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Celsr3 is required in motor neurons to steer their axons in the hindlimb

Nature Neuroscience volume 17, pages 1171–1179 (2014)Cite this article

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Abstract

The cadherin Celsr3 regulates the directional growth and targeting of axons in the CNS, but whether it acts in collaboration with or in parallel to other guidance cues is unknown. Furthermore, the function of Celsr3 in the peripheral nervous system is still largely unexplored. Here we show that Celsr3 mediates pathfinding of motor axons innervating the hindlimb. In mice, Celsr3-deficient axons of the peroneal nerve segregate from those of the tibial nerve but fail to extend dorsally, and they stall near the branch point. Mutant axons respond to repulsive ephrinA-EphA forward signaling and glial cell–derived neurotrophic factor (GDNF). However, they are insensitive to attractive EphA-ephrinA reverse signaling. In transfected cells, Celsr3 immunoprecipitates with ephrinA2, ephrinA5, Ret, GDNF family receptor α1 (GFRα1) and Frizzled3 (Fzd3). The function of Celsr3 is Fzd3 dependent but Vangl2 independent. Our results provide evidence that the Celsr3-Fzd3 pathway interacts with EphA-ephrinA reverse signaling to guide motor axons in the hindlimb.

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Figure 1: Defective hindlimb innervation in Celsr3 mutant mice.
Figure 2: Reduction of dorsal limb innervation in Celsr3 mutant mice.
Figure 3: Celsr3-deficient LMCL axons fail to extend in the dorsal limb.
Figure 4: Celsr3-deficient LMC axons respond to GDNF and forward signaling but not to reverse signaling.
Figure 5: Celsr3 is required for attraction of LMC axons by EphA7 and GDNF in the axon turning assay.
Figure 6: Interactions between Celsr3 and EphA-ephrinA signaling.
Figure 7: Peroneal nerve projections are Fzd3 dependent and Vangl2 independent.

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Acknowledgements

We thank D. Bonanomi for advice on culture of explants; Y.-C. Lai for help with muscle dissection; J. Nathans for discussions; I. Dudanova (Max Planck Institute of Neurobiology), S. Evans (University of California, San Diego), T. Jessell (Columbia University), R. Klein (Max Planck Institute of Neurobiology), P. Vanderhaeghen (Université Libre de Bruxelles) and Y. Yang (US National Institutes of Health) for providing mutant mice; and John Flanagan (Harvard Medical School) for gift of the Myc-ephrinA2 and A5 plasmids. This work was supported by the following grants: Actions de Recherches Concertées (ARC-10/15-026), FRSM 3.4550.11, FNRS T0002.13, Interuniversity Poles of Attraction (SSTC, PAI p6/20 and PAI7/20), Fondation médicale Reine Elisabeth, Fondation JED-Belgique, and WELBIO-CR-2012A-07 from the Région Wallonne, all from Belgium. G.C., F.C. and F.T. are, respectively, research fellow, research associate and senior research associate from the Belgian Fund for Scientific Research (FNRS).

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Authors and Affiliations

  1. Developmental Neurobiology, Institute of Neuroscience, Université catholique de Louvain,, Brussels, Belgium

    Guoliang Chai, André M Goffinet & Fadel Tissir

  2. Guangdong-Hongkong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, China

    Libing Zhou

  3. Unité d'Etude du Mouvement (UEM), Neurologie, Erasme, Université Libre de Bruxelles, Brussels, Belgium

    Mario Manto

  4. Centre National de la Recherche Scientifique, Institut de Biologie du Développement de Marseille Luminy, Aix-Marseille Université, Marseille, France

    Françoise Helmbacher

  5. Laboratory of Neural Differentiation, Institute of Neuroscience, Université catholique de Louvain, Brussels, Belgium

    Frédéric Clotman

  6. Developmental Neurobiology, WELBIO, Brussels, Belgium

    André M Goffinet

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  1. Guoliang Chai
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Contributions

G.C. performed experiments, analyzed the data and wrote the manuscript; L.Z. characterized the locomotor behavior of Celsr3 mutant mice, M.M. conducted the electrophysiology study; F.H. and F.C. contributed analytical tools and commented on the manuscript, A.M.G. conducted the data analyses; F.T. supervised the project, analyzed the data and wrote the manuscript.

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Correspondence to Fadel Tissir.

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Integrated supplementary information

Supplementary Figure 1 Conditional inactivation of Celsr3.

(a–c) Sections of lumbar spinal cord of Rosa26–Tomato;Isl1::Cre embryos at E12.5. Isl1::Cre (red) is expressed in all spinal motor neurons and DRG neurons. Foxp1 (green) and Isl1 (blue) antibodies were used to highlight LMCM and LMCL (the staining was performed 3 times). (d–g) Cross sections of lumbar spinal cord of wild type (d), Celsr3f/–;Wnt1::Cre (e), Celsr3f/–;Isl1::Cre (f), and Celsr3f/–;Olig2::Cre (g) embryos at E12.5, hybridized with Celsr3 digoxigenin–labeled probe. Celsr3 expression is down–regulated in in DRG and dorsal interneurons in Celsr3f/–;Wnt1::Cre (e); in motor columns and most DRG neurons in Celsr3f/–;Isl1::Cre (f) and in motor columns in Celsr3f/–;Olig2::Cre (g). The experiments were performed twice. (h–k) Whole–mount neurofilament staining of hindlimbs from embryos at E12.5. Compared to wild type (h) and Celsr3f/–;Wnt1::Cre (i), the peroneal nerve (arrow) is reduced in Celsr3f/–;Isl1::Cre (j) and Celsr3f/–;Olig2::Cre (k) (mean thickness of the dPN = 41.4 µm in the wild type (n = 8); 40.9 µm in Celsr3f/–;Wnt1::Cre (n = 10); 27.4 µm in Celsr3f/–;Isl11::Cre (n = 12); and 20.7 µm in Celsr3f/–;Olig2::Cre (n = 12). p=0.8843 for Celsr3f/–;Wnt1::Cre vs wild type; p=0.0021 for Celsr3f/–;Isl1::Cre vs wild type; p<0.001 for Celsr3f/–;Olig2::Cre vs wild type; Mann–Whitney test) DRG: dorsal root ganglia, LMC: lateral motor column, MMC: medial motor column, MC Motor columns, PN: peroneal nerve (arrows), TN: tibial nerve (arrowhead), D and V: dorsal and ventral horns of the spinal cord. Scale bar: 10 µm (a–c); 100 µm (d–f); 200 µm (g–j).

Supplementary Figure 2 Motor neurons are normally specified in Celsr3 mutant mice.

(a,b) Transverse section of the lumbar spinal cord at E11.5 from wild type (a) and Celsr3–/– (b) stained with Isl1 (green), Foxp1 (red) and cleaved caspase–3 (white) antibodies (the staining was performed at least 4 times). (c,d) Quantification of LMCL and LMCM neurons expressed as LMCL/LMCM ratio at E11.5 (c) and E13.5 (d). The LMCL/LMCM ratio is calculated as the number of Foxp1–positive and Isl1–negative neurons divided by the number of Foxp1–positive and Isl1–positive neurons. This ratio is similar in both genotypes at E11.5 (c; mean number of LMC neurons is 4311 for wild type, 4452 for Celsr3–/– and 4377 for Celsr2–/–;Celsr3–/–, 3 embryos each genotype, p = 0.7000; p = 0.8971, Mann–Whitney test). It is significantly reduced in Celsr3–/– mutant at E13.5 (d; mean number of LMC neurons 3855 for wild type and 2776 for Celsr3–/–, 3 embryos each genotype; p = 0.0079, Mann–Whitney test). (e,f) Transverse sections of the lumbar spinal cord at E13.5 from wild type (e) and Celsr2–/–;Celsr3–/– (f), stained for cleaved caspase 3 (green) and Isl1 (red). The experiments were repeated 3 times. (g) Quantification of the number of cleaved caspase 3–positive cells. This number is significantly higher in Celsr3–/– and Celsr2–/–;Celsr3–/– mutants than in wild type (n = 4 embryos for each genotype; **: p = 0.0054; ***: p = 0.0002. Mann–Whitney test). Error bars in c,d,g are mean ± s.e.m. Scale bar: 100 µm (a,b); 50 µm (e,f).

Supplementary Figure 3 Expression of axon guidance molecules in wild type and mutants.

(a–i) Transverse sections of lumbar spinal cord and hindlimb at E11.5 from wild type (a,d,g), Celsr3–/– (b,e,h) and Fzd3–/– (c,f,i) embryos, stained with EphA4 (a–c), Ret (d–f) and Neuropilin1 (g–i) antibodies. Dashed lines define boundary between ventral (V) and dorsal (D) limbs. The experiments were repeated 3 times. (j–r) Transverse sections of lumbar spinal cord at E11.5 from wild type (j,m,p), Celsr3–/– (k,n,q) and Fzd3–/– (l,o,r) embryos, hybridized with ephrinA2 (j–l), ephrinA5 (m–o) and Semaphorin 3A (p–r) digoxigenine–labelled probes. Expression of these cues is not perturbed in absence of Celsr3 or Fzd3. The experiments were repeated twice. Scale bar: 100 µm.

Supplementary Figure 4 Lack of Celsr3 does not affect survival or axon growth of motor neuron.

(a,b) Illustration of LMC neurons from wild type (a) and Celsr3–/– (b) after 3 days in vitro. (c) Quantification of motor neuron survival. Hb9::GFP–positive LMC neurons were dissociated from E12.5 embryos and plated as duplicates. Motor neurons were counted after 5 hours and 3 days. The percentage of survival was 56.4% in wild type and 56.8% in Celsr3 mutants (n = 1464 neurons for wild type and 1630 Celsr3 mutants; p = 0.8971, 3 embryos for each genotype, Mann–Whitney test) (d) Mean axon length after 3 days in culture (n = 64 neurons for wild type and 72 neurons for Celsr3 mutants; p = 0.6665, Mann–Whitney test) (e,f) LMC explants from wild type (e) and Celsr3–/– (f) cultured on laminin coated coverslips (100 µg/ml). (g) Quantification of neurite growth (GFP pixels; n = 32 explants for the wild type and 36 for Celsr3 mutants; p = 0.7642; 3 embryos for each genotype, Mann–Whitney test) Error bars in c,d,g are mean + s.e.m. Scale bar: 50 µm (a,b); 100 µm (e,f).

Supplementary Figure 5 Normal innervation of axial muscles in Celsr3–/– mice.

(a,b) Whole mount views of axon projections labeled by Hb9::GFP in the back muscles of wild type (WT, a) and Celsr3–/– (b) embryos at E12.5. The medial anterior thoracic nerve (N. cut) innervates the superficial cutaneous maximus, and the thoracodorsalis nerve (N. th, arrow) innervates the latissimus dorsi. Both genotypes have a similar innervation pattern. Scale bar: 200 µm.

Supplementary Figure 6 Celsr3 mutant axons respond to HGF in the turning assay.

(a–d) Scatter plots of turned angles β versus initial angles α (5o<α<175o) in the indicated conditions. Celsr3–/– motor axons in EphA7–Fc (n = 46); GDNF (n = 44); HGF (n = 68), Wild type in HGF (n = 67). 3 independent experiments were performed for each condition. (e) Mean angle turned β (mean ± SEM) of wild type and Celsr3–/– motor axons in different conditions (ns: p>0.05, **: p<0.01; p = 0.0086 for wild type in HGF; 0.6851 for Celsr3-/- in EphA7; 0.7906 for Celsr3-/- in GDNF; and 0.0094 for Celsr3-/- in HGF; unpaired t–test versus wild type in IgG–Fc gradient; error bars are mean + s.e.m).

Supplementary Figure 7 Genetic interaction between Celsr3 and Epha4.

(a–d) Lateral views of sciatic nerves stained by anti–neurofilament 160 at E12.5 in wild type (a), Celsr2+/–; Celsr3+/–;Eph a4+/– (b), Celsr3–/– (c) and Celsr3–/–;Eph a4+/– (d) embryos. Peroneal nerve (arrows), Tibial nerve (arrowhead). (e) Quantification of the deep peroneal nerve width at E12.5. Mean thickness of the dPN = 47.6 µm in the wild type (n = 12), 47.4 µm in Celsr2+/–; Celsr3+/–;Eph a4+/– (n = 14), 31.6 µm in Eph a4–/– (n = 22), 16.5 µm in Celsr3–/– (n = 20), 12.8 µm in Celsr3–/–;Eph a4+/– (n = 18) and 10.2 µm in Celsr3–/–; Eph a4–/– (n = 12) embryos. ns: not significant, p = 0.8637; *: p = 0.026; **: p = 0.008; ***: p < 0.0001, unpaired t–test. Error bars are mean + s.e.m. Scale bar: 200 µm.­­

Supplementary Figure 8 Generation of Fzd3 mutant mice.

(a) Schematic representation of the knockout first allele (modified from EUCOMM). A cassette containing FTR–Engrailed–2 exon–IRES–LacZ–loxP–neo–FRT–loxP was inserted in intron 2. In Fzd3ko/ko mice, three mRNAs isoforms are produced. In the first, the engrailed2 splice acceptor is “ignored” leading to the production of the wild type transcript. In the second, the engrailed–2–LacZ fusion cassette is transcribed as expected. In the third, a cryptic splice donor in the engrailed–2 exon results in a premature and aberrant splicing of the engrailed–2–LacZ cassette with the insertion of 115 nucleotides between exons 2 and 3 of the wild type mRNA (red box). (b) The conditional allele was obtained by removal of the FTR–Engrailed–2 –IREs–LacZ–loxP–neo–FRT cassette upon crossing with ROSA26–Flp. Exon 3 is flanked by two loxP sites thereby allowing its conditional excision. (c) In the null allele, exon 3 was deleted in the germline by crossing the conditional allele with PGK::Cre. (d) Sequence of the 115 nucleotides found in transcript 3. (e–h) Lateral views of sciatic nerves stained by anti–neurofilament 160 at E12.5 in wild type (e), Celsr3+/–;Fzd3+/– (f), Celsr2+/–;Celsr3+/–;Fzd3+/– (g) and Fzd3f/–;Isl1::Cre (h) embryos. Peroneal nerve (arrows), Tibial nerve (arrowhead). (i) Quantification of the deep peroneal nerve width at E12.5. Mean thickness of the dPN = 43.1 µm in the wild type (n = 10); 43.8 µm in Celsr3+/–;Fzd3+/– (n = 16); and 44.4 µm in Celsr2+/–;Celsr3+/–;Fzd3+/– (n = 10). ns: not significant, p=0.6337 for Celsr3+/–;Fzd3+/– vs wild type; p=0.2250 for Celsr2+/–; Celsr3+/–;Fzd3+/– vs wild type, unpaired t–test. Error bars are mean + s.e.m. (j) Incidence of different phenotypes in wild–type and mutant mice (green: normal; blue: thinning; red: severe reduction). Wild type: n = 32; Celsr3+/–;Fzd3+/–: n = 16; Celsr2+/–; Celsr3+/–;Fzd3+/–: n = 10; Fzd3f/–;Isl1::Cre: n = 12. E2 and E3: exons 2 and 3 of the Fzd3 gene. En2: Engrailed–2 exon, pA: polyadenylation site SA: splice acceptor, SD: splice donor. Scale bar: 200 µm.

Supplementary Figure 9 Celsr3 and Fzd3 interact with GFRα1, Ret and ephrinA5.

(a) Co–IP assays of Myc–ephrinA2/5 and Vangl2–DsRed in transfected HEK293T cells. Myc–ephrinA2 and Myc–ephrinA5 do not interact with Vangl2–DsRed. (b,c) Co–IP assays between Myc–GFRα1, Ret, ephrinA5 and Celsr3–eGFP(b) or Fzd3–eGFP (c) in transfected HEK293T cells. Myc– GFRα1, Ret, ephrinA5 can interact with Celsr3–eGFP and Fzd3–eGFP. (d,e) Co–IP assays between Myc–EphA7 and Celsr3–eGFP(d) or Fzd3–eGFP (e) in transfected HEK293T cells. Myc–EphA7 does not interact with Celsr3–eGFP and Fzd3–eGFP. Full length blots are shown in Supplementary Fig. 11.

Supplementary Figure 10 Schematic summary of the function of Celsr3 in pathfinding of LMCL axons.

(a,b) In the wild type (a), LMCL axons are repelled by EphA forward signaling from ventral limb and attracted dorsally by GDNF and ephrinA reverse signaling. In Celsr3 mutants (b), LMCL axons are still able to respond to EphA forward and GDNF, but not to attractive ephrinA signaling. Hence they segregate from axons innervating the ventral limb, engage in the common peroneal nerve, but fail to progress beyond the location where the peroneal nerve branches. (c) A model for the interaction between Celsr3/Fzd3 and ephrinA reverse signaling. EphAs in dorsal mesenchymal cells bind to ephrinAs in axon growth cones, which recruits Ret, Celsr3, and Fzd3, and attracts axons into the dorsal limb.

Supplementary Figure 11 Full-length pictures of western blots.

(a) Full length blots for Fig. 6f. The membrane was cut according to the molecular weight and incubated with indicated antibodies. (b) Full length blots for Fig. 7j. Fzd3–eGFP was further detected by a chick α–GFP antibody due to a high background generated by the same rabbit α–GFP antibody used in IP. (c) Full length blots for Supplementary Fig. 7k. (d) Full length blots for Supplementary Fig. 9a. (e) Full length blots for Supplementary Fig. 9b. (f) Full length blots for Supplementary Fig. 9c. (g) Full length blots for Supplementary Fig. 9d. (h) Full length blots for Supplementary Fig. 9e.

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Chai, G., Zhou, L., Manto, M. et al. Celsr3 is required in motor neurons to steer their axons in the hindlimb. Nat Neurosci 17, 1171–1179 (2014). https://doi.org/10.1038/nn.3784

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