Vertebral Development Research Articles

Correction of Vertebral Bone Development in Ectodysplasin A1-Deficient Mice by Prenatal Treatment With a Replacement Protein.

X-linked hypohidrotic ectodermal dysplasia with the cardinal symptoms hypodontia, hypotrichosis and hypohidrosis is caused by a genetic deficiency of ectodysplasin A1 (EDA1). Prenatal EDA1 replacement can rescue the development of skin appendages and teeth. Tabby mice, a natural animal model of EDA1 deficiency, additionally feature a striking kink of the tail, the cause of which has remained unclear. We studied the origin of this phenomenon and its response to prenatal therapy. Alterations in the distal spine could be noticed soon after birth, and kinks were present in all Tabby mice by the age of 4 months. Although their vertebral bones frequently had a disorganized epiphyseal zone possibly predisposing to fractures, cortical bone density was only reduced in vertebrae of older Tabby mice and even increased in their tibiae. Different availability of osteoclasts in the spine, which may affect bone density, was ruled out by osteoclast staining. The absence of hair follicles, a well-known niche of epidermal stem cells, and much lower bromodeoxyuridine uptake in the tail skin of 9-day-old Tabby mice rather suggest the kink being due to a skin proliferation defect that prevents the skin from growing as fast as the skeleton, so that caudal vertebrae may be squeezed and bent by a lack of skin. Early postnatal treatment with EDA1 leading to delayed hair follicle formation attenuated the kink, but did not prevent it. Tabby mice born after prenatal administration of EDA1, however, showed normal tail skin proliferation, no signs of kinking and, interestingly, a normalized vertebral bone density. Thus, our data prove the causal relationship between EDA1 deficiency and kinky tails and indicate that hair follicles are required for murine tail skin to grow fast enough. Disturbed bone development appears to be partially pre-determined in utero and can be counteracted by timely EDA1 replacement, pointing to a role of EDA1 also in osteogenesis.

FAM20C plays a critical role in the development of mouse vertebra

Background ContextFamily with sequence similarity 20-member C (FAM20C) is a protein kinase that is responsible for the phosphorylation of many secretory proteins; however, its roles in spine or vertebra development have not be studied. PurposeThe aim of this investigation is to analyze the roles of FAM20C in vertebra development. Study Design/SettingA mouse study of the Fam20c gene using conditional knockout to assess the effects of its inactivation on vertebra development. MethodsBy breeding Sox2-Cre mice with Fam20cflox/flox mice, Sox2-Cre;Fam20cflox/flox mice (abbreviated as cKO mice) are created. X-ray radiography, resin-casted scanning electron microscopy, Hematoxylin and Eosin staining, safranin O staining, Goldner's Masson trichrome staining, Von Kossa staining, tartrate-resistant alkaline phosphatase staining, immunohistochemistry staining, Western Immunoblotting and real-time PCR were employed to characterize the vertebrae of cKO mice compared to the normal control mice. ResultsInactivation of Fam20c in mice results in remarkable spine deformity, severe morphology and mineralization defects, altered levels of osteoblast differentiation markers, reduction of activity of the Wnt/β-catenin signaling pathway and reduced level of osteoclastogenesis in the vertebrae. ConclusionsFAM20C plays an essential role in vertebral development; it may regulate vertebral formation through the Wnt/β-catenin signaling pathway. Clinical significanceMutations in the human FAM20C gene are associated with Raine syndrome. The findings of this study provide valuable clues for the clinical management of Raine syndrome regarding spine manifestations in patients.

Risk Factors for New Vertebral Fracture After Percutaneous Vertebroplasty for Osteoporotic Vertebral Compression Fractures.

PurposeTo analyze the risk factors for new vertebral fractures after percutaneous vertebroplasty (PVP) for osteoporotic vertebral compression fractures (OVCFs).Patients and MethodsWe retrospectively reviewed the records of patients with symptomatic OVCFs who underwent PVP in our hospital, from January 2014 to January 2019. Demographic and lifestyle data on the presence of underlying chronic disease, preoperative bone mineral density, details of vertebral fractures, postoperative osteoporosis treatment, and new fracture development were collected. Patients were divided into postoperative fracture and non-fracture groups. To identify the independent risk factors for new vertebral fracture development, variables significant on univariate analysis were included in a multivariate regression model.ResultsOf the 2202 patients treated with PVP, 362 (16.43%) had a new postoperative vertebral fracture. All patients were followed up for >12 months (mean 14.7 months). Univariate analysis revealed no significant difference in height; body weight; preoperative bone mineral density; number of fractured vertebrae; injection volume of bone cement in a single vertebra; leakage rate of bone cement; or presence of hypertension, coronary heart disease, and chronic obstructive pulmonary disease between the fracture and non-fracture groups (P>0.05). Age, sex, smoking, alcohol consumption, diabetes mellitus, postoperative exercise, and postoperative osteoporosis treatment were associated with new vertebral fractures (all P<0.05). A multivariate analysis showed that age (odds ratio [OR]=1.212, P<0.0001), female sex (OR=1.917, P<0.0001), smoking (OR=1.538, P=0.026), and diabetes (OR=1.915, P<0.0001) were positively correlated with new vertebral fracture development, whereas postoperative exercise (OR=0.220, P<0.0001) and osteoporosis treatment (OR=0.413, P<0.0001) were negatively correlated.ConclusionElderly patients, females, and those with a history of smoking and diabetes are at high risk of new vertebral fracture after PVP. Patients should be encouraged to stop smoking and consuming alcohol, control blood glucose level, participate in sufficient physical activity, and adhere to osteoporosis treatment to prevent new vertebral fractures.

Prenatal muscle forces are necessary for vertebral segmentation and disc structure, but not for notochord involution in mice.

Embryonic muscle forces are necessary for normal vertebral development and spinal curvature, but their involvement in intervertebral disc (IVD) development remains unclear. The aim of the current study was to determine how muscle contractions affect (1) notochord involution and vertebral segmentation, and (2) IVD development including the mechanical properties and morphology, as well as collagen fibre alignment in the annulus fibrosus. Muscular dysgenesis (mdg) mice were harvested at three prenatal stages: at Theiler Stage (TS)22 when notochord involution starts, at TS24 when involution is complete, and at TS27 when the IVD is formed. Vertebral and IVD development were characterised using histology, immunofluorescence, and indentation testing. The results revealed that notochord involution and vertebral segmentation occurred independently of muscle contractions between TS22 and TS24. However, in the absence of muscle contractions, we found vertebral fusion in the cervical region at TS27, along with (i) a displacement of the nucleus pulposus towards the dorsal side, (ii) a disruption of the structural arrangement of collagen in the annulus fibrosus, and (iii) an increase in viscous behaviour of the annulus fibrosus. These findings emphasise the important role of mechanical forces during IVD development, and demonstrate a critical role of muscle loading during development to enable proper annulus fibrosus formation. They further suggest a need for mechanical loading in the creation of fibre-reinforced tissue engineering replacement IVDs as a therapy for IVD degeneration.

Expression analysis and single-nucleotide polymorphisms of SYNDIG1L and UNC13C genes associated with thoracic vertebral numbers in sheep (Ovis aries).

The objective of the current study was to analyze expression levels of synapse differentiation inducing 1-like (SYNDIG1L) and unc-13 homolog C (UNC13C) genes in different tissues, while single-nucleotide polymorphisms (SNPs) of two genes were associated with multiple thoracic vertebrae traits in both Small-tailed Han sheep (STH) and Sunite sheep (SNT). The expression levels of SYNDIG1L and UNC13C were analyzed in the brain, cerebellum, heart, liver, spleen, lung, kidney, adrenal gland, uterine horn, longissimus muscle, and abdominal adipose tissues of two sheep breeds with different thoracic vertebral number (TVN) sheep (T13 groups and T14 groups) by real-time quantitative polymerase chain reaction (RT-qPCR). Meanwhile, the polymorphisms of UNC13C gene g.52919279CT and SYNDIG1L gene g.82573325CA in T14 and T13 were genotyped by the Sequenom MassARRAY® SNP assay, and association analysis was performed with the TVN. The results demonstrated that UNC13C gene was extensively expressed in 11 tissues. The expression of UNC13C gene in longissimus muscle of T14 groups of STH was significantly higher than that of T13 groups (). SYNDIG1L gene was overexpressed in brain and cerebellum tissues, and the expression level of UNC13C gene in the brain and cerebellum of T13 groups in SNT was significantly higher than that of T14 groups (). Association analysis showed that SNPs found in the UNC13C gene had no significant effects on TVN for both two genes. The polymorphism of SYNDIG1L g.82573325CA was significantly correlated with the TVN in both STH () and SNT (). Taken together, the SYNDIG1L gene was related to thoracic vertebral development, and this variation may be potentially used as a molecular marker to select the multiple thoracic vertebrae in sheep.

Assessment of maturation indicators in individuals with different skeletal malocclusion.

The aim of this study was to comparatively evaluate hand-wrist bones, cervical vertebrae and tooth development stages according to skeletal classification. The orthodontic initial records of 297patients were used and separated into three groups according to the skeletal malocclusion. Three groups including 99people each were sampled representing malocclusions with Angle classesI, II and III, respectively. The panoramic, cephalometric and hand-wrist radiographs of all patients included in this study were used to compare dental and skeletal maturation indicators. Calcification of teeth was rated according to the system of Demirjian. To evaluate the stage of skeletal maturation hand-wrist radiographs were analyzed according to the Grave and Brown method. Also, Hassel and the Farman method was used to classifying vertebral developmental stages. Spearman rank correlation tests, as well as Fisher exact χ2 tests with r×c tables, were used for the comparison of categorical variables. Hand-wrist, vertebral and dental development stages showed astatistically significant relationship (p < 0.01) for both genders and in all malocclusions. The association between the different maturation indicators used in this study and the type of malocclusion was also statistically significant for both genders. It was observed that the peak period of skeletal maturation according to the hand-wrist radiograph findings correlated with the cervical vertebrae stage C3 in girls (63.2%) and C2-C3 in boys (43.5-43.5%). The weakest correlation was seen between the dental development stages and the skeletal developmental stages in the classII group (r = 0.443-0.220 [girls]; r = 0.604-0.410 [boys]). The use of the dental development stage as areliable indicator of maturation was limited. According to the Demirjian method, the calcification stage of the second molar might indicate that the individual is in the pubertal peak period.

Gastrulation : Current Concepts and Implications for Spinal Malformations.

It has been recognised for over a century that the events of gastrulation are fundamental in determining, not only the development of the neuraxis but the organisation of the entire primitive embryo. Until recently our understanding of gastrulation was based on detailed histological analysis in animal models and relatively rare human tissue preparations from aborted fetuses. Such studies resulted in a model of gastrulation that neurosurgeons have subsequently used as a means of trying to explain some of the congenital anomalies of caudal spinal cord and vertebral development that present in paediatric neurosurgical practice. Recent advances in developmental biology, in particular cellular biology and molecular genetics have offered new insights into very early development. Understanding the processes that underlie cellular interactions, gene expression and activation/inhibition of signalling pathways has changed the way embryologists view gastrulation and this has led to a shift in emphasis from the ‘descriptive and morphological’ to the ‘mechanistic and functional’. Unfortunately, thus far it has proved difficult to translate this improved knowledge of normal development, typically derived from non-human models, into an understanding of the mechanisms underlying human malformations such as the spinal dysraphisms and anomalies of caudal development. A paediatric neurosurgeons perspective of current concepts in gastrulation is presented along with a critical review of the current hypotheses of human malformations that have been attributed to disorders of this stage of embryogenesis.

A twin‑pair analysis indicates congenital scoliosis is associated with allele‑specific methylation in the SVIL gene.

Congenital scoliosis (CS) is a congenital disease resulting in abnormal vertebral development. Several studies have indicated that both genetic and environmental factors during pregnancy increase the risk of CS development. However, the exact mechanisms underlying CS pathogenesis remain unknown. To address this issue, both genetic (by whole‑exome sequencing) and epigenetic (by methylated DNA immunoprecipitation sequencing) maps from CS disease‑discordant monozygotic twins were generated in the present study. The differences in the presence of common and rare single nucleotide polymorphisms and in methylation patterns between the twins were investigated. The results indicated that rare mutations were more likely to underlie CS development compared with common mutations. Furthermore, differences in the allele‑specific methylation pattern in the supervillin (SVIL) gene between the twins were identified. It has been reported that SVIL exerts a number of functions associated with CS, indicating its role as a novel mechanism promoting CS pathogenesis.

Oral bisphosphonate treatment in patients with Duchenne muscular dystrophy on long term glucocorticoid therapy

Osteoporosis is a major problem in patients with Duchenne muscular dystrophy (DMD), due to glucocorticoid therapy and muscle weakness. Evidence on which to base optimal prevention and treatment strategies, including bisphosphonate use, in DMD are limited. Our objective was to describe bone health outcomes of oral alendronate treatment in patients with DMD and glucocorticoid-induced osteoporosis. We retrospectively studied 54 patients treated between 2005 and 2017, and assessed changes in dual-energy x-ray absorptiometry (DXA) whole body and lumbar spine bone mineral density and content, and lateral distal femur bone mineral density. We also examined vertebral fracture development in a subset with serial spine radiographs. Pre-alendronate DXA Z-score trajectories decreased progressively. Over three years post-alendronate initiation, Z-score trajectories improved (p<0.01) at most sites compared with pre-alendronate trajectories. Height-adjusted Z-score trajectories for lumbar spine bone mineral density (p = 0.01) and whole body bone mineral content (p = 0.0004) also improved. The positive trajectories did not seem to be sustained long term in those treated up to 6 years. Radiographic vertebral findings in 43 patients appeared stable. In conclusion, oral bisphosphonate therapy using alendronate was associated with improvement of DXA bone health indices during the first three years of treatment, and may help mitigate progression of osteoporosis in glucocorticoid-treated patients with DMD.

Mechanisms of Wnt5a‐Ror Signaling in Development and Disease

Wnt5a‐Ror signaling is a conserved developmental pathway that regulates morphogenetic processes during vertebral embryonic development, and dysfunction of the pathway causes a number of human disorders, including Robinow syndrome. The mechanisms of Wnt5a‐Ror signaling remain poorly understood. Using a large‐scale proteomic screen, we identified the E3 ubiquitin ligase Pdzrn3 as a new regulatory target that is degraded upon pathway activation in a beta‐catenin independent, ubiquitin‐proteasome system dependent manner. Using this discovery, we developed a flow cytometry‐based reporter to monitor Pdzrn3 abundance and delineated a signaling cascade involving Frizzled, Dishevelled, Casein kinase 1, and Glycogen synthase kinase 3 that regulates Pdzrn3 degradation. Genetic epistasis analysis suggests that Pdzrn3 resides downstream of Dishevelled and independently of Kif26b, a previously identified Wnt5a‐Ror regulatory target. Further, we discovered that Pdzrn3 degradation requires Wnt5a‐dependent phosphorylation of its C‐terminal LNX3H domain, which is conserved in several Pdzrn3 homologs and likely functions as a novel Wnt5a‐responsive domain. Collectively, this work establishes a new Wnt5a‐Ror signaling cascade involving Pdzrn3 phosphorylation and degradation.Support or Funding InformationNIH Grant 1R35GM119574

Prenatal ultrasound diagnostic clues of congenital dislocation of the knee

Objective To explore prenatal ultrasound diagnostic clues of congenital dislocation of the knee(CDK). Methods The prenatal ultrasonographic features of 13 CDK fetuses diagnosed from January 2013 to December 2018 in the Third Affiliated Hospital of Zhengzhou University were retrospectively analyzed, combined with the results of gene detections and pathological findings. Results Thirteen fetuses were diagnosed by prenatal ultrasound during the second trimester. Two-dimensional and three-dimensional ultrasound showed dislocation of the knee(unilateral in 3 cases and bilateral in 10 cases). All cases were accompanied with multiple abnormalities, including talipes(5 cases), overlapping fingers(2 cases), short nasal bone(1 case), thicken nuchal fold(1 case), narrow thorax(1 case), abnormal vertebral development(1 case), etc. Ultrasound diagnosis included Larsen syndrome in 2 cases, arthrogryosis multiplex congenital in 3 cases, asphyxiating thoracic dysplasia in 1 case, and congenital dislocation of the knee in 7 cases. CDK was confirmed in all fetuses after termination of pregnancy. Three of them were known to undergo chromosome examinations with normal chromosomal microarray analysis (CMA). One case was confirmed to be Larsen syndrome by further second-generation sequencing combined with first-generation sequencing screening, suggesting there was FLNB gene mutation. Conclusions CDK can exist isolated, and it can also be a common manifestation of various diseases. Therefore, attention should be paid to other associated abnormalities in the prenatal detection of knee flexion, and further detection of related genes can provide valuable information for genetic counseling. Key words: Prenatal ultrasonography; Fetal; Congenital dislocation of the knee; Gene

DENTAL IMAGING DILEMMAS IN KLIPPEL-FEIL ANOMALY

<h3>Background</h3> Because of the broad range of findings, Klippel-Feil syndrome has also been referred to as <i>Klippel-Feil sequence</i> and <i>Klippel-Feil anomaly</i>. It is classified into 3 types that differ in degree and location of vertebral fusion. Another contemporary classification also includes patterns of inheritance. <h3>Case Report</h3> A 19-year-old male needed panoramic imaging to evaluate his third molars. However, because of Klippel-Feil disfigurement, his head and neck posture did not fit into various panoramic imaging units. With an altered oblique head positioning in a cone beam computed tomography (CBCT) scanner (NewTom VGi), his jaws were scanned. The CBCT volumetric data were then realigned and reconstructed in CBCT viewing software (InVivoDental 6.0). These reconstructions were appropriate to assess the indicated dental status and to update the assessment of the cervical vertebral development—a limiting condition of Klippel-Feil syndrome. <h3>Definitive Interpretation</h3> The scan data were able to provide the indicated periapical assessment, particularly of the third molars. Postural compromises were also noted in the anterior alignment of the odontoid process–C1 articulation and their alignment to the clivus of the occipital bone. There was also cervical vertebral aplasia with complete fusion of C2-C7. These findings were contributory to his altered posture. <h3>Discussion of Imaging</h3> The availability of large volume CBCT scanners, with their larger footprint, scanning circumference, and larger scanning size area, provide an imaging option for patients with mental and physical disabilities. In the past, a single large field of view (FOV) CBCT scan has been purported by CBCT manufacturers to be a one-stop imaging solution for all extraoral imaging. Dosimetry data and FDA selection criteria have since contraindicated the use of large volume FOV scanning to replace standard periapical, panoramic, and cephalometric imaging. This case report supports the alternative use of CBCT to acquire standard extraoral images when standard imaging is not possible.

Dominant-negative SOX9 mutations in campomelic dysplasia.

Campomelic dysplasia (CD) is an autosomal dominant, perinatal lethal skeletal dysplasia characterized by a small chest and short long bones with bowing of the lower extremities. CD is the result of heterozygosity for mutations in the gene encoding the chondrogenesis master regulator, SOX9. Loss-of-function mutations have been identified in most CD cases so it has been assumed that the disease results from haploinsufficiency for SOX9. Here, we identified distal truncating SOX9 mutations in four unrelated CD cases. The mutations all leave the dimerization and DNA-binding domains intact and cultured chondrocytes from three of the four cases synthesized truncated SOX9. Relative to CD resulting from haploinsufficiency, there was decreased transactivation activity toward a major transcriptional target, COL2A1, consistent with the mutations exerting a dominant-negative effect. For one of the cases, the phenotypic consequence was a very severe form of CD, with a pronounced effect on vertebral and limb development. The data identify a novel molecular mechanism of disease in CD in which the truncated protein leads to a distinct and more significant effect on SOX9 function.

Age estimation from anterior cervical vertebral ring apophysis ossification in South Africans.

Age estimation in living individuals around the age of 18years remains a difficult challenge. In this study, the anterior inferior vertebral ring apophysis development of cervical vertebrae C2, C3, and C4 of 496 white and 478 black South African individuals aged between 15 and 22years was assessed from cephalometric radiographs. Apophysis development was scored according to a four-stage scoring system. Ancestry and sex differences in apophysis maturation were assessed and likelihood values determined for individuals in each population group being 18years, based on developmental stages. Regression equations were developed for each ancestry and sex group. The results indicated that the median ages for attainment of stages 0, 1, and 2 were below the 18-year threshold for all ancestry and sex groups. Additionally, WSA males and BSA females attained stage 3 for C2, and WSA females attained stage 3 for C2, C3, and C4, below the 18-year threshold. The presence of stage 0 for black and white males in all three observed vertebrae and stage 1 for black males for C2, C3, and C4, white females for C2 and C3, and white males for C4 indicates an age below 18years (with a 95% or higher probability). The results indicate that anterior inferior apophysis ossification stages of C2, C3, and C4 can be used as a reliable indicator to determine the likelihood of being 18years of age at a 95% confidence index level. Apophysis development provides a valuable addition to the methods that can be used to assess age in the adolescent years.

Whole-Genome Resequencing Reveals Loci Associated With Thoracic Vertebrae Number in Sheep.

The number of vertebrae, especially thoracic vertebrae, is an important economic trait that may influence carcass length and meat production in animals. However, the genetic basis of vertebrae number in sheep is still poorly understood. To detect the candidate genes, 400 increased number of thoracic vertebrae (T14L6) and 200 normal (T13L6) Kazakh sheep were collected. We generated and sequenced 60 pools of genomic DNA (each pool prepared by mixing genomic DNA from 10 sheep with the same thoracic traits), with an average depth of coverage of 25.65×. We identified a total of 42,075,402 SNPs and 11 putatively selected genomic regions, including the VRTN gene and the HoxA gene family that regulate vertebral development. The most prominent areas of selective elimination were located in a region of chromosome 7, including VRTN, which regulates spinal development and morphology. Further investigation indicated that the expression level of the VRTN gene during fetal development was significantly higher in sheep with more thoracic vertebrae than in those with a normal number of thoracic vertebrae. A genome-wide comparison between sheep with increased and normal numbers of thoracic vertebrae showed that the VRTN gene is the major selection locus for the number of thoracic vertebrae in sheep and has the potential to be utilized in sheep breeding in the future.

NFAT5/TonEBP controls early acquisition of notochord phenotypic markers, collagen composition, and sonic hedgehog signaling during mouse intervertebral disc embryogenesis

High osmolarity, bound water, and hydrostatic pressure contribute to notochord mechanics and its morphogenesis into the nucleus pulposus (NP) compartment of the intervertebral disc. Indeed, the osmoadaptive transcription factor, nuclear factor of activated T-cells 5 (NFAT5 aka TonEBP), is robustly expressed by resident cells of the notochord and NP. Nevertheless, the molecular mechanisms that drive notochord osmoregulation and the functions of NFAT5 in disc embryogenesis remain largely unexplored. In this study, we show that deletion of NFAT5 in mice results in delayed vertebral column development and a reduced NP aspect ratio in the caudal spine. This phenotype is associated with lower levels of the T-box transcription factor, Brachyury, delayed expression of notochord phenotypic markers, and decreased collagen II deposition in the perinotochordal sheath and condensing mesenchyme. In addition, NFAT5 mutants showed a stage-dependent dysregulation of sonic hedgehog (Shh) signaling with non-classical expression of Gli1. Generation of mice with notochord-specific deletion of IFT88 (ShhcreERT2;Ift88f/f) supported this mode of Gli1 regulation. Using isolated primary NP cells and bioinformatics approaches, we further show that Ptch1 and Smo expression is controlled by NFAT5 in a cell autonomous manner. Altogether, our results demonstrate that NFAT5 contributes to notochord and disc embryogenesis through its regulation of hallmark notochord phenotypic markers, extracellular matrix, and Shh signaling.

How informative is vertebral development for the origin of lissamphibians?

AbstractThe origin of lissamphibians remains unresolved and different origins within Paleozoic early tetrapods have been proposed. Both the vertebral morphology as well as the ossification sequence of the vertebrae are potentially important character traits to test these different hypotheses. Here, we aim to determine if vertebral formation and ossification sequence in lissamphibians and early tetrapods are valuable indicatives of phylogenetic relationships. To answer this question, we examined the vertebral development in growth series of different salamander taxa and compared the results with literature data on vertebral development in frogs, caecilians and early tetrapods. In salamanders, caecilians and early tetrapods, the vertebral centrum develops by chordacentral and perichordal centrum formation, whereas in frogs, no chordacentral centrum formation has been observed so far. In the salamander taxa studied here, the following chondrification and ossification sequences are observed: chondrification first of the neural arches and then of the centra followed by ossification first of the centra and then of the neural arches. In frogs, in contrast, the neural arches ossify prior to the centra and it can be assumed that this developmental sequence represents the ancestral condition for tetrapods, as it characterizes some stem‐tetrapods and all temnospondyls from which growth series are known. However, the ossification sequence of the vertebral column is only incompletely known in stem‐amniotes which makes a comparison with extant lissamphibians difficult. This indicates that the mode of centrum formation and the ossification sequence are highly variable and cannot be used to determine the origin of lissamphibians within early tetrapods.

Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model

ObjectivesTo investigate the effect of endogenous Cas9 on genome editing efficiency in transgenic zebrafish.ResultsHere we have constructed a transgenic zebrafish strain that can be screened by pigment deficiency. Compared with the traditional CRISPR injection method, the transgenic zebrafish can improve the efficiency of genome editing significantly. At the same time, we first observed that the phenotype of vertebral malformation in early embryonic development of zebrafish after ZFERV knockout.ConclusionsThe transgenic zebrafish with expressed Cas9, is more efficient in genome editing. And the results of ZFERV knockout indicated that ERV may affect the vertebral development by Notch1/Delta D signal pathway.

Genome-wide meta-analysis and replication studies in multiple ethnicities identify novel adolescent idiopathic scoliosis susceptibility loci.

Adolescent idiopathic scoliosis (AIS) is the most common musculoskeletal disorder of childhood development. The genetic architecture of AIS is complex, and the great majority of risk factors are undiscovered. To identify new AIS susceptibility loci, we conducted the first genome-wide meta-analysis of AIS genome-wide association studies, including 7956 cases and 88459 controls from 3 ancestral groups. Three novel loci that surpassed genome-wide significance were uncovered in intragenic regions of the CDH13 (P-value_rs4513093=1.7E-15), ABO (P-value_ rs687621=7.3E-10) and SOX6 (P-value_rs1455114=2.98E-08) genes. Restricting the analysis to females improved the associations at multiple loci, most notably with variants within CDH13 despite the reduction in sample size. Genome-wide gene-functional enrichment analysis identified significant perturbation of pathways involving cartilage and connective tissue development. Expression of both SOX6 and CDH13 was detected in cartilage chondrocytes and chromatin immunoprecipitation sequencing experiments in that tissue revealed multiple HeK27ac-positive peaks overlapping associated loci. Our results further define the genetic architecture of AIS and highlight the importance of vertebral cartilage development in its pathogenesis.

Expression profiles of RA synthases and catabolic enzymes in newly hatched and metamorphosing larvae of Japanese flounder, Paralichthys olivaceus

Retinoic acid (RA) plays various embryogenesis and post-embryogenesis roles in vertebrates. As exposure of metamorphosing flounder larvae to RA has teratogenic effects on skin color and vertebral column development, harmonized RA synthesis and catabolism are likely essential in metamorphic development. To approach understanding of the roles of RA in flounder metamorphic development, we here examined the tissue mRNA expression of RA synthases (aldh1a1, aldh1a2, aldh1a3) and catabolic enzymes (cyp26a1, cyp26b1, cyp26c1) in newly hatched and metamorphosing larvae, and three-month-old juveniles by in situ hybridization (ISH). No ISH signal was detected for any genes from the skin and vertebral column susceptible to the teratogenic effects by RA. Since the intestine expressed aldh1a2 at high level in larvae but not in juvenile, it is a possibility that the larval intestine serves as a source of RA, and RA catabolic enzymes function at the level below sensitivity of ISH at vertebral column and skin development. We found that aldh1a2 and aldh1a3 were expressed along the margin of the tectum and the neurohypophysis of pituitary, respectively, both in contact with the cerebrospinal fluid (CSF), and cyp26b1 at the posterior tectum and cerebellum. We hypothesize that RA is supplied from the tectum and pituitary via the CSF for brain growth and maintenance, and cyp26b1 locally regulates RA contents in the brain.