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, the UPMC Charles Darwin Ethics Committee. Embryos of either sex were used. Tamoxifen injection. Ptf1a:cre ERT2
, T-5648) dissolved in corn oil (Sigma-Aldrich, C-8267). The embryos were collected at E11. EdU labelling. Pregnant females were injected intraperitoneally with EdU (1 mg per 10 g body weight) and killed three hours later, The proliferating cells were visualized after immunohistochemistry using the Alexa Fluor 647 Click-iT EdU Imaging Kit
, Antisense riboprobes were labelled with digoxigenin-11d-UTP (Roche Diagnostics) as described elsewhere 12 , by in vitro transcription of cDNA of mouse Ntn1 (ref. 7) or mouse Ntn1 exon 3. DiI tracing. E12-E13 hindbrains fixed in 4% PFA in an open book configuration were injected using a glass micropipette with DiI crystals or small drops of DiI (Invitrogen)
, Brightness and contrast were adjusted using Adobe Photoshop. Whole-mount labelling and 3DISCO clearing. Whole-mount immunostaining and 3DISCO optical clearing procedure has been described previously 32. 3D imaging was performed with an ultramicroscope using Inspector Pro software (LaVision BioTec). Western blotting. HEK-293T cells (from ATCC, not authenticated, tested for mycoplasma contamination with a negative result) were transfected with pCDNA3, pCDNA3-human NTN3, pCDNA3-human NTN1 or pCDNA3-mouse Ntn1 plasmids using Fugene HD transfection reagent (Promega) according to the manufacturer's instructions. Cells were harvested and lysed 36 h after transfection. Cells were lysed using RIPA buffer (150 mM NaCl, 50 mM Tris pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, complete protease inhibitor cocktail (Roche Diagnostics)) and incubated for 1 h at 4 °C. Floor plates were micro-dissected from hindbrains and spinal cords from Ntn1 fl/ fl , Shh:cre;Ntn1 fl/fl and Ntn1 ?/? E11 embryos. Floor plates were lysed in RIPA buffer and incubated for 20 min at 4 °C. Protein content was determined by a BCA assay, Sections were then incubated overnight with the following primary antibodies: goat anti-human Robo3 (1:250, R&D Systems AF3076), goat anti-Dcc (1:500, Santa Cruz sc-6535), goat anti-Robo1 (1:500, R&D Systems AF1749), rat anti-mouse netrin-1 (1:500, R&D Systems MAB1109), mouse anti-nestin-Alexa488 (1:1000, Abcam ab197495), mouse anti-neurofilament (1:300, DSHB 2H3), goat anti-Foxp2 (1:1000, Santa Cruz sc-21069), rabbit anti-Foxp2 (1:1000, Abcam ab16046), rabbit anti-Sox2 (1:500, Abcam ab97959), rabbit anti-? gal, p.500
, Tween (TBS-T) for 1 h at room temperature and incubated for 90 min at room temperature with primary antibodies: anti-actin (Sigma-Aldrich, A5060, rabbit polyclonal, 1:1,500), anti-HPRT (Abcam, ab109021, rabbit monoclonal EPR5299, 1:10,000), anti-Ntn1 (R&D Systems, MAB1109, rat monoclonal, 1:500), anti-NTN3 (Abcam, ab185200, rabbit polyclonal, 1:1,000) and anti-Slit2 (Abcam, ab134166, rabbit monoclonal, 1:400). After three washes in TBS-T, membranes were incubated with the appropriate HRP-conjugated secondary antibody (1:5,000, Jackson ImmunoResearch). Detection was performed using Pierce ECL Western Blotting Substrate (ThermoScientific)
, For western blot, at least three independent cases were quantified from independent experiments using densitometric analysis (ImageJ) by normalizing phosphorylation signals to total protein levels. The control cases were normalized to 1 and for the mutants, data were presented as mean values ± s.e.m. (0.1133 ± 0.05 for Shh:cre; Ntn1 fl/fl 1 for Ntn1 fl/fl and 0 for Ntn1 ?/? ), Data quantification and statistics. All data quantification was done by an observer blinded to the experimental conditions. We did not perform randomization into groups. No statistical methods were used to predetermine sample size. Data are presented as mean values ± s.e.m. Statistical significance was calculated using onesided unpaired tests for non-parametric tendencies
, The data that support the findings of this study are available from the corresponding author upon reasonable request
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, Extended Data Figure 2 | Floor-plate-specific deletion of netrin-1 in Shh:cre;Ntn1 fl/fl mutants. 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 Ntn1 fl/fl embryos (a) Ntn1 mRNA is highly expressed in floor plate (Fp)
, 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 Ntn1 fl/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;Ntn1 fl/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 Ntn1 fl/fl , Shh:cre, Weak Ntn1 expression is still detected in the floor plate (arrowhead) of Ntn1 ?geo/?geo hypomorphs (b) (n = 5), whereas no signal is seen in Ntn1 ?/? (c) embryos (n = 6)
, Wild-type values were normalized to 1 and mutant values were compared using a non-parametric MannWhitney test. Mutant values are represented as the mean ± s.e.m. (* P < 0.05; Ntn1 fl/fl to Shh:cre;Ntn1 fl/fl or Ntn1 ?/? , Mann-Whitney test (P = 0.0022 for both)). Scale bars, 100 ? m, except a, b, c and d higher magnifications, 50 ? m. LETTER RESEARCH 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 Ntn1 ?geo/?geo hypomorphs (b; arrowheads; n = 3) and almost absent in Ntn1 ?/? embryos (c; n = 6), Netrin-1 is undetectable in Ntn1 ?/? and reduced 90% in Shh:cre;Ntn1 fl/fl mice. i, Western blot quantification
, Coronal sections at three rostro-caudal levels of the spinal cord of an E10 Shh:cre;Ntn1 fl/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
, Scale bars, 100 ? m