In addition to DINE-deficient mice  and C760R knock-in mice , newly developed G607S and C760G knock-in mice were used in this study (see below for detailed information). Homozygous mutant mice were obtained by intercrossing heterozygous parents. All mutant mice were crossbred with Hb9::eGFP mice  to label embryonic motor nerves with GFP. Noon on the day of vaginal plug detection was considered embryonic day 0.5 (E0.5). The genotype of DINE-deficient mice, Hb9::eGFP mice and C760R knock-in mice was determined using allele-specific primers as previously described .
Generation of DINE knock-in mice with a missense mutation
DINE knock-in mice with a pathogenic mutation (G607S: replacement of Gly607 with Ser) were generated using the CRISPR/Cas9 system. The plasmid vector pX330 was a gift from Feng Zhang (Addgene plasmid # 42230) , expressing single-guide RNA (sgRNA), as well as Cas9. The CRISPR Design Tool (http://tools.genome-engineering.org) was used to design sgRNA in silico. The target sequence and protospacer adjacent motif (PAM) sequence was TCTCTGAACTACGGGGGTATTGG (The PAM sequence is shown in bold letters). The sgRNA and Cas9 mRNA were synthesized in vitro using commercially available kits as previously described . The sgRNA (10 ng/μl) and Cas9 mRNA (10 ng/μl) were injected into C57BL/6 mouse zygotes in the presence of 90 bp single-stranded oligonucleotides (ssODN) (10 ng/μl) using a microinjection system under standard conditions. The ssODN sequence was: CAGTCGTCATAGCCATGGGTCAGTTCGTGCCCAATGATGGTGCtAATACCCCCGTAGTTCAGAGACCTGGGCCCACAGCAGCAGGTTAAA (the mutation site is shown as a lowercase letter). The zygotes were cultured in culture medium at 37 °C in 5% CO2 up to the two-cell embryo stage and then the embryos were transferred into the oviducts of recipient mice. Tail genomic DNA was amplified using a specific primer set: forward 5′-ACTATCCGTCCTTCCCCTCC-3′ and reverse 5′-AGATCTCTGGGGCCTCTCTG-3′. The PCR product was used to confirm the mouse genotype by sequencing with the forward primer or restriction fragment length polymorphism analyses with Ban I.
Similar methods were used for generation of another DINE knock-in mouse with an artificial mutation (C760G: replacement of Cys760 with Gly) with appropriate modification of the target sequence as well as the ssODN sequence: the target and PAM sequences were CCCAGTTTGAGGAATTCGGCCGG. The ssODN sequence was: CACCCCAGGGTCCTGGGCAGCGTATCCCAGTTTGAGGAATTCGGCCGaGCCTTCCACgGTCCCAAGGACTCTCCCATGAACCCCGTCCAT. Two mutation sites are shown as lowercase letters and the first mutation (a) is a synonymous mutation to inhibit re-cutting by Cas9.
Off-target effect analysis
We chose five potential off-target sites (OT1-OT5) of the target sequence for G607S knock-in generation (TCTCTGAACTACGGGGGTATTGG) using the CRISPR design tool (http://tools.genome-engineering.org): OT1 TCTaaGAACTACtGGGGTATGGG, OT2 agTgTGAACTACGGGGGTAaCAG, OT3 TCTtTGAACcACGGGGGgATGAG, OT4 TCTaaGtACTACaGGGGTATAAG, OT5 aCaCTGAAgTACaGGGGTATGAG. Mutation sites and PAM sequences are shown as lowercase letters and bold letters, respectively. The surveyor assay was performed to detect the CRISPR/Cas9-induced mutations, as previously described . Target fragments on the off-target sites were amplified using specific primer sets: OT1 forward 5′-TGTTAACAAAATGGAAATGATTCAA-3′ and OT1 reverse 5′-TCAGAGTTCCATGTGGCAGTA-3′, OT2 forward 5′-TCCTTCTCAGATCCCTTGTCA-3′ and OT2 reverse 5′-TGCCATGGATGTAAATCATCA-3′, OT3 forward 5′-CGGTGGGTGGTGTTTCTTAT-3′ and OT3 reverse 5′-GGTGGCAGGAGTTCCTTCTT-3′, OT4 forward 5′-GGCTGCAGGCAGGTAGTTCT-3′ and OT4 reverse 5′-TCCCAAACAGTTAATGAATCAGTG-3′, OT5 forward 5′-TTCTTCTGGAGTCCCCAATG-3′ and OT5 5′-reverse 5′- GCACAGGTTTTTGGAGGAAA-3′.
E12.5 mouse embryos were collected, fixed in 4% paraformaldehyde (PFA) at 4 °C for 2 h, then immersed in PBS containing 30% sucrose for two additional days. The tissue samples were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA), and stored at −80 °C until use. Serial 20 μm sections were cut using a cryostat microtome. Immunohistochemistry was performed as described previously , with minor alternations. Briefly, the sections were rinsed three times in PBS, permeabilized by immersion in absolute methanol for 6 min at −30 °C, rinsed in PBS for 30 min, and blocked for 30 min in 0.3% Triton X-100 and 0.2% bovine serum albumin (BSA) in PBS. The sections were then incubated with goat anti-DINE primary antibody (1:500; Santa Cruz Biotechnology, Dallas, TX, USA) in the blocking solution at room temperature overnight. After washing, anti-goat secondary antibody conjugated with Alexa Fluor 546 (1:500; Invitrogen, Carlsbad, CA, USA) was applied for 50 min and then tissues were rinsed three times in PBS. The sections were visualized using confocal microscopy (FV1200; Olympus, Tokyo, Japan).
Whole-mount immunohistochemistry was performed as previously described . Embryonic tissues were incubated with rabbit anti-GFP primary antibody (1:500; Life Technologies, Carlsbad, CA, USA) to detect the motor nerves. For detection of cranial nerves, embryonic heads were incubated with mouse monoclonal antibody 2H3 (1:500; Developmental Studies Hybridoma Bank, University of Iowa, IA, USA). Subsequently, tissues samples were incubated with Alexa Fluor 488 goat anti-rabbit secondary antibody (1:500; Invitrogen) and Alexa Fluor 546 goat anti-mouse secondary antibody (1:500; Invitrogen). After PBS washes, tissue samples were dehydrated through a methanol series (30%, 50%, 80%, and 100% for 30 min each), cleared with benzyl alcohol-benzyl benzoate (BABB), and imaged using confocal microscopy FV1200 (Olympus).
An FV1200 laser-scanning confocal microscope (Olympus) was used to acquire the confocal images. We used 10x and 20x dry objective lenses to visualize motor nerves. Multiple adjacent regions of embryonic mouse limb, head, or individual muscles were imaged using a motorized xy stage module. Image analyses were performed using IMARIS software (Bitplane, Zurich, Switzerland). To evaluate the extent of motor innervation in each individual skeletal muscle, all stacked images were converted into 3D images and the motor nerve terminals were semiautomatically traced using the filament tracer function. The number of terminal points was automatically calculated. The measurement of the nerve length of ocular motor nerves was performed using the measurement point function. To appropriately compare results between samples, all data were processed using the same criteria.
Total RNA was isolated from embryonic spinal cords using the RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. For quantification of DINE mRNA expression, total RNA (1 μg aliquots) was converted to cDNA by ReverTra Ace (Toyobo, Osaka, Japan) and an oligo (dT) primer. The resultant cDNA was diluted 1:50 with distilled water. The quantitative PCR (qPCR) procedures were performed as previously described . TaqMan Gene Expression Assays (Applied Biosystems, Waltham, MA, USA) for mouse actin beta (ACTB) (Mm00607939_s1) and ECEL1 (Mm00469610_m1) were used for specific target amplification. Relative mRNA expression was calculated by using the comparative cycle threshold (CT) method, and then normalized to endogenous ACTB mRNA expression for each sample. The CT value was obtained from the amplification plot with the aid of SDS software (Applied Biosystems).
For qualitative analysis of the mutant DINE transcript, total RNA (1 μg aliquots) was converted to cDNA using ReverTra Ace (Toyobo, Osaka, Japan) with random primers. The cDNA was amplified using the following specific primers: forward 5′-CCACCCTGTATGACCCAGAC-3′, reverse 5′-ATAGAGGCGAACGATGCACT-3′.
Preparation of membrane fractions from embryonic mouse spinal cord
E17.5 mice spinal cords were homogenized in 50 mM Tris-HCl (pH 7.4) containing protease inhibitor cocktail complete mini (Roche Diagnostics, Indianapolis, IN, USA) (homogenizing buffer). After one centrifugation, the supernatant was centrifuged at 20,000 g for 30 min at 4 °C. The pellet fraction was collected and then solubilized with homogenizing buffer containing 1% Triton X-100 for 30 min at 4 °C and centrifuged again at 20,000 g for 30 min at 4 °C. The supernatants served as protein samples for further analyses.
Protein samples (100 μg) were boiled in Glycoprotein Denaturing Buffer (NEB, Ipswich, MA, USA) for 10 min. For Endoglycosidase H (Endo H) treatment, samples were added to a reaction containing GlycoBuffer 3 (NEB) and endo H (1000 units, NEB) and incubated at 37 °C for 1 h. For PNGase F treatment, samples were added to a reaction containing GlycoBuffer 2 (NEB), 1% Nonidet P-40 (NEB), and PNGase F (1000 units, NEB) and incubated at 37 °C for 1 h.
Western blotting analysis
Protein samples (50 μg) with or without glycosidase treatment were separated by 5–20% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to polyvinylidene fluoride (PVDF) membranes. After incubating with 2% enhanced chemiluminescence (ECL) blocking reagent (GE Healthcare, Buckinghamshire, UK), the membranes were incubated with goat anti-DINE antibody (1:500; Santa Cruz Biotechnology) at 4 °C overnight. The membranes were repeatedly washed then incubated with horseradish peroxidase-conjugated anti-goat IgG secondary antibody (1:5000; Vector, Burlingame, CA, USA). Anti-GAPDH antibody (1:5000; Trevigen, Gaithersburg, MD, USA) was used for the control experiments. Each set of experiments was repeated at least three times to confirm results.
Data were first analyzed for normal distribution and equal variance. When normally distributed, two independent samples were statistically analyzed using a two-tailed Student’s t test or Welch’s t test. If the data did not pass normality testing, the Mann-Whitney U test was used. For three independent samples, the data was statistically analyzed using one-way ANOVA for normal distributions or the Kruskal-Wallis test followed by the Steel-Dwass test for non-normal distributions, with p < 0.05 considered significant. All analyses were completed with Statcel 3 (add-in software for Excel, Microsoft, USA).