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PlexinA1 is a new Slit receptor and mediates axon guidance function of Slit C-terminal fragments

Abstract

Robo-Slit and Plexin-Semaphorin signaling participate in various developmental and pathogenic processes. During commissural axon guidance in the spinal cord, chemorepulsion by Semaphorin3B and Slits controls midline crossing. Slit processing generates an N-terminal fragment (SlitN) that binds to Robo1 and Robo2 receptors and mediates Slit repulsive activity, as well as a C-terminal fragment (SlitC) with an unknown receptor and bioactivity. We identified PlexinA1 as a Slit receptor and found that it binds the C-terminal Slit fragment specifically and transduces a SlitC signal independently of the Robos and the Neuropilins. PlexinA1–SlitC complexes are detected in spinal cord extracts, and ex vivo, SlitC binding to PlexinA1 elicits a repulsive commissural response. Analysis of various ligand and receptor knockout mice shows that PlexinA1-Slit and Robo-Slit signaling have complementary roles during commissural axon guidance. Thus, PlexinA1 mediates both Semaphorin and Slit signaling, and Slit processing generates two active fragments, each exerting distinct effects through specific receptors.

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Figure 1: Plxna1, Robo1 and Robo2 triple deficiency recapitulates midline guidance defects induced by loss of Slit1, Slit2 and Slit3.
Figure 2: PlexinA1 and Slit transheterozygotes exhibit a midline recrossing phenotype.
Figure 3: PlexinA1 interacts with the Slit C-terminal fragment in vitro.
Figure 4: Slits bind PlexinA family members.
Figure 5: PlexinA1 is a functional receptor for Slits in commissural neurons.
Figure 6: PlexinA1 mediates a SlitC repulsive signal in commissural neurons.
Figure 7: Slits and Semaphorins mediate specific PlexinA1 activation.

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Acknowledgements

V.C. is supported by grants from the French National Research Agency (ANR-2010-BLANC-1430-01) and the European Research Council under the European Union's Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement 281604-YODA. C.D.-B. is supported by a post-doc fellowship from the 'Ligue Contre le cancer'. This work was performed within the framework of the LABEX CORTEX and the LABEX DevWeCan of Université de Lyon within the program 'Investissements d'Avenir' (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). A.C. is supported by grants from the Fondation pour la Recherche Médicale (Programme 'équipe FRM') and the Agence Nationale de la Recherche (ANR-08-MNP-030) to A.C. We acknowledge M. Tessier-Lavigne (Laboratory of Brain Development and Repair, Rockefeller University) and A.B. Huber (Institute for Developmental Genetics, GSF-Research Center for Environment and Health) for sharing mouse lines. We thank T. Toyofuku (Osaka University) and A. Püschel (Abt. Molekularbiologie, Institut für Allgemeine Zoologie und Genetik, Westfälische Wilhelms-Universität) for sharing plasmids.

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Contributions

C.D.-B. designed and conducted the majority of experiments. A.J. constructed expression vectors and participated in the experiments in Figure 7. C.C., F.R., H.N., K.T., K.K. and J.F. contributed to experiments shown in the figures and performed genotyping. Y.Y. provided Plxna1 embryos and mouse line. Y.K. and Y.E.J. contributed to the binding experiments and purified Slit2C protein. Y.Z. and A.C. provided various constructs and mouse lines and validated various antibodies. A.C. brought scientific input and advice for manuscript preparation. V.C. designed the experimental plan, supervised the project and wrote the manuscript with C.D.-B.

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Correspondence to Valérie Castellani.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Drawing of spinal cord open-book preparations and DiI tracing.

Supplementary Figure 2 In ovo electroporation of dominant-negative PlexinA1 in commissural neurons induces midline guidance defects.

a, b. Microphotographs of chick open-books (a) and quantitative analysis (b) after control/GFP or PlexinA-DN/GFP co-electroporations (Ctl; n = 8 embryos, 107 fields; PlexA-DN; n = 8 embryos, 107 fields). Abnormal turn (*) and recrossing (arrowhead) phenotypes are outlined. Scale bar: 25 μM. c, d. Immunofluorescent labeling (c) and quantification of grey matter entry phenotype in chick open books (d) co-electroporated with Ctl/GFP (n = 7 embryos) or PlexA-DN/GFP (n = 8 embryos). c shows GFP fluorescence, Robo3 and Hoechst staining. Lower panels are grayscale enlargements of GFP fluorescence. Arrowheads point axons abnormally entering the grey matter. Scale bar: 100 μM. For b and d, error bars indicate mean + SEM; *p<0.05; **p<0.01; ***p<0.001; Student t-test.

Supplementary Figure 3 SlitC interacts with PlexinA1 in vitro and in vivo.

a. Co-immunoprecipitation performed in COS7 cells transfected with Flag-tagged PlexinA1 construct together with Myc-tagged Slit1 or Slit2. b. Scheme of PlexinA1 protein, and PlexinA1 fragment used to map interaction domain with Slit PlexinA1IPT1-4 and PlexinA1IPT3-4. Sema: Semaphorin domain; PSI: Plexin, Semaphorin and Integrin domain; IPT: Ig-like, Plexin and Transcription factor domain; TM: Transmembrane domain; C1/2: Segmented GAP domains. c. Co-immunoprecipitations in COS7 cells transfected with Flag-tagged PlexinA1, PlexinA1IPT1-4 or PlexinA1IPT3-4 or together with Myc-tagged Slit2C-AP construct. d. Co-immunoprecipitation performed in E12.5 spinal cords using a Slit2C immunoprecipitating antibody. 2 samples for each condition are presented (s1 and s2). In Slit2C immunoprecipitate, the lower band corresponds to the immunoglobulin heavy chain. e. Co-immunoprecipitation performed in spinal cords from E12.5 Robo1/2+/+ and Robo1/2—/— embryos using a PlexinA1 immunoprecipitating antibody. PlexinA1 and Slit1 are detected using antibodies directed against PlexinA1 and a C-terminal Slit1 epitope respectively. For Slit1 immunoblot, two different exposures are shown, to detect both the full-length protein (10’) and the C-terminal cleavage fragment (10’’), pointed by arrowhead.

Supplementary Figure 4 Experimental settings and PlexinA1 status at the growth cone surface in collapse assays.

Schemes of the experimental settings (left panels) and PlexinA1 surface status. Right panels: microphotographs of Hoechst and phalloidin labeling of cultured commissural neurons, with enlarged panels illustrating collapsed and not collapsed observations. Scale bar: 15 μm.

Supplementary Figure 5 PlexinA1 mediates a SlitC collapse response independently of the Robos.

a. In vitro validation of PlexinA1 siRNA efficiency. Western Blot analysis of PlexinA1 protein expression was performed on COS7 cells transfected with PlexinA1-Flag expressing construct together with scramble siRNA or PlexinA1 siRNA. b. Collapse assay on E12.5 PlexinA1 wild-type (+/+), heterozygous (+/—), and null (—/—) commissural neurons incubated with Slit2-FL supernatant or Slit2C supernatant alone or together with GDNF. c. Collapse assay performed on E12.5 Robo1/2 wild-type (+/+) and null (—/—) commissural neurons incubated with Slit1C supernatant alone or together with GDNF. For b and c, n = 4 experiments per condition; number of analyzed growth cones per condition > 200; graphs show mean collapse rate + SD; ***p<0.001; Student t-test. d. Collapse assay on E12.5 commissural neurons incubated with increasing concentrations of Slit2 alone or together with GDNF (n = 3 experiments per condition; number of analyzed growth cones per condition > 100). Graph shows mean collapse rate + SD; *p<0.05, Mann-Whitney U-test.

Supplementary Figure 6 SlitC triggers activation of Rac1 and phosphorylation of ERK.

a. Rac1 activation assay performed on HEK293T cells transfected with PlexinA1 and incubated with indicated supernatants for 15 min. Rac1 active form (bound to GTP) is pulled-down with a GST-CRIB (Cdc42/Rac Interactive Binding) fusion protein. Lower diagram shows quantification of Rac1-GTP/total Rac1 ratio. Graph shows mean + SD; *p<0.05; Mann-Whitney test; 3 independent experiments. b. Time course analysis of ERK phosphorylation in HEK293T cells transfected with PlexinA1 and incubated with supernatants from cells transfected with vectors encoding Slit1-FL, Slit2-FL, Slit2N or Slit2C. Western Blot analysis shows detection of both phosphorylated and total p42 and p44 forms.

Supplementary Figure 7 Model of spatiotemporal engagement of receptors for midline repellents during floor plate crossing.

In the FP Slit is processed into N-terminal (SlitN) and C-terminal (SlitC) fragments. PlexinA1 is processed by calpain in precrossing commissural growth cones. Upon crossing, FP-released GDNF suppresses calpain1 activity, triggering PlexinA1 availability at the growth cone surface. Thus, from the crossing stage, two independent Slit signaling, PlexA1/SlitC and Robo/SlitN are triggered to mediate complementary effects of Slits midline repellents. In parallel, PlexinA1/Nrp2 complexes process the repulsive Sema3B signaling.

Supplementary Figure 8 Full-length pictures of the blots presented as cropped panels in main-text Figure 3.

Supplementary Figure 9 Full-length pictures of the blots presented as cropped panels in main-text Figure 7.

In Fig. 7h, the same membrane was used to detect PlexinA1 (α-Flag) and Nrp2 (α-HA) successively,

Supplementary Figure 10 Western blot validation of antibodies used in the study.

a-d: Western blot analysis of E12.5 embryos tissue lysates from wild-type, Slit2—/— (a), Robo1/2—/— (b, c) and Nrp2—/— (d) using Slit2C (a, Epitomics 2864-1), Robo1 (b, abcam ab85312), Robo2 (c, abcam ab64158) and Nrp2 (d, R&D AF567) antibodies. Arrowheads point predicted endogenous protein size.

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Delloye-Bourgeois, C., Jacquier, A., Charoy, C. et al. PlexinA1 is a new Slit receptor and mediates axon guidance function of Slit C-terminal fragments. Nat Neurosci 18, 36–45 (2015). https://doi.org/10.1038/nn.3893

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