Distinct functional consequences of ECEL1/DINE missense mutations in the pathogenesis of congenital contracture disorders
© The Author(s). 2017
Received: 25 September 2017
Accepted: 26 October 2017
Published: 13 November 2017
Endothelin-converting enzyme-like 1 (ECEL1, also termed DINE in rodents), a membrane-bound metalloprotease, has been identified as a gene responsible for distal arthrogryposis (DA). ECEL1-mutated DA is generally characterized by ocular phenotypes in addition to the congenital limb contractures that are common to all DA subtypes. Until now, the consequences of the identified pathogenic mutations have remained incompletely understood because of a lack of detailed phenotypic analyses in relevant mouse models. In this study, we generated a new knock-in mouse strain that carries an ECEL1/DINE pathogenic G607S missense mutation, based on a previous study reporting atypical DA hindlimb phenotypes in two siblings with the mutation. We compared the morphological phenotypes of G607S knock-in mice with C760R knock-in mice that we previously established. Both C760R and G607S knock-in mouse embryos showed similar axonal arborization defects with normal trajectory patterns from the spinal cord to the target hindlimb muscles, as well as axon guidance defects of the abducens nerves. Intriguingly, distinct phenotypes in DINE protein localization and mRNA expression were identified in these knock-in mouse lines. For G607S, DINE mRNA and protein expression was decreased or almost absent in motor neurons. In the C760R mutant mice DINE was expressed and localized in the somata of motor neurons but not in axons. Our mutant mouse data suggest that ECEL1/DINE G607S and C760R mutations both lead to motor innervation defects as primary causes in ECEL1-mutated congenital contracture disorders. However, the functional consequences of the two mutations are distinct, with loss of axonal transport of ECEL1/DINE in C760R mutants and mRNA expression deficits in G607S mutants.
Distal arthrogryposis (DA) is a group of congenital movement disorders causing contracture phenotypes mainly in distal joints of patients’ limbs . In addition to the severe phenotypes in distal limb joints, DA also affects the movement of proximal limb joints and other body parts from birth onward, and can be divided into at least 10 different subtypes depending on the affected regions . In most cases, the disease is autosomal dominantly inherited and the patients often have a mutation in genes encoding the muscle contraction apparatus. Endothelin-converting enzyme-like 1 (ECEL1, also termed DINE in rodents [13, 16]), a membrane-bound metalloprotease, has recently been identified as a gene responsible for the autosomal recessive type 5 form of DA (DA5) [MIM 615065] [8, 21], originally characterized by its ocular phenotypes . In contrast to other causal genes of DA, ECEL1 is predominantly expressed in fetal and adult motor neurons [8, 34], suggesting a unique neurogenic pathogenesis in patients with ECEL1 mutations. In addition to limb contractures, patients with ECEL1 mutations have ptosis and strabismus with variable expressivity and penetrance. Based on the ophthalmic abnormality, previous clinical studies have pointed out that ECEL1 could be considered as a causal gene of another congenital contracture disorder termed congenital cranial dysinnervation disorder (CCDD) [14, 30]. This is a heterogeneous group of syndromes resulting from aberrant wiring of motor nerves in the head muscles [6, 26], and not from malformations of the eye itself. Further, some patients with ECEL1 mutations have been shown to develop a restrictive pulmonary insufficiency .
The highly conserved amino acid sequences of ECEL1/DINE between humans and rodents and the dominant expression of ECEL1/DINE in neuronal tissues of both species [22, 34] make it possible to utilize mouse models to gain a basic understanding of the etiology of ECEL1 mutations that cause DA. Previous gene disruption studies showed that DINE-deficient mice die immediately after birth due to respiratory failure [23, 29]. Morphological analyses at embryonic stages revealed that disruption of DINE leads to abnormal axonal arborization of phrenic motor nerves, which innervate the diaphragm muscle and play a crucial role in respiratory control . More recently, systematic examination of motor innervation in more than 10 hindlimb muscles revealed that DINE deficiency also affected the axonal arborization of motor nerves, especially in foot muscles, partially consistent with the severe contracture phenotype seen in the distal limb joints of DA patients . Importantly, DINE knock-in mice with a pathogenic missense mutation (p.C760R) identified in DA patients (Fig. 1) , reproduced the insufficient arborization of motor nerves in diaphragm muscle and limb muscles .
Previous mutant mouse studies clearly suggested that axonal arborization defects of motor nerves in respiratory muscle and limb muscles could at least partially explain the pathogenesis of respiratory failure and limb contractures in ECEL1-mutated DA, although there is some discordance in the severity of respiratory failure between the patients and the mouse models. However, it remains unclear whether patients with different ECEL1 mutations develop their symptoms by a common pathogenic mechanism. Further, it raises the question of how each identified mutation affects ECEL1/DINE function. The present study aimed to understand the etiology of ECEL1-mutated congenital contracture disorders in more detail by using two distinct, relevant knock-in mouse lines with different pathogenic missense mutations.
Materials and methods
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).
Generation of DINE knock-in mice with a pathogenic mutation (G607S)
Axonal arborization defects of motor nerves in G607S mutant hindlimb muscles
Axon guidance defects in abducens nerves in DINE mutant mice
Drastic reduction of DINE expression in G607S mutant spinal cord
Axonal transport of C760R DINE protein is impaired in spinal cord
The homologous residue in a family member protein to cysteine 760 in the DINE protein has been shown to form a disulfide bond , but there is no direct evidence showing that cysteine 760 of DINE actually forms a disulfide bond. Thus, it remains unclear whether the pathogenic effect of C760R results from the lack of a disulfide bond or an arginine-induced conformational change. To further discriminate the pathogenic possibilities, we generated a new DINE knock-in mouse with an artificial substitution of cysteine 760 with glycine, another small non-charged amino acid. This C760G mutant produced identical immunohistochemical results to the C760R mutant (Fig. 6g–i, p–r). These results indicate that cysteine 760 contributes to the appropriate conformation of DINE protein, possibly via a disulfide bond, and this conformational change possibly promotes the axonal transport of DINE.
Information of three distinct DINE mutant mice
DINE mRNA level
DINE protein level
Motor nerves in hindlimb muscles b
Abducens motor nerves
axonal arborization defect
axonal arborization defect
axon guidance defects c(stalled or wandering)
strongly reduced (due to abnormal splicing)
axonal arborization defect
axon guidance defects c(stalled or wandering)
Although ECEL1 was originally identified as a gene responsible for DA, a previous clinical study noted the presence of dominant ocular phenotypes in addition to the absence of hindlimb contracture phenotypes in patients with the ECEL1 G607S mutation, resulting in another congenital contracture disorder termed CCDD. However, further experimental studies were needed to validate the genotype-phenotype relationship of the G607S mutation and CCDD, not only because the clinical study evaluated only two siblings with the mutation, but also because the phenotypic expressivity often differs among patients with ECEL1 mutations. In this study, we utilized our two distinct knock-in mouse strains as two different congenital contracture disorder models (i.e. C760R for DA, G607S for CCDD), and compared morphological phenotypes of both cranial and spinal motor nerves. Consistent with the abnormal ocular phenotype observed in the patients with ECEL1 mutations, our morphological analyses in embryonic head revealed that the two different mutant lines similarly affected axon guidance of abducens nerves. Notably, our mutant mice reproduced the variable expressivity as well as the low penetrance seen in patients with ECEL1 mutations in a previous clinical study . These data provide the first evidence that axon guidance defects of abducens nerves could be a primary cause of CCDD with ECEL1 mutations, and supports the possibility that the overlapping phenotypes of the ECEL1 mutation causing DA and that causing CCDD could be explained by abnormal motor innervation of ocular motor nerves.
Our previous study demonstrated that both DINE-deficient mice and homozygous C760R mutant mice showed axonal arborization defects of motor nerves in the diaphragm muscle and the limb muscles without affecting the axon guidance toward their target muscles [23, 24]. In contrast to the phenotypes seen in spinal motor neurons, two distinct axon guidance defects, i.e. stalled and wandering, were newly identified in homozygous mutant abducens nerves. Because no clear abnormal nerve trajectory phenotypes could be detected in the other ocular motor nerves, the axon guidance defects seemed to have specifically occurred in the mutant abducens nerves. A number of adhesion molecules and signaling molecules have been shown to play crucial roles in axon guidance, but it remains incompletely understood how axon guidance can be correctly achieved in each cranial and spinal motor nerve. Although it is likely that the axon guidance mechanisms in the abducens nerve are different from those of other ocular nerves, the exact molecular mechanisms remain to be elucidated. Recently, Nugent et al. reported that knock-in mice with an α2-chimerin gain-of-function missense mutation, identified in CCDD patients, showed a similar stalled phenotype in the abducens nerves, with variable penetrance . More importantly, they also demonstrated that axons in α2-chimerin-knockout mice, a loss-of-function model, and mice with knockout of the upstream adhesion molecule, EphA4, exhibited the wandering phenotype in abducens nerves as also detected in our DINE mutant mice. These two similar phenotypes suggest the possibility that DINE might contribute to proper axon guidance of abducens nerves via directly affecting the signaling pathway from EphA4 to α2-chimerin.
A previous clinical study reported a missense mutation c.1819G > A (p.G607S) in the ECEL1 gene as a causal mutation of congenital contracture syndromes, as their in silico analysis predicted the amino acid change to be damaging for the function of the ECEL1 protein . In order to validate and further explore the functional consequences, we generated a DINE knock-in mouse with the c.1819G > A (p.G607S) mutation and unexpectedly found a drastic reduction of DINE expression at the transcriptional level, probably via an abnormal splicing process. Recently, it has been revealed that human exons function as sequences necessary for correct splicing in addition to coding information, and disruption of the splice regulatory information in exons leads to pathogenic outcomes [32, 33]. It is often hard to precisely evaluate how identified disease-causing missense mutations affect the splicing process, mainly due to the functional overlap between protein-coding sequences and the splice regulatory information , but the rapid progress in development of in silico tools to identify the disruption of critical information for correct splicing sometimes enables prediction of the possible mechanism. In fact, one commonly used prediction tool  identified that the c.1819G > A (p.G607S) mutation inhibits the binding of a splicing enhancing factor on exonic sequences (Additional file 4: Figure S4). These results emphasize the importance of in vivo characterization with relevant animal models, in addition to in silico analyses, to precisely identify the biological basis of pathogenic missense mutations.
Our biochemical and immunohistochemical analyses provided the first evidence that C760R mutant protein accumulates in neuronal cell soma, possibly due to the loss of appropriate protein conformation and subsequent impairment of protein transportation into axons. The newly generated knock-in mouse with an artificial mutation (C760G) in which arginine was replaced with glycine reproduced the result, supporting the idea that altered localization of C760R mutant protein could result from the loss of a disulfide bond, as a previous study showed for the homologous cysteine in a family member protein . In contrast to the present data, previous cell culture experiments could not detect altered localization of the mutant protein and suggested that the pathogenic mutation possibly affected enzymatic activity . This discordance might depend on the different experimental paradigms used in the previous study and in ours: whereas the previous study overexpressed the mutant protein in non-neuronal cells, we explored the targeting of the endogenous mutant protein in an in vivo situation, i.e. in mouse spinal motor neurons. Although our in vivo analyses strongly suggest that the mutation affects the targeting of ECEL1/DINE, because of the lack of an established activity evaluation system we cannot exclude the possibility that the mutations also affected enzymatic activity. To date, no physiological substrates of ECEL1/DINE have been identified, but recent in vivo overexpression experiments with DINE-deficient mice have shown that mutation of the predictive protease domain of the DINE protein failed to rescue the motor innervation defects resulting from deficiency of DINE . Identification of the physiological substrates of ECEL1/DINE and subsequent development of an in vitro enzymatic assay are needed to further explore the functional consequences of the pathogenic mutations.
Based on analyses of clinically relevant mouse models, we suggest that impaired axonal arborization of spinal motor nerves, as well as axon guidance defects in abducens nerves, are common primary causes in two distinct ECEL1-mutated congenital contracture disorders. Another important finding was that the functional consequences of the two pathogenic mutations differed: G607S and C760R led to a drastic reduction of DINE expression and altered localization of DINE protein, respectively. Further in vivo experimental studies with the relevant animal models, working in tandem with clinical genetic studies, would contribute to elucidating the pathogenic mechanisms of ECEL1-mutated congenital contracture disorders, including DA and CCDD.
We thank Yukiko Nagai for secretarial assistance and Saido lab members for assistance. We are grateful to the RIKEN BSI-Olympus Collaboration Center for imaging equipment and software, the RIKEN BSI-Research Resource Center for injection of the CRISPR/Cas9 system into mouse zygotes as well as for the technical help with DNA sequencing analyses. This work was financially supported by the Japan Society for the Promotion of Science KAKENHI Grant Number 26860141, the Special Postdoctoral Researchers Program in RIKEN, the Japan foundation of Applied Enzymology and the RIKEN Brain Science Institute. We thank Ann Turnley, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
KN, HK and TCS conceived the study. KN designed the experiments. KN and MT performed the experiments. KN analyzed the data and prepared the figures. KN, SKS, HK and TCS wrote and edited the manuscript. All authors provided feedback and agreed on the final manuscript.
The authors declare that they have no competing interests.
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