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. 2018 Jun 18;14(6):e1007432.
doi: 10.1371/journal.pgen.1007432. eCollection 2018 Jun.

A conserved role for Syntaxin-1 in pre- and post-commissural midline axonal guidance in fly, chick, and mouse

Oriol Ros  1   2 , Pablo José Barrecheguren  1   3 , Tiziana Cotrufo  1   2 , Martina Schaettin  4 , Cristina Roselló-Busquets  1   2 , Alba Vílchez-Acosta  1   2 , Marc Hernaiz-Llorens  1   2 , Ramón Martínez-Marmol  1   2 , Fausto Ulloa  1   2 , Esther T Stoeckli  4 , Sofia J Araújo  3   5   6 , Eduardo Soriano  1   2   7   8
Affiliations

A conserved role for Syntaxin-1 in pre- and post-commissural midline axonal guidance in fly, chick, and mouse

Oriol Ros et al. PLoS Genet. .

Abstract

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Axonal guidance defects found in the VNC of Syx1A mutant embryos.
(A, B) Stage 16 embryos were stained with anti-Syx1A to detect Syntaxin 1 localization and with HRP to visualize the axon scaffold. A, Syx1A is detected in all axonal tracts, both commissural and longitudinal. B, Syx1A is not detected at these stages in Syx1AΔ229 mutant embryos. (C-E) Stage 16 embryos were stained with the anti-FasII mAb 1D4 to mark all FasII-positive axons and with HRP to visualize the axon scaffold. C, wt embryo showing the FasII-positive longitudinal connectives and the anterior and posterior commissures. D, Syx1AΔ229 mutant embryo representative of the strongest CNS phenotypes encountered; many commissures and longitudinal connectives are collapsed (arrowhead) and midline crosses (arrow) are observed. E, Syx1AΔ229 mutant embryo representative of the milder CNS phenotypes encountered; commissures are generally unaffected and defects are detected only in the longitudinal connectives, such as defasciculation (long arrow) and collapse (arrowhead). Anterior is up in all panels. (F) Quantification of the number of axon guidance defects encountered in Syx1AΔ229 mutants in comparison with the background defects in the wt. (G) Quantification of the diverse axonal guidance defects encountered in FasII positive axons in Syx1AΔ229 mutants in comparison with the background defects in the wt. (H) Higher magnification of Syx1AΔ229 mutant embryos representative of the wt and the strongest CNS phenotypes encountered by staining all axons with HRP; most commissures (98%) are thinner than in the wt; many segments (36%) show “fuzzy” commissures with a clear lack of separation between anterior commissure (AC) and posterior commissure (PC) (arrows and asterisk); longitudinal connectives are thinner between segments (arrowheads; 57% of cases) n = 120 segments; anterior is up. (I) Quantification of the axonal guidance defects encountered in all axons of Syx1AΔ229 mutants in comparison with the background defects in the wt when all axons are stained using anti-HRP antibody.
Fig 2
Fig 2. Diverse axonal pathway defects encountered in the VNC of n-syb, snap-25, and Ti-VAMP mutant embryos.
(A-D) Stage 16 embryos were stained with anti-FasII to mark all FasII-positive axons and to better observe longitudinal axonal pathway defects. A, wt embryo showing the three FasII-positive longitudinal connectives. B, SNAP-25 mutant embryos, representative of defasciculation (arrow) and fascicle collapse (arrowhead) phenotypes. C, nSybd02894 mutant embryos, representative of midline crosses (arrow) and defasciculation phenotypes (arrowhead). D, Vamp7 mutant embryos showing mild defasciculation phenotypes (arrow). (E) Quantification of the number of axon guidance defects encountered in nSyb, SNAP-25, and Vamp7/Ti-VAMP mutants in comparison with the background defects in the wt.(F) Quantification of the diverse axonal guidance defects encountered in n-syb, Snap-25, and Vamp7/Ti-VAMP mutants in comparison with the background defects in the wt. (G-J) Stage 16 embryos were stained with BP102 antibody to mark all axons in the VNC. G, wt embryo showing a detail of 6 segments with its anterior and posterior commissures. H, Snap25 mutant embryos, representative of fuzzy commissures (arrow) and thinning of longitudinals (arrowhead) phenotypes. I, nSybd02894 mutant embryos, representative of thinning of longitudinals (arrow). J, Vamp7 mutant embryos showing mild thinning of longitudinals phenotype (arrow). (K) Quantification of the two axonal guidance defects encountered in SNAP-25, n-syb and Vamp7/Ti-VAMP mutants in comparison with the background defects in the wt.
Fig 3
Fig 3. Silencing of SNARE proteins affected the guidance of commissural axons in chicken embryos.
Commissural axons stained with the lipophilic dye DiI in open-book preparation of stage HH26 chicken embryos. In untreated control embryos (A) and in control embryos expressing EGFP (B) axons cross the midline and turn rostrally along the contralateral floor-plate border. In contrast, axonal tracing in embryos electroporated with dsRNA derived from STX-1A (C), SNAP-25 (D), VAMP-2 (E), or Ti-VAMP (F) revealed axonal stalling at the floor-plate entry site (green arrows), axonal stalling in the floor plate (red arrows), and aberrant or no turning into the longitudinal axis at the contralateral floor-plate border (black arrows). (G) After perturbation of SNARE signaling the percentage of DiI injection sites with normal axonal navigation per embryo was decreased: One-way ANOVA F5,226 = 11.99. p<0.0001. Newman-Keuls multiple comparison test: GFP-STX1 p<0.01; GFP-SNAP25 p<0.05; GFP-VAMP2 p<0.001; and GFP-TI-VAMP p<0.01. (H) Downregulation of individual SNARE proteins at E3 did not significantly enhance stalling at the floor-plate entry site. (I) In contrast, commissural axons failed to cross the floor plate after silencing SYT1A, VAMP2, or Ti-VAMP. In all these groups, axonal stalling in the floor plate was significantly increased in comparison to the EGFP-expressing control group. One way ANOVA F5, 199 = 8.232. p<0.0001. Newman-Keuls multiple comparison test: GFP-STX-1 p<0.05; GFP-SNAP25 p>0.05; GFP-VAMP2 p<0.001; and GFP-TI-VAMP p<0.05. Because a strong stalling phenotype (I) prevents independent analysis of the turning phenotype (J), ANOVA analyses were not done separately for the different phenotypes. Rather we compared the average percentage of DiI injection sites per embryo exhibiting aberrant axon navigation (G). When electroporation of dsRNA derived from STX1 was carried out already at E2 (K) the number of DiI injection sites per embryo with normal axonal trajectories was much lower compared to electroporation at E3. Scale bar in A: 50 μm.
Fig 4
Fig 4. Syntaxin1 A/B mutant mouse embryos have aberrant axonal guidance phenotypes.
(A-F) E12 mouse spinal cords; commissural axons immunostained with α-TAG-1 (A, C and E) and superposition with DAPI staining (B, D and F). (A) and (B) correspond to wt genotypes. The double KO genotype for STX1 (C-F) results in aberrant axonal guidance towards the floor plate. Axons are defasciculated and invade the motor column but still enter the floor plate. Arrows point at commissural axon bundles leaving the spinal cord ectopically through the motor exit point. (Scale bar: 75 μm). (G) Histogram illustrating the axon bundle width at three levels of the transversal section of the spinal cord: width 1, width 2 and width 3 were measured in wt and KO spinal cords. Significant differences are labelled by asterisks (*p≤0.05), (***p≤0.001). In (C) an example of the measurements taken for the quantification of the commissural bundle width is shown. Data are presented as the mean ± SEM. Statistical significance was determined using two-tailed Student’s t-test. Differences were considered significant at p<0.05.
Fig 5
Fig 5. Commissural axon navigation in open-book preparations of mouse spinal cords.
(A,B) Tracing of dI1 commissural axons in open-book preparations of spinal cords dissected from E12 wild-type embryos revealed axonal extension towards the floor plate, midline crossing and rostral turning along the contralateral floor-plate border. In general, we found more labeled axons per DiI injection site in wild-type compared to mutant embryos. We added a second image from a wild-type embryo with fewer labeled axons (B) for direct comparison with the mutant embryos (C-E). (C) In mutant embryos lacking both alleles of STX1A, axons extended towards the midline but failed to enter or stalled within the floor plate (green arrow). Axons that manage to reach the contralateral floor-plate border mostly failed to turn. (D,E) These aberrant phenotypes were even seen more often in double KO embryos. Furthermore, we also found more DiI injection sites where fibers failed to enter the floor-plate area in double KO embryos (G). (F) The quantitative analysis of normal trajectories in the different groups indicated significant differences between all mutant groups compared to wild-type mice: normal trajectories in wild-type at 75.6±5.9% (n = 8, 70 injection sites) of the DiI injection sites compared to 21.7±11.1% in STX1A-/-/STX1B+/+ (n = 5; 40 injection sites) and 10.2±5.4% in STX1A-/-/STX1B+/- (n = 9; 96 injection sites) mice. Only 1.0±0.9% of the injection sites were normal in double knock-out mice (n = 7; 74 injection sites). **p<0.01, ***p<0.001, ****p<0.0001 compared to wild-type. (G) The detailed analysis of the individual guidance steps demonstrated mainly floor-plate stalling (green arrows) and failure to turn rostrally into the longitudinal axis (red arrow) as navigation defects in mutant mice. At many injection sites axons were unable to enter the floor plate (purple arrow). Bar: 70 μm.
Fig 6
Fig 6. Colocalization of SNARE proteins and DCC/Robo3 in mouse commissural axons.
Dissociated mouse commissural neurons were stained for different combinations of DCC and Robo3, and SNARE proteins (Syntaxin1, VAMP2, SNAP25 and Ti-VAMP). Analyses of confocal images from growth cones revealed partial colocalization between: (A) DCC and Syntaxin1; (B) DCC and VAMP2; (C) DCC and SNAP25; and (D) DCC and Ti-VAMP. Colocalization was also found between (E) Robo3 and Syntaxin1; (F) Robo3 and VAMP2; (G) Robo3 and SNAP25; and (H) Robo3 and Ti-VAMP. Arrowheads point to areas of colocalization. Scale bar: 5 μm.
Fig 7
Fig 7. Syntaxin1A is required for axonal sensitivity to Slit-2 and Netrin-1.
(A-D) Explants of dI1 neurons dissected from chick embryos grown on laminin substrate extend neurites readily in the absence of Slit-2 (A). In the presence of Slit-2, neurite length is strongly reduced (B). In contrast, neurite growth from explants taken from embryos electroporated with dsRNA derived from STX1A did not differ in the absence (C) or presence (D) of Slit-2. Bar: 200 μm. (E-H) Dorsal spinal cord explants obtained from E11 wild-type and STX1A/B knock-out mouse embryos were confronted with HEK293T cells aggregates expressing or not Netrin-1. (E, F) Axons from wild-type explants showed a marked attraction when confronted to Netrin-1 expressing cell aggregates (F), in contrast to explants confronted with control cells which exhibited a radial axonal growth (E). Mutant spinal cord explants (STX1A(-/-)B(-/-)) exhibited a radial pattern of axonal growth in all conditions (G, H). Scale bar: 100μm. (I) Plots showing average neurite lengths in explants of dl1 neurons incubated with Slit-2. Significant difference is exclusively seen in wild-type explants. (two-way ANOVA analysis; * p<0.05, ** p = 0.0011, ****p<0.0001). At least, 37 explants per condition were used for quantification. The average neurite length in control explants was 214 μm in the absence of Slit-2 (A,I; n = 42 explants) and 43 μm in the presence of Slit-2 (B,I; n = 51 explants). Explants taken from embryos after silencing STX1A extended neurites with an average length of 214 μm in the absence (C,I; n = 37) and 158 μm in the presence of Slit-2 (D,I; n = 41). J) Quantification of Proximal/Distal (P/D) ratios in spinal cord mouse explants confronted to Netrin-1 expressing cell aggregates (two-way ANOVA analysis; *p<0.05).
Fig 8
Fig 8. Drosophila Syx1A genetically interacts with to robo2 and fra.
(A-D) Stage 16 embryos stained with anti-FasII to mark all FasII-positive axons. A, wt embryo showing the three FasII-positive longitudinal connectives. B, Syx1A mutant embryo showing the three longitudinal connectives closer to the midline. C, fra3 mutant embryo showing an increased distance between fascicles. D, robo2 mutant embryo showing midline crosses and fascicles closer to the midline. (E) Quantification of the relative distance between fascicles in control (n = 20), Syx1A (n = 20), fra (n = 37) and robo2 (n = 20) VNCs. Significant differences are labelled by asterisks (***p≤0.001). (F-I) Stage 16 embryos stained with anti-FasII to mark all FasII-positive axons and to better observe axonal pathway defects. F, robo2 mutant embryo showing midline crosses (arrows) and fascicle collapses. G-I, robo2;Syx1A double mutant embryos representative of the three phenotypes encountered; G: Weak, H: Intermediate and I: Strong. Asterisks show fascicle collapses in the VNC in all cases. Anterior is up. (J) Quantification of the total number of axon guidance defects encountered in Syx1A (n = 38), robo2 (n = 39) and robo2;Syx1A (n = 31) mutant embryos. Significant differences are labelled by asterisks (***p≤0.001). (K-N) Stage 16 embryos stained with anti-FasII to mark all FasII-positive axons and to better observe axonal pathway defects. K, fra3 mutant embryo showing fascicle defasciculation and collapse phenotypes (arrows). L-N, fra;Syx1A/+ mutant embryos representative of the phenotypes encountered. Arrows point at defasciculation and collapsed phenotypes. (O) Quantification of the total number of axon guidance defects encountered in fra (n = 37), Syx1A (n = 31), fra/+;Syx1A (n = 15), fra;Syx1A/+ (n = 8) and fra;Syx1A (n = 8) mutant embryos. Significant differences are labelled by asterisks (*p≤0.05,***p≤0.001). (P) Quantification of the relative distance between medial fascicles in control (n = 20), Syx1A (n = 20), robo2 (n = 20) and robo2;Syx1A (n = 17) VNCs. Only accounted the cases like panel G, where fascicles had not collapsed.
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This work was funded by MINECO (to ES: SAF2013-42445-R, SAF2016-7426 and BFU2010-21507; FU: RYC-2007-00417; and SJA: RYC-2009-05510), ISCIII (to ES: CIBERNED), Ministerio de Educación, Cultura y Arte (to OR: AP2005-1662), Fundació La Caixa (to PJB) and the Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (to ETS: 3100A_130730 and 31003A_166479). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.