF ) domains enhances the repulsive function of slit [41] .', 'Expression of a slit construct lacking the EGF domains also disrupted longitudinal axon guidance , suggesting the EGF domains might be the Dscam1 binding site [41] .', 'We expressed EGF domains 1–3 in cell culture and were able to immunoprecipitate the EGF domains with anti-Dscam1 , but not with anti-Robo1 ( Fig 7A and 7B ) .', 'We confirmed the interaction using a cell overlay assay ( Fig 7C ) .', 'To test the involvement of Robo in Slit-N signaling , a slit construct with the Robo binding site of leucine-rich repeat 2 ( LRR2 ) deleted was generated ( Slit-D ) .', 'Slit-D was found to still be capable of binding Dscam1 in an immunoprecipitation assay ( Fig 7D ) .', 'Interestingly , the bound Slit-D protein is the uncleaved form , indicating that Slit-D can bind to Dscam1 in the absence of proteolytic processing .', 'Slit-D also bound Dscam1 in a cell overlay assay ( Fig 7E ) , confirming that the Dscam1 binding site is distinct from the Robo binding site ( schematic in Fig 7F ) .', 'We expressed slit-D at the CNS midline in a slit mutant , and as expected , slit-D was unable to rescue the collapse of axons onto the CNS midline ( Fig 7G ) .', 'However , pan-neural expression of slit-D disrupted the guidance of all longitudinal tracts and caused the appearance of a large fascicle wandering the midline that was reminiscent of slit-U , Dscam1 robo1 , and lola mutants ( Fig 7H ) .', 'Slit-D may be acting as a dominant-negative binding to Dscam receptors and/or endogenous Slit .', 'We expressed slit-D in muscles to test whether removal of the Robo binding site could unmask a positive activity of Dscam1-Slit signaling .', 'However , overexpression of slit-D actually inhibited innervation at muscle 6/7 to the same extent as Dscam1 mutants , suggesting that Slit-D may act as a dominant negative to inhibit Dscam1 function ( Figs 6H and 7I ) .', 'Similar results were obtained for a construct lacking all the leucine-rich repeat ( LRR ) domains ( ΔL1–L4; Fig 6H ) , which has no effect when expressed in the CNS [41] .', 'Deletion of the Robo binding site therefore appears to inactivate Slit’s biological activity even though Slit-D remains capable of binding Dscam1 , suggesting that in the embryo Dscam1-Slit signaling is Robo dependent .', 'The pCC axon behavior in slit-UC is distinct from that in robo1 mutants .', 'A possible explanation is that cleavage of Slit allows Slit-N to suppress attraction to the CNS midline [14] .', 'If this model is true , then it should be possible to suppress the slit mutant phenotype by expressing slit-N at places other than the midline ( midline expression would not allow us to distinguish between repulsion and suppression of attraction ) .', 'In slit mutants , the longitudinal fascicles are attracted to and collapse onto the CNS midline , with separation between the fascicles limited to occasional very small circles ( Fig 8C ) , similar to those seen in robo1 mutants but much smaller .', 'We used the 24B-GAL4 muscle driver to express slit-N lateral to the CNS , as muscle precursors are present to the sides of the developing CNS ( a subset lie on top of the CNS as well ) .', 'Expression of slit-N in slit mutants induced separation of fascicles such that the fascicles formed small circles ( Fig 8D ) .', 'Quantification of the phenotype revealed it to be statistically significant ( Fig 8E ) .', 'The CNS phenotype of homozygous slit mutants is very strong and challenging to suppress , and most experiments typically use slit/+ heterozygotes to detect effects .', 'Netrin mutants can weakly suppress slit homozygotes [19] , and our results with ectopic Slit-N are similar in strength of suppression .', 'If Dscam1 is acting to suppress attraction to midline Netrins , then NetAB mutants should partially suppress the Dscam1 longitudinal phenotype .', 'NetAB mutants have mature longitudinal tracts that , although largely intact , often show breaks in the outermost fascicle and defasciculation ( Fig 9B ) .', 'When combined with a Dscam1 mutation , the longitudinal tracts show much greater disruption of the outermost fascicles and occasionally the medial fascicle , as well as a lot of axon stalling ( Fig 9C ) .', 'When stained with BP102 to visualize the entire CNS axon scaffold , NetAB mutants display missing longitudinal connectives ( Fig 9E; Table S2 in [42] ) .', 'When stained with BP102 , NetAB; Dscam1 double mutants have dramatic disruptions of the longitudinal connectives and commissures ( Fig 9F ) .', 'The NetAB phenotype is quite variable , and this is reflected in a wide range of phenotypes observed in NetAB; Dscam1 double mutants ( S6 Fig ) .', 'We quantified these defects and found statistically significant difference between NetAB and NetAB; Dscam1 for both commissures ( p < 0 . 0005 , Tukey HSD; S6 Data ) and longitudinal tracts ( p < 0 . 005 , Tukey HSD; S7 Data ) .', 'This result makes it extremely unlikely that Dscam1 is required to suppress attraction to midline-derived Netrins in longitudinal axons .', 'Dscam1 enhances both the midline crossing and longitudinal connective defects of NetAB , and we favor a model in which an axon growth-promoting activity of Dscam1 is responsible ( Fig 10 and below ) .'], ['Pioneer longitudinal axons need to traverse segment boundaries .', 'Previous work has established that crossing the boundary requires a combination of pathway cues and growth cone motility for axons to connect adjacent segments [12] .', 'Longitudinal glia and a mesh of neuronal filopodia provide signals to guide pioneers to and across the segment boundary [12 , 43] .', 'Netrins guide the positioning of the longitudinal glia [44] , are also trapped by the Fra receptor on the neuronal mesh , and are presented to the navigating pioneers during their initial trajectories [13] .', 'In NetAB and fra mutants , the dMP2 descending pioneer axon makes guidance errors , and this may underlie the defects seen in the longitudinal connectives at the end of embryogenesis [45–47] .', 'Our finding that the NetAB; Dscam1 double mutant strongly disrupts longitudinal connectives confirms that these genes play important roles in this process .', 'It is noteworthy that the lola transcription factor regulates expression of fra , as well as robos1-3 , slit , and Dscam1 , potentially explaining why lola has such a profound longitudinal axon phenotype [9] .', 'The significance of the segment boundary as a barrier to axon growth is well established in the homologous grasshopper CNS , in which the growth cones of the ascending and descending longitudinal pioneers pause at the segment boundary for 5 h until selective fasciculation between the pioneers is established .', 'Ablation studies revealed that selective fasciculation is absolutely critical for longitudinal growth [48] .', 'In the fly CNS , the physical distances are significantly smaller , and fasciculation may play a lesser role [25] .', 'For example , filopodia from the pCC pioneer can contact glia on the other side of the segment boundary before the arrival of dMP2/MP1 axons [43] .', 'Mutants in Fas2 that prevent fasciculation of the pioneers do not disrupt pioneer guidance [49] .', 'Nevertheless , the descending MP1 and dMP2 growth cones pause at the segment boundary and wait for the ascending pCC and vMP2 axons to arrive [25] .', 'In addition , mutants in the adhesion molecule N-cadherin ( CadN ) can display breaks in the MP1 pathway that are suggestive of axon stalling [50] .', 'The mature CadN phenotype closely resembles that of Dscam1 mutants , and our observation that NetAB mutants have fasciculation defects suggests that both Dscam1 and NetAB are required in axons that follow the pioneers .', 'It was predicted that longitudinal axon phenotypes might be more subtle in the fly , as the shorter distances would allow guidance defects to self-correct over time through the use of parallel pathways [25] .', 'This may explain why pioneer defects in mutants such as CadN and NetAB only result in subtle defects in outermost fascicle of the mature Fas2 pattern .', 'Detecting strong defects in pioneer axons will likely require knockdown of parallel pathways at the same time , and we are actively testing this hypothesis with Dscam1 .', 'In flies , growth cone motility appears to be another critical requirement for crossing the segment boundary .', 'Temperature-shifted Notch mutants display strong segment crossing defects .', 'Activation of noncanonical Notch signaling in the pioneer axons is sufficient to rescue the mutant defects because of an increase in growth cone motility .', 'Notch signaling promotes the formation of filopodia , which may allow the growth cone to sample growth-promoting cues on the other side of the segment boundary [12] .', 'In vertebrates , the simultaneous and conflicting actions of midline attractants and repellents on longitudinal growth cones have been proposed to drive forward growth parallel to the midline , in the so-called “push-pull” model [5] .', 'Interestingly , part of the chemorepellent activity of Slit is the ability to increase axon outgrowth of cultured axons [5 , 51] .', 'Expression of Slit and Netrin in the spinal cord is continuous [52 , 53] , but the segmented nature of the fly ventral nerve cord means that expression occurs at discrete locations .', 'It may therefore be necessary to increase motility at the segment boundary to ensure crossing .', 'We propose that this is the function of the Dscam1-Slit-N-Robo1 complex , and we discuss this in detail below .', 'It is important to note that alternative mechanisms are also possible .', 'Slit-N bound to axons after diffusing from the midline could be acting as a contact-dependent attractant , either neutralizing Dscam1-mediated homophilic repulsion between axons [54] or promoting attachment between the longitudinal axons and their substrate as has been proposed for Slit-N in muscles [27] .', 'The complex could be trapping Slit-N for presentation to additional receptors as has been proposed for Robo1 and Syndecan [37 , 55] .', 'Slit-N signaling could also modulate fasciculation via regulation of cadherins [56] .', 'While most prior studies on fly Dscam1 have focused on dendrite inhibition , Dscam1 clearly also has a positive role in axon outgrowth , as Dscam1 is required for the growth of presynaptic arbors and overexpression increases the size , branch length , and branching of arbors [57 , 58] .', 'Differences in elongation rates for longitudinal pioneers have been described in robo1 mutants [14] .', 'Our work suggests that Dscam1∆C disrupts axon growth in both commissures and longitudinal tracts ( Fig 4E and 4H ) .', 'The Dscam1 robo1 and slit-UC mutants have significant breaks in the longitudinal fascicles suggestive of stalling at the segment boundary ( Fig 1J and 1K ) , and examination of the pCC growth cones in the slit-UC mutant reveals different rates of outgrowth ( Fig 3B ) .', 'Pan-neural expression of the slit-D construct also causes longitudinal breaks ( Fig 7H ) .', 'Finally , lateral expression of Slit-N causes a modest promotion of growth away from the midline in slit mutants ( Fig 8 ) .', 'Taken together , these results suggest that Dscam1 , Robo1 , and Slit-N all play a positive role in axon outgrowth , especially in longitudinal axons .', 'Lack of a growth-promoting signal in slit-UC or Dscam1 robo1 mutants would prevent pioneer axons from crossing the segment boundary .', 'In previous studies , the longitudinal pioneers have been observed to stall , exit the CNS , or grow in the opposite direction when guidance is disrupted [12 , 13 , 48] .', 'The slit-UC allele used in our work encodes a protein with decreased stability [22] , so it is likely to be acting as a hypomorphic allele with reduced repulsive function .', 'The reduction in repulsion in slit-UC or Dscam1 robo1 mutants allows axons that do not stall to inappropriately enter the midline , resulting in a large central fascicle that wanders back and forth across the midline ( Fig 1J and 1K ) .', 'A priority for us is to construct additional slit-UC alleles with hopefully greater protein stability through CRISPR-Cas9 based approaches .', 'This may allow clearer visualization of axon growth phenotypes .', 'Our interpretation of the role of the Dscam1-Robo1-Slit-N complex relies heavily on prior studies of Dscam1 and limited observations of growth rate differen
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['The Slit protein is a major midline repellent for central nervous system ( CNS ) axons .', 'In vivo , Slit is proteolytically cleaved into N- and C-terminal fragments , but the biological significance of this is unknown .', 'Analysis in the Drosophila ventral nerve cord of a slit allele ( slit-UC ) that cannot be cleaved revealed that midline repulsion is still present but longitudinal axon guidance is disrupted , particularly across segment boundaries .', 'Double mutants for the Slit receptors Dscam1 and robo1 strongly resemble the slit-UC phenotype , suggesting they cooperate in longitudinal axon guidance , and through biochemical approaches , we found that Dscam1 and Robo1 form a complex dependent on Slit-N .', 'In contrast , Robo1 binding alone shows a preference for full-length Slit , whereas Dscam1 only binds Slit-N .', 'Using a variety of transgenes , we demonstrated that Dscam1 appears to modify the output of Robo/Slit complexes so that signaling is no longer repulsive .', 'Our data suggest that the complex is promoting longitudinal axon growth across the segment boundary .', 'The ability of Dscam1 to modify the output of other receptors in a ligand-dependent fashion may be a general principle for Dscam proteins .']
['Most complex nervous systems have an anterior-posterior axis of symmetry: the midline of the central nervous system .', 'Longitudinal nerves , formed by bundled axons , connect the brain to other parts of the body and grow long distances parallel to the central nervous system midline .', 'In the fruit fly , the nerve cord is segmented , and the boundary of each segment represents a challenge for the longitudinal axons to cross .', 'What cues promote growth of axons across these boundaries remains unknown .', 'The Slit protein is classically known as a repulsive signal that prevents axons from crossing the midline by binding to its receptor Robo on the surface of axons .', 'However , it is known that Slit is cleaved into two fragments , but the significance of this process remains to be elucidated .', 'In this study , we find that cleavage of Slit allows the large fragment ( Slit-N ) to bind to another receptor called Dscam1 , which then forms a complex with Robo1 .', 'Our experiments confirm that Slit processing is required for the longitudinal growth of axons across the segment boundary .', 'Our work suggests that the Slit-N-Dscam1-Robo1 complex signals axons to grow , but Slit-N is not a specific navigational cue .', 'Dscam proteins have evolutionarily conserved ligand-binding activities; therefore , they may be able to alter the signaling output of other receptors .', 'The Slit fragments play diverse roles , from tumor metastasis to HIV replication; thus , identifying a receptor that is specific for Slit-N provides a molecular explanation for why cleavage occurs .']
['medicine and health sciences', 'nervous system', 'neuroscience', 'developmental biology', 'immunoprecipitation', 'nerve fibers', 'embryos', 'research and analysis methods', 'chemoattractant axon guidance', 'embryology', 'developmental neuroscience', 'animal cells', 'axons', 'precipitation techniques', 'pioneer axons', 'cellular neuroscience', 'cell biology', 'axon guidance', 'anatomy', 'central nervous system', 'phenotypes', 'neurons', 'genetics', 'biology and life sciences', 'cellular types']
During the development of the insect central nervous system, cleavage of the secreted signaling protein Slit allows the formation of a complex with the receptors Dscam1 and Robo1 that is required for longitudinal axon growth across body segment boundaries.