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.

Knockdown of L1camb decreases the number of mature oligodendrocytes and inhibits myelination. (a) Fertilized eggs from Tg(mbp:Dendra2-CAAX) zebrafish, expressing a membrane targeted version of the fluorescent protein Dendra2 under control of the mbpa promoter, were injected with control morpholino (cMo), or morpholinos targeting splicing (sMo) or translation (tMo) of l1camb. The extent of myelination was assessed at 96 hpf. Micrographs of the posterior part of the fish are displayed (left panels) with the total length from the end of the yolk sac extension to the tail marked by the black line and the extend of myelinated fibers by the white line. Enlarged views of the area of the spinal cord surrounding the yolk sac extension for embryos injected with cMo, or sMo are displayed in the right panels. Note the reduced extent of myelinated fibers and the reduced myelination in both the ventral and dorsal tract of the spinal cord in larvae from l1camb_sMo injections compared to larvae from cMo injections. (b) The extent of myelinated fibers was quantified by measuring the percentage of the caudal spinal cord containing myelinated fibers and the mean values +/− SEM are plotted. The analyzed larvae were from at least three independent injections. (c) Fertilized eggs from a transgenic zebrafish line, Tg(mbp:Dendra2), expressing the fluorescent protein Dendra2 under control of the mbpa promoter, were injected with control morpholinos (cMo), morpholinos targeting splicing (sMo) or translation (tMo) of l1camb. The posterior part of the spinal cord caudal to the yolk sac extension at 76 hpf is displayed. (d) Quantification of the number of Dendra2-positive cells caudal to the yolk sac extension at 76 hpf. Mean values +/− SEM for analyzed larvae from at least three independent injections are plotted. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test.