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. 2019 Mar 6;9:3716. doi: 10.1038/s41598-019-39884-x

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© The Author(s) 2019

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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L1cam proteolysis is not required for axonal outgrowth from motor neurons, or development of the ventricular system. (a) Fertilized eggs from wild-type AB zebrafish, were injected with control morpholino (cMo), or morpholinos targeting splicing (sMo) or translation (tMo) of l1camb. The overall morphology of the larvae was assessed at 24 (left panel) and 48 (central panel) hpf. Enlarged view of the area of the mid- and hindbrain at 48 hpf (right panel). (b) Quantification of the penetration of the hydrocephalus phenotype, the percentage of larvae displaying an enlargement of the fourth ventricle was calculated for at least three independent injections. Mean values +/− SEM are plotted. The total number of larvae assessed in each group was cMo (149), sMo (215) and tMo (50). (c) Axonal outgrowth from motor neurons was assessed in fertilized embryos injected with morpholinos as in a, and following immunostaining for znp-1. (d) Quantification of axonal outgrowth from motor neurons. The number of motor neurons with visible axonal outgrowth at 24 hpf was counted. Mean values +/− SEM are displayed. Measurements are from at least three independent injections. (e) Fertilized eggs from wild-type AB zebrafish were injected with control morpholino (cMo) or a morpholino targeting splicing of l1camb alone or in combination with mRNA encoding wild-type (WT), proteinase-resistant (L1ΔFN45), or soluble (L1ECD) L1cam. The extend of hydrocephalus was evaluated at 48 hpf and the penetration of the hydrocephalus phenotype, the percentage of larvae displaying an enlargement of the fourth ventricle was calculated for at least three independent injections. Mean values +/− SEM are plotted. The total number of larvae assessed in each group was cMo (161), sMo (205), sMo- L1ECD (60), sMo-L1 WT (48), and sMo-L1ΔFN45 (51). Example pictures are displayed in Supplemental Fig. 6(f) Axonal outgrowth from motor neurons was assessed in fertilized embryos injected with morpholinos and mRNA as in e, following immune-staining with znp-1.The number of motor neurons with visible axonal outgrowth at 24 hpf was counted. Mean values +/− SEM are displayed. Measurements are from at least three independent injections. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test.