Netrin-1 is an evolutionarily conserved, secreted extracellular matrix protein involved in axon guidance at the central nervous system midline1,2. Netrin-1 is expressed by cells localized at the central nervous system midline, such as those of the floor plate in vertebrate embryos1,3. Growth cone turning assays and three-dimensional gel diffusion assays have shown that netrin-1 can attract commissural axons2,4,5,6. Loss-of-function experiments further demonstrated that commissural axon extension to the midline is severely impaired in the absence of netrin-1 (refs 3, 7, 8, 9). Together, these data have long supported a model in which commissural axons are attracted by a netrin-1 gradient diffusing from the midline. Here we selectively ablate netrin-1 expression in floor-plate cells using a Ntn1 conditional knockout mouse line. We find that hindbrain and spinal cord commissural axons develop normally in the absence of floor-plate-derived netrin-1. Furthermore, we show that netrin-1 is highly expressed by cells in the ventricular zone, which can release netrin-1 at the pial surface where it binds to commissural axons. Notably, Ntn1 deletion from the ventricular zone phenocopies commissural axon guidance defects previously described in Ntn1-knockout mice. These results show that the classical view that attraction of commissural axons is mediated by a gradient of floor-plate-derived netrin-1 is inaccurate and that netrin-1 primarily acts locally by promoting growth cone adhesion.
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We thank C. Wright for the Ptf1a:creERT2 line and P. Charnay for the Krox20:cre line. We also thank A. Kolodkin and R. Vigouroux for critical reading of the manuscript and S. Fouquet of the Vision Institute imaging facility for its technical support. This work was supported by grants from the Agence Nationale de la Recherche (ANR-14-CE13-0004-01) (A.C.). It was performed in the frame of the LABEX LIFESENSES (reference ANR-10-LABX-65) supported by French state funds managed by the ANR within the Investissements d’Avenir programme under reference ANR-11-IDEX-0004-02 (A.C.). This work was also supported by grants from INCA, ERC, ANR and Fondation Bettencourt (P.M.). C.D. was recipient of a PhD fellowship from the Fondation pour la recherche médicale.
The authors declare no competing financial interests.
Reviewer Information Nature thanks T. Gomez and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Coronal cryostat sections of the hindbrain and spinal cord (brachial level) of E11 and E13 embryos. a, At E11, the floor plate (Alcam+ positive cells in green) and commissural axons are immunoreactive for netrin-1 (n = 6). b, At E13 Ntn1 mRNA is still expressed in the floor plate (Fp) and ventricular zone of the basal plate (n = 6). c, In a E11 Ntn1βgeo/+ mice, Robo3+ commissural neurons in the dorsal hindbrain (arrowheads) are not immunoreactive for βgal, unlike the basal plate neuroepithelium (arrows; n = 6). d, Western blot analysis of HEK-293T cells overexpressing human NTN3, NTN1 or mouse Ntn1 proteins (n = 3). Left, the monoclonal anti-Ntn1 antibody (MAB1109) specifically recognizes netrin-1 proteins (human and mouse) and not netrin-3. Right, netrin-3 is specifically recognized by the polyclonal anti-NTN3 antibody (ab185200), unlike netrin-1. e, At E11, netrin-1 is expressed in the spinal cord by floor plate (arrowhead) and ventricular zone progenitors (n = 6). f, The floor plate (Alcam+), commissural axons, radial processes of neural progenitors and basal lamina are immunoreactive for netrin-1 (n = 6). g, At E13 (n = 7), netrin-1 is still highly expressed in nestin+ radial processes of neural progenitors and at the pial surface. h, i, Netrin-1 is absent from the hindbrain of Ntn1−/− at E13 (h) and the spinal cord at E11 (i) (n = 6 for each). Floor-plate cells (arrowhead) still express Alcam (green). j, Netrin-1 immunostaining without permeabilization at E11. Commissural axons are still labelled (arrowheads) including those that have crossed the midline (arrow). Commissural axons are also stained with anti-Robo3 on the left panel. V, ventricle. k, Shows the absence of Dcc immunoreactivity on a hindbrain section from a Dcc−/− E11 embryo (DAPI counterstaining, n = 6). l, The radial processes of neural progenitors are present in Ntn1−/− embryos (n = 6). Scale bars, 100 μm except in g, 50 μm.
Coronal cryostat sections of the hindbrain and spinal cord of E10 and E11 embryos. a–d, In situ hybridization for Ntn1 on E11 spinal cord (brachial level). In Ntn1fl/fl embryos (a) Ntn1 mRNA is highly expressed in floor plate (Fp) and ventricular zone (n = 6). Weak Ntn1 expression is still detected in the floor plate (arrowhead) of Ntn1βgeo/βgeo hypomorphs (b) (n = 5), whereas no signal is seen in Ntn1−/− (c) embryos (n = 6). In Shh:cre;Ntn1fl/fl embryos (d), Ntn1 mRNA is not expressed in the floor plate (arrowhead) but is still present in the ventricular zone (n = 6). e, E10 Shh:cre;Ntn1fl/fl spinal cord sections at brachial, thoracic and lumbar levels. At all levels, netrin-1 is found in the ventricular zone, with the highest levels in the p3 progenitor domain, but is absent from the floor plate (arrowheads, n = 6). f, g, In Ntn1fl/fl commissural axons, basal lamina (Pia) and floor plate (arrowhead in f) are immunoreactive for netrin-1 (f). By contrast, the floor plate is not labelled in Shh:cre;Ntn1fl/fl embryos (arrowhead in g) whereas netrin-1 remains expressed along neural progenitor processes and basal lamina (pia; n = 6/6). h, Western blot with anti-Ntn1 antibody on floor plate extracts from Ntn1fl/fl, Shh:cre;Ntn1fl/fl and Ntn1−/− E11 embryo hindbrain and spinal cord (at least 3 cases for each from 3 independent experiments). Netrin-1 is undetectable in Ntn1−/− and reduced 90% in Shh:cre;Ntn1fl/fl mice. i, Western blot quantification. Wild-type values were normalized to 1 and mutant values were compared using a non-parametric Mann–Whitney test. Mutant values are represented as the mean ± s.e.m. (*P < 0.05; Ntn1fl/fl to Shh:cre;Ntn1fl/fl or Ntn1−/−, Mann–Whitney test (P = 0.0022 for both)). Scale bars, 100 μm, except a, b, c and d higher magnifications, 50 μm.
Extended Data Figure 3 Floor-plate-derived netrin-1 is not necessary for midline crossing in hindbrain and spinal cord.
Coronal cryostat sections of the hindbrain and spinal cord (brachial level) of E10, E11 and E13 embryos. a–d, E11 and E13 hindbrain sections (upper and middle panels) and E11 spinal cord sections (lower panels). In wild-type mice (a), Robo3+ and Dcc+ commissural axons cross the floor plate (n = 6). Midline crossing is reduced in Ntn1βgeo/βgeo hypomorphs (b; arrowheads; n = 3) and almost absent in Ntn1−/− embryos (c; n = 6). By contrast, no midline crossing defects are present in Shh:cre;Ntn1fl/fl embryos (d) (n = 9). e, Coronal sections at three rostro-caudal levels of the spinal cord of an E10 Shh:cre;Ntn1fl/fl embryo labelled with anti-Robo3. Commissural axons cross the floor plate at all levels. The dashed lines on the left panel indicate the level of the sections. Brach, brachial; Hind, hindbrain; Thor, thoracic; Lumb, lumbar. Scale bars, 100 μm, except e left panel, 400 μm.
Coronal cryostat sections of the hindbrain of E11 and E13 embryos and spinal cord (brachial level) of E11 embryos. a, b, In Foxg1:cre;Ntn1fl/+ embryos, as in wild type, netrin-1 is expressed in the hindbrain ventricular zone (arrowhead) and commissural axons (arrow). This is not the case in Foxg1:cre;Ntn1fl/fl mutants; however, the floor plate (Fp) is still labelled for netrin-1. Note that netrin-1 is present in the vicinity of the Fp (n = 6). c, Foxg1:cre drives Cre expression in E13 (left) and E11 (right) hindbrain cells (tdTomato+ cells in red) but not in the floor plate (arrowheads; n = 3/3). A few Alcam+ floor-plate cells are tdTomato+. d, e, Netrin-1 distribution is similar in the spinal cord of Foxg1:cre;Ntn1fl/+ (d) and Foxg1:cre;Ntn1fl/fl (e) embryos (n = 5). f, In Ntn1βgeo/βgeo, Foxp2+ olivary neurons fail to migrate ventrally (arrowheads) and only few of them are able to reach to the floor plate (arrowheads; n = 6). g, Quantification of the size of hindbrain commissures in the different mutants compared to controls. Six embryos of each genotype and nine sections from each were quantified. Data are normalized to wild type and are represented as mean ± s.e.m. (one-way Kruskal–Wallis with Mann–Whitney post-test, *P < 0.05; NS, not significant). Comparison between wild type and the different conditions for Dcc, Robo3 and neurofilament, P < 0.05, except the comparison between wild type and Shh:cre;Ntn1fl/fl where: Dcc, P = 0.0649; Robo3, P = 0.1797; neurofilament, P = 0.0649. h, Netrin-1 guidance mechanisms of hindbrain commissural axons, past and current models. In the initial model, soluble netrin-1 secreted by floor plate (FP) forms a ventral–dorsal gradient, which attracts ventrally travelling commissural axon (CN) growth cones (GC). In the revised model, pioneer CN axons form in a superficial region containing high levels of netrin-1 produced by neural progenitor cells (NPCs) extending from the ventricular zone (VZ) to the basal lamina (BL) at the surface of the hindbrain. Commissural axons might also capture netrin-1 and establish a netrin-1-rich pathway guiding follower axons. Their ventral extension might be facilitated by chemorepellents produced in the dorsal hindbrain (indicated with a question mark). Scale bars, 100 μm.
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Dominici, C., Moreno-Bravo, J., Puiggros, S. et al. Floor-plate-derived netrin-1 is dispensable for commissural axon guidance. Nature 545, 350–354 (2017). https://doi.org/10.1038/nature22331
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