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. 2018 Oct 23;8:15647. doi: 10.1038/s41598-018-34092-5

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Hippocampal neurons from Sema3a^−/− embryos extend supernumerary axons in culture. (a–c) Cultures of hippocampal or cortical neurons from E17 Sema3a^+/− or Sema3a^−/− embryos were analyzed at 3, 4, 5 and 6 d.i.v. by staining with an anti-MAP2 (green, dendrites) and the Tau-1 (a, red, axons) or an anti-AnkG antibody (b, red, AIS marked by arrow heads). Representative images of hippocampal neurons at the indicated times in culture are shown. The scale bar is 25 μm. (c) The percentage of unpolarized hippocampal neurons without an axon or AIS (0, red), polarized neurons with a single axon or AIS (1, blue), neurons with multiple axons or AIS (>1, green) and neurons with neurites that are positive for both axonal and dendritic markers (in, yellow) is shown (Student’s t-test and two-way ANOVA; n = 3 independent experiments with >150 neurons per genotype; values are means ± s.e.m., **p < 0.01, ***p < 0.001 compared to control as indicated).