A conserved role for Syntaxin-1 in pre- and post-commissural midline axonal guidance in fly, chick, and mouse
[['Axonal growth and guidance are responsible for the correct formation of neural circuits .', 'These processes rely on the tightly regulated response of the growth cone to both diffusible and membrane-bound guidance cues .', 'In response to such cues , several intracellular signaling cascades are activated within the growth cone , leading to directional growth .', 'For instance , the chemoattractant Netrin-1 binds to the receptor Deleted in Colorectal Cancer ( DCC ) at growth cones , activating several kinases and small GTPases , cyclic nucleotides , and calcium cascades , as well as cytoskeletal rearrangements [1–8] .', 'In contrast , few reports have addressed the cross-talk mechanisms between axonal guidance and membrane dynamics and turnover , other than the fact that growth cones are filled by vesicles and express most SNARE ( Soluble NSF Attachment Protein REceptor ) and exocyst proteins [9–13] .', 'A growing number of reports using in vitro approaches indicate that axon guidance mechanisms require the participation of SNARE-mediated exocytosis for chemoattraction and endocytosis for repulsion [14–18] .', 'Thus , it has been demonstrated that the vSNARE ( vesicular SNARE ) VAMP2 is required for L1-mediated chemoattraction [19] and for Sema3A-induced chemorepulsion [17] , that the vSNARE Ti-VAMP and the tSNARE ( target SNARE ) SNAP25 are necessary for neurite outgrowth [20–22] , and that Syntaxin-1 ( STX1 ) is required for Netrin-1-mediated attraction of axons and migrating neurons [15 , 16] .', 'However , the participation of these proteins in neural circuit formation in vivo is still controversial .', 'For instance , mice deficient for the SNAP25 and VAMP2 proteins show virtually no neural circuitry defects but do present a severe alteration of evoked synaptic activity [23–25] .', 'Ti-VAMP-deficient mice display behavioral defects but no alterations in gross brain morphology [26] .', 'STX1A knock-out ( KO ) mice show only mild cognitive defects and a normal brain structure [27] and axonal defects have not been described in STX1B KO [28] .', 'In a previous study we showed that STX1A is required for the navigation of dorsal commissural in the chick spinal cord [16] .', 'Syntaxin-1 loss-of-function in Drosophila and chick embryos results in motor axonal defects [29] .', 'Drosophila melanogaster displays neural expression of a synaptobrevin ( VAMP ) gene , namely n-synaptobrevin ( n-syb ) [30] and a SNAP25 homolog , Snap25 [31] .', 'Mutations in these components of the core SNARE complex give rise to neurotransmitter release phenotypes [32] .', 'A single D . melanogaster STX1 homolog , Syntaxin1A ( Syx1A ) , has been identified that shows strong homology to rodent STX1A [33–35] .', 'Mutations in this gene are homozygous lethal , with severe alleles resulting in early embryonic death .', 'In D . melanogaster , loss of Syx1A abolishes synaptic transmission [33 , 36] , and other secretion phenotypes , such as soft cuticle and undigested yolk , have also been reported [33] .', 'In addition , Syx1A is involved in cell membrane formation during cellularization [37] and in the condensation of the embryonic CNS [33] .', 'In addition , Syx1A has been reported to affect the properties of neuronal membranes and influence membrane dynamics throughout development [38] .', 'However , in D . melanogaster , in contrast to vertebrates , Syx1A has yet to be directly implicated in axonal guidance .', 'Here we systematically inactivated SNARE genes in three species , Drosophila melanogaster , chick , and mouse , to determine the role of SNARE proteins in CNS midline axonal guidance in vivo .', 'We show the involvement of this protein complex , in particular STX1 , in D . melanogaster and chicken axonal guidance , reporting aberrant phenotypes .', 'Furthermore , we provide the first description of abnormal midline axonal defects in mouse embryos double mutant for STX1A and STX1B .', 'Our results point to an evolutionarily conserved mechanism of SNARE complex proteins in midline axonal guidance .'], ['To determine whether Syx1A affects axon guidance at the midline of D . melanogaster embryos , we started by analyzing the CNS phenotypes of Syx1A mutant embryos .', 'For this purpose , we used zygotic mutants , since Syx1A has a maternal contribution and its function is required for proper cellularization [37] .', 'In D . melanogaster , CNS axons of wild-type ( wt ) embryos are found in a stereotypic ladder-like arrangement .', 'Within each neuromere , two commissures link the two halves of the nervous system .', 'Individual neuromeres are connected by axons running in discrete fascicles in the lateral connectives .', 'An antiserum that specifically recognizes D . melanogaster Syx1A revealed that , like Frazzled ( Fra , the D . melanogaster DCC homolog ) [39] , Syx1A is expressed in developing axons in the embryo from stage 13 .', 'At later stages , this isoform is expressed at high levels on commissural and longitudinal axons in the developing CNS ( Fig 1A ) .', 'Embryos of the null Syx1AΔ229 genotype [33] displayed no detectable Syx1A protein in the ventral nerve cord ( VNC ) when stained with the same antibody ( Fig 1B ) .', 'To analyze axonal midline phenotypes , we stained embryos with HRP , which marks all axons in the CNS , and anti-Fasciclin II antibodies ( anti-FasII ) , which label lateral fascicles ( Fig 1C ) .', 'At stage 15 or later , FasII identifies three major axonal tracts , which are visible as parallel straight lines: the medial ( FasII-m or 1st ) , intermediate ( FasII-i or 2nd ) and lateral ( FasII-l or 3rd ) fascicle .', 'In wt conditions , these FasII-positive axons do not cross the midline .', 'Examination of the commissural and longitudinal pathways labelled by HRP and anti-FasII in the VNC of Syx1A mutants revealed specific defects in diverse axonal pathways .', 'From stage 14 onwards , by analyzing FasII-positive axons , we detected guidance defects in the VNC of 50% of the embryos ( n = 38 ) .', 'These defects included aberrant midline crossing , as well as abnormal arrangement of ipsilateral fascicles ( Fig 1D , 1E and 1F ) .', 'Of these embryos , 18% displayed strong defects ( Fig 1D and 1F ) and 32% weak defects ( Fig 1E and 1F , see Materials and Methods for quantification methods and definition of weak and strong phenotypes ) .', 'FasII-positive fibers never crossed the midline in wt conditions .', 'In contrast , Syx1A embryos displayed clear abnormal crossing of FasII-positive axons , thereby suggesting that longitudinal fibers aberrantly cross the midline in Syx1A mutants ( Fig 1G ) .', 'When HRP staining ( a pan-axonal marker ) was used , control embryos presented regularly spaced commissures ( Fig 1C and 1H ) .', 'However , embryos with the strong Syx1A phenotype showed collapsed commissures with no clear separation between anterior and posterior ones ( Fig 1D and 1H ) .', 'We analyzed and quantified commissural phenotypes in the VNC of Syx1A embryos displaying phenotypes ( weak and strong phenotypes , 50% of embryos ) .', 'Collapsed commissures ( 98% Fig 1H , arrows , and 1I ) and “fuzzy” commissures were detected in many segments ( 36% , Fig 1H , asterisk , and 1I ) , as well as thinning of longitudinal fascicles between segments ( 57% , Fig 1H , arrowhead , and 1I ) .', 'On average , Syx1A mutant embryos displayed more than one defect in VNC axonal guidance per embryo ( Fig 1G ) .', 'Among these defects , the strongest were axonal midline crosses ( Fig 1D , short arrow , 13% ) , commissural thinning ( Fig 1H and 1I , 55% ) , and thinner longitudinal fascicles ( Fig 1H and 1I , 32% ) , which were not detected in controls .', 'The most penetrant phenotypes were fasciculation defects ( Fig 1E , long arrow , 58% ) , but Syx1A mutant embryos also showed fascicle collapse ( Fig 1D and 1E , arrowhead , 42% ) .', 'Variability among these axonal phenotypes between individuals possibly reflects the differential contribution of the maternally deposited gene product .', 'These results show that loss of Syx1A function induces axonal guidance defects in both commissural and longitudinal axons at embryonic stages of fly CNS development .', 'We next used the same approach to study whether genetic loss of additional components of the SNARE core complex ( SNAP25 , VAMP2 and Ti-VAMP/VAMP7 ) also alters the commissural and longitudinal pathways in D . melanogaster .', 'To do so , we examined mutants for nSyb ( human VAMP2 ortholog ) [40] and Snap25 [41] , and analyzed the D . melanogaster ortholog of Ti-VAMP/VAMP7 ( Vamp7 source:Flybase ) .', 'Overall , the nSyb , Snap-25 , and Vamp7 phenotypes were weaker than the Syx1A one ( Fig 2A–2D ) .', 'When analyzing FasII positive axons for fasciculation defects , the strongest axonal guidance phenotypes were found in Snap25 mutants , in which 74% embryos displayed fascicle collapse , defasciculation , or both ( Fig 2B and 2F , n = 35 ) .', '57% of nSyb mutants showed fascicle collapse , defasciculation , or both ( Fig 2C and 2F , n = 41 ) .', 'In addition , a small percentage of nSyb embryos ( 4% , n = 41 ) showed axonal midline crosses of FasII positive axons ( Fig 2C , arrow ) .', 'Vamp7 mutants exhibited the weakest phenotypes ( Fig 2D and 2E n = 31 ) .', 'We could detect stronger phenotypes when all axons were visualized with BP102 antibody .', 'Again , the strongest phenotypes were detected in SNAP25 mutant embryos ( Fig 2H and 2K ) where 6 . 9% of embryonic segments showed fuzzy commissures and 27 . 5% showed thinning of longitudinal fibers ( n = 80 segments ) .', 'nSyb mutants displayed intermediate phenotypes , fuzzy commissures in only 3 . 1% of segments and thinning of longitudinal fibers in 10 . 6% ( n = 80 ) ( Fig 2I and 2K ) .', 'In Vamp7 embryos we could only detect thinning of commissures ( in 5% of embryonic segments , n = 80 ) ( Fig 2J and 2K ) .', 'These results indicate that Vamp7 , nSyb , and Snap25 can also influence D . melanogaster axonal guidance at the midline , but to a lesser extent than Syx1A .', 'Next , we used in ovo electroporation of double-stranded RNAs derived from STX1A , SNAP25 , VAMP2 , and Ti-VAMP to study the role of these genes in dI1 commissural axon guidance in the chicken spinal cord .', 'In untreated controls and in EGFP-expressing control embryos , most commissural axons followed a stereotypic trajectory ( Fig 3A and 3B ) .', 'The vast majority of the dI1 axons crossed the floor plate and turned rostrally along the contralateral floor-plate border .', 'See Methods for details on the quantification method .', 'In contrast , we found that the down-regulation of all SNARE-complex proteins ( STX1A , SNAP25 , VAMP2 , and Ti-VAMP ) generated defects in commissural axon guidance , as axons either failed to enter or to cross the floor plate , or failed to turn into the longitudinal axis along the contralateral floor-plate border ( Fig 3C–3G ) .', 'After silencing STX1A aberrant axon pathfinding was found at 36% of the DiI injection sites per embryo ( Fig 3C and 3G ) .', 'We found overall similar percentages of injection sites with aberrant phenotypes when SNAP25 , VAMP2 or Ti-VAMP were down-regulated ( Fig 3G ) .', 'When electroporated at embryonic day 3 ( E3/HH17-18 ) most axons reached the floor plate and entered the midline area in all groups ( Fig 3H ) .', 'A detailed analysis of the aberrant phenotypes indicated a significant increase in floor-plate stalling in embryos where STX1A , VAMP2 , or Ti-VAMP was silenced ( Fig 3I ) .', 'Similarly , turning of post-crossing axons was affected in all experimental groups ( Fig 3J ) .', 'In a separate series of experiments , we compared electroporation of dsSTX1A at HH13/14 ( E2 ) with electroporation at HH17/18 ( E3 ) ( Fig 3K ) .', 'These experiments revealed an effect of timing of gene silencing on the severity of the phenotype .', 'Electroporation at E3 , when axons are starting to grow in the dorsal spinal cord , resulted in normal axon guidance at 56 . 4% of all DiI injection sites per embryo .', 'In contrast , electroporation of neural precursors at E2 resulted in normal axon navigation only at 23 . 8% of all DiI injection sites .', 'Importantly , failure of some axons to enter the floor plate was observed in 3/8 embryos electroporated early but was never observed in the embryos electroporated late .', 'These data support the notion that the silencing of SNARE proteins in the chicken spinal cord leads to various commissural axon guidance defects .', 'To study the involvement of STX1 in commissural axon guidance in mammals , we generated double KO mice for the two STX1 isoforms , STX1A and STX1B .', 'Double KO embryos died just after birth and displayed strong motor abnormalities , thereby suggesting severe alterations in nervous system organization .', 'To address axonal phenotypes in the midline , we examined the commissural pathway in the spinal cord in E12 embryos [42 , 43] .', 'In wt embryos , commissural axons stained with TAG-1 antibodies were organized as a narrow axonal bundle extending from the dorsal spinal cord towards the floor plate without invading the motorneuron area ( Fig 4A and 4B ) .', 'In contrast , in STX1A/B ( -/- ) mice , TAG-1-positive commissural axons were still directed towards the ventrally located floor plate , although their organization differed from that of controls .', 'Instead of forming a narrow bundle , axons in STX1A/B ( -/- ) mice were clearly defasciculated , invading the entire mantle zone .', 'Individual fibers or bundles invading lateral motorneuron areas were frequently observed ( Fig 4C–4F ) .', 'However , axons appeared to reach the floor plate .', 'To support these observations , we quantified the width of the commissural pathway at three dorso-ventral levels ( Fig 4C–4G ) .', 'At each one , we found a significant difference in the width of commissural axon bundles between wt and double STX1 KO mice .', 'To confirm these findings , we stained spinal cord sections with Robo3 antibodies , a marker of pre-crossing and post-crossing commissural fibers .', 'In wt embryos , Robo3-stained fibers formed a tight fascicle directed towards the floor plate .', 'Moreover , post-commissural fibers in the ventral spinal cord were heavily stained ( S2A and S2B Fig ) .', 'In contrast , commissural fibers in STX1A/B ( -/- ) embryos exhibited wider ipsilateral fascicles , often invading the lateral ( motorneuron ) domains in the ventral spinal cord .', 'Many ipsilateral commissural axons in mutant embryos were tipped with growth cones indicating that they failed to reach the floor plate .', 'Consistent with this observation , the bundles of post-crossing commissural axons ( located in the ventral spinal cord ) were markedly decreased in STX1A/B null-mutant embryos ( S2C and S2D Fig ) .', 'Taken together , our observations indicate that the lack of STX1 results in aberrant commissural axon growth on both ipsilateral and contralateral sides .', 'To confirm the above observations , we also examined commissural axon navigation in open-book preparations of E12 spinal cords ( Fig 5 ) .', 'Comparable to our observation in the chicken spinal cord , we found aberrant navigation of dI1 axons at the floor plate .', 'In the double STX1 KO embryos , almost no axons were found to cross the midline and to turn properly along the contralateral floor-plate border ( Fig 5D and 5E ) .', 'Axons mainly failed to reach the contralateral border of the floor plate .', 'In embryos lacking STX1A but expressing STX1B from one or two wt alleles , axon guidance was still compromised , exhibiting intermediate phenotypes ( Fig 5C , 5F and 5G ) .', 'A quantitative analysis showed a pronounced decrease in normal axonal trajectories in mutant compared to wt embryos ( Fig 5F ) .', 'In wild-type mice we found normal axon trajectories at 75 . 6±6 . 3% of the DiI injection sites per embryo .', 'In contrast , in mice lacking STX1A but expressing one or two wild-type allele ( s ) of STX1B normal trajectories were only seen at 10 . 2±5 . 4% or 21 . 7±12 . 3% of the DiI injection sites , respectively .', 'In double KO mutants axon navigation was affected even more strongly , as normal trajectories were only seen at 1% of the injection sites ( Fig 5F and 5G ) .', 'A detailed analysis of the different guidance defects revealed a problem in floor-plate stalling in all mutants in addition to a failure to turn rostrally along the contralateral floor-plate border .', 'Failure to enter the floor plate was only found in mutants but never in wild-type mice ( Fig 5G ) .', 'Embryos having at least one wt allele of either STX1A or STX1B exhibited weaker axonal defects than the double KO mice ( Fig 5G ) .', 'To confirm the expression of SNARE proteins in spinal cord commissural axons we performed immunocytochemical analyses in neuronal cultures .', 'Dissociated mouse commissural neurons were identified with two antibody markers: DCC and Robo3 antibodies .', 'Cultures were co-immunostained for different SNARE proteins including STX1A , VAMP2 , SNAP25 and Ti-VAMP .', 'Confocal images revealed that commissural growth cones , identified by the expression of DCC and Robo3 , co-expressed all the studied SNARE proteins ( Fig 6 ) .', 'We also observed variable degrees of colocalization between DCC/Robo3 receptors and SNARE proteins .', 'These results indicate that embryonic commissural axons express the SNARE proteins analyzed in the present study .', 'We next addressed whether STX1A is required for Slit-2 and Netrin-1 actions .', 'Tissue explants of chick dorsal commissural neurons dissected from control embryos and grown on laminin are known to extend neurites readily in the absence of Slit-2 ( Fig 7A ) .', 'In the presence of Slit-2 , neurite length was strongly reduced ( Fig 7B ) .', 'In contrast , neurite growth from explants taken from embryos electroporated with dsRNA derived from STX1A did not differ in the absence or presence of Slit-2 ( Fig 7C , 7D and 7I ) , indicating that the absence of STX1A results in a markedly reduced Slit-2 response of chick commissural axons .', 'We have previously shown that the blockade of STX1 with Botulinum Toxin C1 reduces Netrin-1 induced chemoattraction in mouse spinal cord explants [16] .', 'To confirm this finding , we co-cultured in collagen gels dorsal spinal cord explants from STX1 KO embryos with Netrin-1 expressing cell aggregates .', 'In comparison with explants cultured with control cells ( exhibiting radial growth , Fig 7E ) , wild-type explants confronted to aggregates of Netrin-1 expressing cells showed strong chemoattraction ( Fig 7F and 7J ) .', 'In contrast , Netrin-1 induced chemoattraction was absent in spinal cord explants derived from double STX1A/B KO embryos ( Fig 7G , 7H and 7J ) .', 'Our previous studies have shown that exposition to Netrin-1 increases DCC surface expression in growth cones and that such increase is not diminished after blockade of STX1 [15 , 16] .', 'Here we performed experiments to determine whether STX1 regulates Robo3 surface expression in the presence of Netrin-1 .', 'Mouse embryonic commissural neurons were cultured and stained for the differential immunolabeling of surface and intracellular Robo3 protein pools ( S3 Fig ) .', 'The data show that in control commissural growth cones , Robo3 trafficking and distribution is not substantially altered by Netrin-1 incubation .', 'In contrast , STX1A/B knock-out growth cones incubated with Netrin-1 exhibited a decrease in surface Robo3 signals ( S3D and S3E Fig ) .', 'These findings suggest that the inactivation of STX1 alters Robo3 trafficking and/or surface expression , probably by increasing Robo3 internalization .', 'During our analysis of midline guidance defects in Syx1A mutant embryos , we detected that the arrangement of the longitudinal fascicles was shifted towards the CNS midline compared to controls ( Fig 8A and 8B; n = 20 ) .', 'In order to clarify whether the FasII–positive axonal fascicles were closer to the midline in Syx1A mutants , we quantified these distances at embryonic stage 16 and compared them to wt , frazzled ( fra ) and robo2 mutants ( Fig 8C–8E ) .', 'Overall , Syx1A and robo2 mutations induce a shift of FasII-positive fibres ( both FasII-m and FasII-i ) towards the midline .', 'Regarding both FasII-m and FasII-I , the opposite occurs in fra mutant embryos , thereby suggesting that Syx1A interferes with a midline repression pathway ( Fig 8A–8E ) [44] .', 'In order to address whether Syx1A is involved in the Slit/Robo pathway , we generated double mutants for Syx1A and robo2 and examined the positioning of the longitudinal tracts in robo2;Syx1A embryos .', 'As shown in Fig 8F–8J , the longitudinal fascicle distance to the midline decreased in the double mutant embryos ( Fig 8I and 8P ) , where the strongest phenotypes showed a complete collapse of the tracts in the midline ( Fig 8I , asterisk ) , as reported for slit ( sli ) mutants [45] .', 'This result indicates that Syx1A and robo2 interact genetically .', 'In molecular terms , our above results suggest that Syx1A is involved in axonal repulsion in the midline .', 'In vertebrates , it has been reported that Netrin1/DCC axonal guidance is coupled to exocytosis through STX1 [15] .', 'Therefore , we examined whether differences in Syx1A levels could affect the Drosophila fra axonal guidance phenotypes .', 'We found that the fra mutant phenotype was significantly aggravated by removing one copy of Syx1A ( Fig 8K–8O ) , thereby suggesting that Syx1A levels severely interfere with the Netrin/Fra guidance pathway .'], ['Here we used loss-of-function models in three species ( fly , chick and mouse embryos ) to examine the role of SNARE proteins in midline crossing of commissural axons .', 'We report aberrant axonal phenotypes in the D . melanogaster midline and in the chicken spinal cord .', 'Furthermore , we generated double mutant STX1A/B mice that show abnormal commissural phenotypes in the murine spinal cord .', 'These findings indicate an evolutionarily conserved role of SNARE complex proteins in midline axonal guidance .', 'The guidance of commissural axons at the midline of the spinal cord is a complex process regulated by many molecules .', 'For instance , the interaction between Netrin-1 and DCC attracts pre-crossing axons toward the floor plate [42 , 43] .', 'Axon growth towards the floor plate is also altered in mice lacking VEGF/Flk1 signaling [46] .', 'Midline crossing was shown to depend on attractive ( Axonin-1/TAG1 with NrCAM; [47] ) and repulsive interactions ( Slit/Robo; [48]; Sema3B/NrCAM/PlexinA1/Neuropilin; [49] [50] [51 , 52] ) .', 'The rostral turn of post-crossing commissural axons was shown to depend on morphogen gradients formed by Wnts [53 , 54] and Shh [55 , 56] , although the latter is also involved in attraction of commissural axons toward the ventral midline in parallel to Netrin-1 [57 , 58] .', 'In addition , an interaction between axonal Semaphorin6B and its ligand PlexinA2 [59] and interactions between the SynCAMs [60 , 61] are also involved in the turning of post-crossing commissural axons into the longitudinal axis .', 'Our results in D . melanogaster indicate that the SNARE complex is involved in midline axonal guidance .', 'We show that Ti-Vamp/Vamp7 , nSyb , and Snap25 influence axonal guidance at the midline , but to a lesser extent than Syx1A .', 'In general , these phenotypes may be incomplete Drosophila KOs , because in this fly there is a maternal contribution for all these components .', 'Additionally , it is known that there is a high level of redundancy in D . melanogaster .', 'In this regard , it has been reported that Syb can replace nSyb and Snap24 can replace Snap25 [40 , 41] .', 'Regarding the Syx1A phenotypes , we can conclude that Syx1A loss of function induces commissural and ipsilateral axonal phenotypes .', 'Interestingly , some of the phenotypes observed in Syx1A mutants resemble the loss of function phenotypes of the Robo/Slit pathway [62 , 63] .', 'We detected midline crosses of FasII-positive axons and a strong genetic interaction with robo2 .', 'These findings suggest that Syx1A is involved in repulsive midline guidance in Drosophila .', 'In addition , most of the ipsilateral phenotypes detected are also common in frazzled ( fra , the D . melanogaster homolog of DCC ) , and fra phenotypes are aggravated by decreased Syx1A levels .', 'Furthermore , similar ipsilateral phenotypes to Syx1A are observed in netrin mutant ventral nerve cords [44] , as well as in embryos mutant for Heparan Sulfate Proteoglycans ( HSPGs ) [64] and Hh [65] , thereby suggesting that Syx1A may participate in various guidance pathways in D . melanogaster not just in axonal guidance but also in fasciculation/defasciculation events .', 'Recent studies suggest that whereas axonal attraction requires exocytosis , chemorepulsion relies on endocytosis [14 , 18 , 66 , 67] .', 'In addition , SNARE proteins have been reported to be involved in both exo- and endocytosis [68] .', 'Our genetic interaction experiments support the notion that Syx1A participates in both Netrin/Fra attraction and Slit/Robo repulsion .', 'The data obtained in chicken reinforce the idea that the silencing of SNARE proteins induces an overall increase in various commissural guidance defects , with the silencing of STX1A leading to defects in all the guidance steps analyzed .', 'Silencing STX1A at E2 , on day before neurons start to extend axons rather than E3 , just before they start to extend axons , also resulted in axons failing to enter the midline area .', 'This phenotype resembled previously reported findings on the role of STX1A in DCC-mediated guidance of pre-crossing commissural axons towards the midline [16] .', 'Based on these results , we conclude that SNARE proteins make a crucial contribution to the navigation of chick commissural axons at the floor plate .', 'SNARE proteins are involved in both attractive [16] and repulsive ( this study ) decision-making steps in axon guidance , as STX1A , VAMP2 and Ti-VAMP silencing leads to a substantial increase in pre-crossing and post-crossing phenotypes .', 'These phenotypes are in agreement with those reported in previous studies [15 , 16] , confirming the involvement of STX1 in the regulation of chemoattractive guidance pathways for commissural axons .', 'The phenotypes seen in the chicken spinal cord resemble those observed after silencing Calsyntenin-1 and RabGDI in dI1 neurons [69] .', 'In the absence of Calsyntenin-1 and RabGDI , Robo1 is not transported to the growth cone surface , resulting in axonal stalling in the floor plate .', 'Furthermore , silencing Calsyntenin-1 , but not RabGDI , prevents the expression of the Wnt receptor Frizzled-3 on the growth cone surface , leading to failure of post-crossing axons to turn rostrally in response to the Wnt gradient [54] .', 'These findings are consistent with the analyses of commissural axon navigation at the midline in STX1 KO mice .', 'In double mutants , the absence of STX1A and STX1B proteins prevented midline crossing .', 'In Netrin-1 [70 , 71] and DCC KO [44 , 70] mice , pre-commissural fibers display strong phenotypes , including defasciculation , aberrant trajectories , and a complete failure to cross the floor plate .', 'Weaker phenotypes have been observed in mouse mutants for other genes involved in commissural axonal guidance , including Sonic Hedgehog and the VEGF/FlK1 pathways [46 , 57] .', 'The phenotype described here in STX1A/B mutant mice is reminiscent of , that in Netrin-1 and DCC KO mice , with fewer axons reaching the floor plate , lateral invasion of motorneuron territories , and fewer post-crossing commissural axons , although the phenotype is much weaker and many fibers still reach the ipsilateral floor-plate border .', 'A likely explanation could be that although STX1A/B might be required for correct sensing of Netrin1/DCC guidance [15 , 16] , compensatory mechanisms may derive from the possible lack of STX1A/B requirement in other commissural attractive pathways ( e . g . , SHH or VEGF/Flk1 ) .', 'Our findings indicate that STX1A/B not only affects pre-crossing axons but strongly affects midline crossing and post-commissural axonal guidance .', 'This conclusion is supported by our findings in Drosophila , which revealed a genetic interaction between Syx1A and the Robo pathway , as well as by the present in vitro experiments showing that STX1 is required for both Netrin-1 attraction and Slit-2 repulsion ( Fig 7 ) .', 'Our previous studies showed that the lack of STX1 did not affect DCC surface expression induced by Netrin-1 [15] .', 'In the present study we show that the lack of STX1 results in a decreased surface expression of the Robo3 receptor in response to Netrin-1 .', 'It is therefore possible that the lack of STX1 may alter the surface expression of other Robo family members ( eg Robo1 ) and Frizzled-3 , the receptors used by post-crossing axons .', 'This idea is supported by our STX1 loss-of-function data , showing that the lack of this SNARE protein abolished the responsiveness of post-crossing commissural axons to Slit , both in vitro ( explant experiments ) and in vivo ( chick and mouse analyses ) .', 'We thus propose that , in contrast to DCC expression , the effect of STX1 loss-of-function on post-crossing axons may be explained in part by preventing the surface expression of axon guidance receptors required for midline crossing and post-crossing navigation .', 'Robo3 is an atypical Robo receptor that has been proposed recently to potentiate DCC-mediated attraction to Netrin-1 , but without binding Slits [72 , 73] .', 'Thus , the observation that Netrin-1 in STX1-deficient growth cones results in decreased Robo3 surface expression , supports the notion of a possible contribution of Robo3 membrane downregulation to explain the reduced Netrin-1 chemoattraction found in STX1 loss-of-function models .', 'Many guidance molecules and their receptors involved in axonal guidance are conserved between vertebrates and invertebrates ( e . g . Robo , Hh , and Netrins ) [57 , 65 , 74] .', 'Our systematic analysis of the phenotypes at the CNS midline of fly , chick , and mouse embryos mutant for STX1 unveils an evolutionarily conserved role for STX1 in midline axonal guidance .', 'Overall , the ipsilateral phenotypes reported are consistent with the participation of STX1 in Netrin-1-dependent axonal guidance , as proposed previously [15 , 16] .', 'In addition , here we describe post-commissural phenotypes that are reminiscent of those found in Robo , NrCAM and VEGF loss-of-function models [46 , 47 , 75 , 76] , thereby suggesting that STX1 underlies various signaling pathways .', 'Furthermore , the phenotypes described herein for other SNARE proteins point to the participation of SNAP25 , VAMP2 and Ti-VAMP in midline axonal guidance , although the precise implication and relevance of individual SNARE proteins to specific axonal guidance signaling complexes remains to be determined .', 'We propose that the coupling of the guidance receptor cell machinery to proteins that regulate exocytosis is a general and conserved mechanism linking chemotropic signaling to membrane trafficking [29] .'], ['The following stocks are described in FlyBase ( http://flybase . org ) : Syx1AΔ229 , SNAP-25 ( Df ( 3L ) 1-16 ) , nSybd02894 , Ti-VAMP ( P[CG1599G7738] ) , fra3 and robo2 .', 'All alleles used are genetic nulls .', 'Wild-type control is yw .', 'D . melanogaster stocks and crosses were kept under standard conditions at 25ºC .', 'D . melanogaster embryos were staged as described by Campos-Ortega and Hartenstein [77] and stained following standard protocols .', 'For immunostaining , embryos were fixed in 4% formaldehyde for 20 min .', 'We used antibodies that recognize FasII ( mAb1D4 , DSHB ) , βGal ( Promega ) , Affinity-Purified Anti-HRP TRITC ( Jackson immunoResearch ) , and mAbBP102 ( DSHB ) .', 'We used biotinylated HRP ( Amersham ) or non-biotinylated HRP ( GE Healthcare ) , Alexa488 , Alexa-555 and Alexa-647 , Cy2 , Cy3 and Cy5 conjugated secondary antibodies ( Jackson ImmunoResearch ) .', 'For HRP histochemistry , the signal was amplified using the Vectastain-ABC kit ( Vector Laboratories ) when required .', 'In addition , the signal for the DAB reaction was intensified with NiCl2 , except for double stainings , where it was omitted from one of the reactions .', 'DIC photographs were taken using a Nikon Eclipse 80i microscope .', 'Fluorescent images were obtained with a confocal microscope ( Leica TCS-SPE-AOBS and TCS-SP2-AOBS systems ) and processed using Fiji [78] and Adobe Photoshop .', 'Images are maximum proje
['Introduction', 'Results', 'Discussion', 'Materials and methods']
['Axonal growth and guidance rely on correct growth cone responses to guidance cues .', 'Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics , little is known about the crosstalk mechanisms between guidance and membrane dynamics and turnover .', 'Recent studies indicate that whereas axonal attraction requires exocytosis , chemorepulsion relies on endocytosis .', 'Indeed , our own studies have shown that Netrin-1/Deleted in Colorectal Cancer ( DCC ) signaling triggers exocytosis through the SNARE Syntaxin-1 ( STX1 ) .', 'However , limited in vivo evidence is available about the role of SNARE proteins in axonal guidance .', 'To address this issue , here we systematically deleted SNARE genes in three species .', 'We show that loss-of-function of STX1 results in pre- and post-commissural axonal guidance defects in the midline of fly , chick , and mouse embryos .', 'Inactivation of VAMP2 , Ti-VAMP , and SNAP25 led to additional abnormalities in axonal guidance .', 'We also confirmed that STX1 loss-of-function results in reduced sensitivity of commissural axons to Slit-2 and Netrin-1 .', 'Finally , genetic interaction studies in Drosophila show that STX1 interacts with both the Netrin-1/DCC and Robo/Slit pathways .', 'Our data provide evidence of an evolutionarily conserved role of STX1 and SNARE proteins in midline axonal guidance in vivo , by regulating both pre- and post-commissural guidance mechanisms .']
['Syntaxin-1 is a core factor in tethering synaptic vesicles and mediating their fusion to the cell membrane at the synapse .', 'Thus , Syntaxin-1 mediates neurotransmission in the adult nervous system .', 'Here we show that this protein is also involved in axonal guidance in the CNS of vertebrates and invertebrates during the development of the nervous system: our systematic analysis of the phenotypes in the nervous system midline of fly , chick , and mouse embryos mutant for Syntaxin-1 unveils an evolutionarily conserved role for this protein in midline axonal guidance .', 'Further , we also dissect the contribution of other proteins regulating neuronal exocytosis in axonal development .', 'We propose that the coupling of the guidance molecule machinery to proteins that regulate exocytosis is a general mechanism linking chemotropism to axonal growth .']
['invertebrates', 'medicine and health sciences', 'nervous system', 'neuroscience', 'animals', 'animal models', 'developmental biology', 'drosophila melanogaster', 'model organisms', 'experimental organism systems', 'nerve fibers', 'embryos', 'drosophila', 'research and analysis methods', 'chemoattractant axon guidance', 'embryology', 'developmental neuroscience', 'spinal cord', 'animal cells', 'axons', 'insects', 'arthropoda', 'cellular neuroscience', 'eukaryota', 'neuroanatomy', 'cell biology', 'axon guidance', 'anatomy', 'phenotypes', 'neurons', 'genetics', 'biology and life sciences', 'cellular types', 'organisms']