Propineb induced notochord deformity, craniofacial malformation, and osteoporosis in zebrafish through dysregulated reactive oxygen species generation.

Dithiocarbamate (DTC) fungicides are contaminants that are ubiquitous in the environment. Exposure to DTC fungicides has been associated with a variety of teratogenic developmental effects. Propineb, a member of DTCs, was evaluated for the toxicological effects on notochord and craniofacial development, osteogenesis in zebrafish model. Embryos at 6 hours post-fertilization (hpf) were exposed to propineb at dosages of 1 and 4 µM. Morphological parameters were evaluated at exposure times of 24, 48, 72, and 120 hpf after propineb exposure. The survival and hatching rates as well as body length decreased at 1 and 4 µmol/L groups. Besides, transgenic zebrafish exposed to propineb showed abnormal vacuole biogenesis in notochord cells at the early stage of development. The expression of collagen type 2 alpha 1a (col2a1a), sonic hedgehog (shh), and heat shock protein family B member 11 (hspb11) measured by quantitative PCR and in situ hybridization experiment of col8a1a gene have consolidated the proposal process. Besides, Alcian blue, calcein, and alizarin red staining profiles displayed craniofacial malformations and osteoporosis were induced following propineb exposure. PPB exposure induced the changes in oxidative stress and reactive oxygen species inhibitor alleviated the deformities of PPB. Collectively, our data suggested that propineb exposure triggered bone abnormalities in different phenotypes of zebrafish. Therefore, propineb is a potential toxicant of high priority concern for aquatic organisms.

Multistage surgical repair for split notochord syndrome with neuroenteric fistula: case report.

Split notochord syndrome (SNS) is a rare congenital defect of the central nervous system and has been associated with several anomalies affecting multiple organ systems. One association has been communication with the gastrointestinal tract and the spine, previously identified as a neuroenteric fistula (NEF). Here, the authors describe the unique case of a female infant with SNS and NEF treated with a multistage surgical repair. The three-stage operative plan included a two-stage repair of the defect and temporary subgaleal shunting followed by delayed ventriculoperitoneal shunt placement. The infant recovered well postsurgery and over a 5-year follow-up. A case description, surgical techniques, and rationale are reported. Additionally, a systematic review of the literature utilizing the MEDLINE database was performed.Treatment of SNS with NEF using a multidisciplinary multistaged approach to repair the intestinal defect, close the neural elements, and divert cerebrospinal fluid to the peritoneum is shown to be a safe and viable option for future cases.

Diastematobulbia type II without associated dermoid tumor: case report.

Split cord malformation (SCM) is a term used for all double spinal cords. It represents 3.8%-5% of spinal dysraphisms. Pang et al.'s embryological theory proposes the formation of an "accessory neurenteric canal" that communicates with the yolk sac and amnion. To the authors' knowledge, only three cases of diastematobulbia (basicranial SCM) associated with a spur or dermoid have been reported in the literature.The case patient is a newborn girl with an occipitocervical meningocele and dermal sinus associated with an anomaly of notochordal development in the transition between the medulla oblongata and the spinal cord (diastematobulbia) without a bony septum or dermoid cyst. The patient also has agenesis of the atlas and an absence of corticospinal tract decussation. This patient underwent reconstruction of the occipital meningocele and dermal sinus excision.To the authors' knowledge, this is the first described case of type II diastematobulbia (basicranial SCM), without a dermoid cyst. The authors analyzed the embryological errors present in the case patient and considered the option of further surgical treatment depending on the evolution of the patient's condition. At the time of this report, the patient had shown normal psychomotor development. However, this fact may only be due to the patient's young age. Considering that after initial untethering the patient remained clinically asymptomatic, conservative and close surveillance has been and continues to be the proposed treatment.

Toxicological assessment and developmental abnormalities induced by butylparaben and ethylparaben exposure in zebrafish early-life stages.

Toxicological effects of butylparaben (BuP) and ethylparaben (EtP) on zebrafish (Danio rerio) early-life stages are not well established. The present study evaluated, using zebrafish embryos and larvae, the toxicity of BuP and EtP through benchmark dose (BMD) approach. BuP was more toxic than EtP to zebrafish larvae. In fact, Lethal Concentration 50 (LC50) values at 96 h post-fertilization (hpf) for BuP and EtP were 2.34 mg/L and 20.86 mg/L, respectively. Indeed, BMD confidence interval (lower bound (BMDL) - upper bound (BMDU) was 0.91-1.92 mg/L for BuP and 10.8-17.4 mg/L for EtP. Zebrafish embryos exposed to 1 mg/L, 2.5 mg/L of BuP and 5 mg/L, 10 mg/L, 20 mg/L, 30 mg/L of EtP showed several developmental abnormalities and teratological effects compared to negative control. Exposed zebrafish developed reduced heartbeat, reduction in blood circulation, blood stasis, pericardial edema, deformed notochord and misshaped yolk sac. Embryos exposed to the highest concentrations of the chemicals (2.5 mg/L of BuP, 10 mg/L, 20 mg/L and 30 mg/L of EtP) showed the developmental abnormalities at 48 hpf while those treated with 1 mg/L of BuP and 10 mg/L of EtP reported behavioral changes at 72 hpf, including trembling of head, pectoral fins and spinal cord. This research identified the lethal and sublethal effects of BuP and EtP in zebrafish early-life stages and could be helpful to elucidate the developmental pathways of toxicity of parabens.

Dstyk mutation leads to congenital scoliosis-like vertebral malformations in zebrafish via dysregulated mTORC1/TFEB pathway.

Congenital scoliosis (CS) is a complex genetic disorder characterized by vertebral malformations. The precise etiology of CS is not fully defined. Here, we identify that mutation in dual serine/threonine and tyrosine protein kinase (dstyk) lead to CS-like vertebral malformations in zebrafish. We demonstrate that the scoliosis in dstyk mutants is related to the wavy and malformed notochord sheath formation and abnormal axial skeleton segmentation due to dysregulated biogenesis of notochord vacuoles and notochord function. Further studies show that DSTYK is located in late endosomal/lysosomal compartments and is involved in the lysosome biogenesis in mammalian cells. Dstyk knockdown inhibits notochord vacuole and lysosome biogenesis through mTORC1-dependent repression of TFEB nuclear translocation. Inhibition of mTORC1 activity can rescue the defect in notochord vacuole biogenesis and scoliosis in dstyk mutants. Together, our findings reveal a key role of DSTYK in notochord vacuole biogenesis, notochord morphogenesis and spine development through mTORC1/TFEB pathway.

Spontaneous cerebrospinal fluid rhinorrhoea with meningitis secondary to ecchordosis physaliphora.

Transclival CSF fistulae are rare. We report a 40-year-old female who presented with meningitis after a short history of CSF rhinorrhoea. The defect resulted from the rare notochordal remnant, ecchordosis physaliphora (EP), and was successfully resected and repaired endoscopically.

Acute toxicity screening of different extractions, components and constituents of Polygonum multiflorum Thunb. on zebrafish (Danio rerio) embryos in vivo.

Polygonum multiflorum Thunb. has been used widely in East Asia in treatment of diseases associated with aging. However, there are many reports referred to the toxicity of P. multiflorum, especially for liver adverse reactions. The toxicity of it is caused by over dosage or by the herb itself remains unclear. The aim of this study was to study the toxicity of different extractions, components and constituents of P. multiflorum, which were assessed in zebrafish embryos. Firstly, the difference of extracting solvent to the toxicity of P. multiflorum was researched to probe the influence of usages to the safety of P. multiflorum. The toxicity of 70% EtOH extract is considerably higher than that of other extracts. Secondly, 70% EtOH extract was subjected to macroporous resin (DM-8) eluting with a gradient of water and EtOH (H2O, 25% EtOH, 40% EtOH and 95% EtOH) to give four components (A-D). The toxicity of the component (D) showed higher than the other components (A-C). Thus, the component (D) was taken more attentions to research. Lastly, study on the chemical constituents of the component (D), 27 compounds, including 7 anthraquinones (1-7), 8 stilbenes (8-15), 7 anthrones (16-22), 3 cinnamic acid amides (23-25), 2 naphthols (26-27) were isolated and assessed in zebrafish embryos. Compounds 1-3, 16-22 and 26-27 showed severe toxicity against the zebrafish embryos while other compounds, such as stilbenes, showed no obvious toxicity.

Copper pyrithione, a booster biocide, induces abnormal muscle and notochord architecture in zebrafish embryogenesis.

The metal pyrithiones, principally zinc (ZnPT) and copper (CuPT), are replacing tributyltin (TBT) as antifouling agents. Zebrafish embryos were exposed within the first hour after fertilization to 12 and 64 µg/L of CuPT for 24 h. Morphological abnormalities in notochord and muscle architecture were observed at 96 h post fertilization (hpf). TEM revealed abnormal electron dense deposits in the notochord sheath and muscle fiber degeneration in animals treated with 12 µg/L of CuPT. Embryos that were exposed to 64 µg/L of CuPT displayed severe muscle fiber degeneration including abnormal A and I band patterning and altered z disk arrangement. Abnormalities in the notochord sheath, swelling of the mitochondria and numerous lipid whorls were also noted. Total antioxidant capacity was significantly decreased in embryos exposed to 12 and 64 µg/L of CuPT. Acridine orange staining revealed an increase in apoptosis particularly in the brain, eye, heart and tail regions of both treatment groups. Apoptosis was confirmed with an increase in caspase 3/7 activity in both treatment groups. Severe alternations in primary motor neuron axon extensions, slow tonic muscle fibers and fast twitch fibers were observed in CuPT treated embryos. There was a significant upregulation in sonic hedgehog and myod1 expression at 24 hpf in the 12 µg/L treatment group. Exposed zebrafish embryos showed ultra-structural hallmarks of peroxidative injury and cell death via apoptosis. These changes question the use of copper pyrithione as an antifouling agent.

Disordered vertebral and rib morphology in pudgy mice. Structural relationships to human scoliosis.

Normal and abnormal vertebral development have been studied over the past 200 years at increasing levels of resolution as techniques for biological investigation have improved. Disordered development of the axial skeleton from the early embryonic period on leads to structurally malformed vertebrae and intervertebral discs and ribs causing the severe deformities of scoliosis, kyphosis, and kyphoscoliosis. Developmental malformation of the axial skeleton therefore has led to considerable biological and clinical interest. This work will detail our studies on the structural deformities of the vertebral column and adjacent ribs in the pudgy mouse [1] caused by mutations in the delta-like 3 (Dll3) gene of the Notch family [2]. While gene abnormalities in the pudgy mouse have been outlined, there has been no in-depth assessment of the histopathology of the pudgy vertebral and rib abnormalities that this study will provide. In addition, although congenital scoliosis has been recognized as a clinical problem since the mid-nineteenth century (1800s) [3] and accurately defined by radiography since the early twentieth century (1900s) [4-6], there have been few detailed histopathologic studies of human cases. We will also relate our histopathologic findings in the pudgy mouse to the histopathology of human vertebral and rib malformations in clinical cases of congenital scoliosis, one of which we defined in detail previously [7].

Neuroendoscopic Trans-Third Ventricular Approach for Surgical Management of Ecchordosis Physaliphora.

BACKGROUND: We sought to report the successful surgical management of a case of ecchordosis physaliphora (EP) using a neuro-endoscopic trans-third ventricular approach (ETTVA) and to provide a current review of literature on EP. CASE DESCRIPTION: A 57-year-old man presenting with a 2-year history of diplopia due to right abducens nerve palsy and paresthesia of the left body underwent magnetic resonance imaging, which revealed a retroclival intracisternal lesion. The cystic lesion was considered to be most likely EP according to neuroradiologic features. The patient underwent an endoscopic trans-third ventricular resection. A pediatric endoscope was passed from a precoronal burr hole through the left lateral into the third ventricle. The floor of the third ventricle was opened by a 2-micron laser. This approach permitted us to expose the lesion in the retroclival cistern and follow up with a subtotal removal. Remnants of the capsule, which were firmly adherent to small pontine arteries and the left abducens nerve, were left. Histology confirmed EP. The patient recovered well from surgery, and symptoms regressed at clinical follow-up. CONCLUSION: The endoscopic approach for third ventriculostomy can also be used for the surgical management of retroclival lesions. However, a small pediatric endoscope with an angled view, which can be passed through the floor of the third ventricle without causing harm, is mandatory to explore all important structures in the narrow surgical space. Limitations in this delicate environment are firm adhesions to vessels and nerves because only 1-instrument manipulation is possible and bleeding must be avoided.

Ecchordosis Physaliphora: Evaluation with Precontrast and Contrast-Enhanced Fast Imaging Employing Steady-State Acquisition MR Imaging Based on Proposed New Classification.

PURPOSE: Ecchordosis physaliphora (EP) is a notochordal remnant typically located at the dorsal surface of the clivus, which has to be distinguished from the other retroclival lesions. Our aim is to investigate the imaging features of intracranial EP using precontrast and contrast-enhanced fast imaging employing steady-state acquisition (FIESTA). METHODS: We retrospectively evaluated the precontrast and contrast-enhanced FIESTA images of 399 patients with temporal magnetic resonance imaging to detect "classical EP" and "possible EP." The classical EP was classified into type A (hyperintense excrescence (cyst-like component) on the dorsal surface of the clivus) and type B (hyperintense excrescence plus a hyperintense lesion within the clivus). Possible EP was subdivided as incomplete EP (T2-hypointense protrusion of the clivus) and EP variant (hyperintense lesion within the clivus alone). RESULTS: We found 31 (31 of 399, 7.7 %) EPs of which 11 were defined as classical EP (2.7 %) and 20 were defined as possible EP (5.0 %). Of the 11 classical EPs, 7 (63.6 %) were diagnosed as type A and 4 (% 36.4) were diagnosed as type B. Of the 20 possible EPs, 19 were classified as incomplete EP (95.0 %) and one was classified as EP variant (5.0 %). CONCLUSIONS: Contrast-enhanced FIESTA images are helpful in the assessment of EP, although we do not define a role in the current classification proposed by Chihara et al. (Eur Radiol 23:2854-2860, 2013).

Notochord manipulation does not impact oesophageal and tracheal formation from isolated foregut in 3D explant culture.

BACKGROUND: Tracheo-oesophageal malformations result from disturbed foregut separation during early development. The notochord, a specialised embryonic structure, forms immediately adjacent to the dividing foregut. In the Adriamycin mouse model of oesophageal atresia, foregut and notochord abnormalities co-exist, and the site and severity of foregut malformations closely correlate to the position and extent of the notochord defects. Notochord and foregut abnormalities also co-exist in the Noggin Knockout mouse as well in a small number of human cases. The notochord is a source of powerful molecular signals during early embryogenesis, being particularly important for neural crest development. The influence of notochord signaling on the adjacent foregut is not known. The purpose of this study was to examine the impact of notochord manipulation on foregut separation using a robust 3D explant method for culturing isolated foregut which permits oeosphageal and tracheal formation in vitro. METHODS: Foregut was micro-dissected from embryonic day 9 mice (License B100/4447 Irish Medicines Board), embedded in collagen and cultured for 48 h with native notochord intact (n = 6), notochord removed (n = 10) or additional notochord transplanted from stage matched controls (n = 8). Specimens were analysed for foregut morphology and molecular patterning using immunohistochemistry for Hnf3b (an endoderm marker) and Sox2 (a notochord and oesophageal marker) on cryosections. RESULTS: Foregut separation into distinct oesophagus and trachea was observed in isolated foregut specimens with or without their native notochord. In specimens with additional notochord transplants, foregut morphology and molecular patterning were comparable to controls whether or not the native notochord was maintained. In particular foregut separation was not disrupted by the transplantation of additional notochord at the dorsal foregut endoderm. CONCLUSION: The relationship between the embryonic foregut and notochord is complex and ill-defined; however, the notochord does not contribute essentially to oesophagus and trachea formation beyond E9 in the mouse, and the transplantation of additional notochord does not disrupt foregut separation in 3D explant culture.

Sulphated glycosaminoglycans and proteoglycans in the developing vertebral column of juvenile Atlantic salmon (Salmo salar).

In the present study, the distribution of sulphated glycosaminoglycans (GAGs) in the developing vertebral column of Atlantic salmon (Salmo salar) at 700, 900, 1100 and 1400 d° was examined by light microscopy. The mineralization pattern was outlined by Alizarin red S and soft structures by Alcian blue. The temporal and spatial distribution patterns of different types of GAGs: chondroitin-4-sulphate/dermatan sulphate, chondroitin-6-sulphate, chondroitin-0-sulphate and keratan sulphate were addressed by immunohistochemistry using monoclonal antibodies against the different GAGs. The specific pattern obtained with the different antibodies suggests a unique role of the different GAG types in pattern formation and mineralization. In addition, the distribution of the different GAG types in normal and malformed vertebral columns from 15 g salmon was compared. A changed expression pattern of GAGs was found in the malformed vertebrae, indicating the involvement of these molecules during the pathogenesis. The molecular size of proteoglycans (PGs) in the vertebrae carrying GAGs was analysed with western blotting, and mRNA transcription of the PGs aggrecan, decorin, biglycan, fibromodulin and lumican by real-time qPCR. Our study reveals the importance of GAGs in development of vertebral column also in Atlantic salmon and indicates that a more comprehensive approach is necessary to completely understand the processes involved.

Fusaric acid induces a notochord malformation in zebrafish via copper chelation.

Over a thousand extracts were tested for phenotypic effects in developing zebrafish embryos to identify bioactive molecules produced by endophytic fungi. One extract isolated from Fusarium sp., a widely distributed fungal genus found in soil and often associated with plants, induced an undulated notochord in developing zebrafish embryos. The active compound was isolated and identified as fusaric acid. Previous literature has shown this phenotype to be associated with copper chelation from the active site of lysyl oxidase, but the ability of fusaric acid to bind copper ions has not been well described. Isothermal titration calorimetry revealed that fusaric acid is a modest copper chelator with a binding constant of 4.4 × 10(5) M(-1). These results shed light on the toxicity of fusaric acid and the potential teratogenic effects of consuming plants infected with Fusarium sp.

Incidental findings of skull-base abnormalities in cone-beam computed tomography scans with consultation by maxillofacial radiologists.

INTRODUCTION: Cone-beam computed tomography (CBCT) gives orthodontists and other dental clinicians 3-dimensional information for planning treatment in the craniofacial region. Often overlooked are incidental findings outside the treatment region of interest. METHODS: Two patients with incidental findings of skull-base abnormalities are presented. The orthodontic patient was tentatively diagnosed with a notochordal remnant in the clivus; the implant patient exhibited an empty sella turcica. RESULTS: For the clivus lesion in the orthodontic patient, an artifact was ruled out after a second CBCT image and further distinguished from a fat-containing tumor after magnetic resonance imaging. The impression after magnetic resonance imaging was a notochordal remnant, although chordoma was also included in the differential, warranting a 6-month follow-up magnetic resonance image to confirm the diagnosis. The CBCT study for the implant patient demonstrated an enlarged sella turcica. The impression after the magnetic resonance imaging was an enlarged and partially empty sella with no evidence of a pituitary mass. CONCLUSIONS: Orthodontists and implant surgeons may come across incidental findings outside their area of expertise on CBCT scans, highlighting the importance of appropriate consultation with maxillofacial radiologists. Notochordal remnants may present as nonexpansile intraosseous low-density areas. The challenge in distinguishing these lesions radiographically with chordomas warrants follow-up to confirm a diagnosis. An empty sella is a noteworthy finding because of its potential for endocrine and neuro-ophthalmological disorders despite an asymptomatic presentation.

Developmental toxicity and neurotoxicity of two matrine-type alkaloids, matrine and sophocarpine, in zebrafish (Danio rerio) embryos/larvae.

Matrine and sophocarpine are two major matrine-type alkaloids included in the traditional Chinese medicine (TCM) Kushen (the root of Sophora flavescens Ait.). They have been widely used clinically in China, however with few reports concerning their potential toxicities. This study investigated the developmental toxicity and neurotoxicity of matrine and sophocarpine on zebrafish embryos/larvae from 0 to 96/120h post fertilization (hpf). Both drugs displayed teratogenic and lethal effects with the EC50 and LC50 values at 145 and 240mg/L for matrine and 87.1 and 166mg/L for sophocarpine, respectively. Exposure of matrine and sophocarpine significantly altered spontaneous movement and inhibited swimming performance at concentrations below those causing lethality and malformations, indicating a neurotoxic potential of both drugs. The results are in agreement with most mammalian studies and clinical observations.

Loss of col8a1a function during zebrafish embryogenesis results in congenital vertebral malformations.

Congenital vertebral malformations (CVM) occur in 1 in 1000 live births and in many cases can cause spinal deformities, such as scoliosis, and result in disability and distress of affected individuals. Many severe forms of the disease, such as spondylocostal dystostosis, are recessive monogenic traits affecting somitogenesis, however the etiologies of the majority of CVM cases remain undetermined. Here we demonstrate that morphological defects of the notochord in zebrafish can generate congenital-type spine defects. We characterize three recessive zebrafish leviathan/col8a1a mutant alleles ((m531, vu41, vu105)) that disrupt collagen type VIII alpha1a (col8a1a), and cause folding of the embryonic notochord and consequently adult vertebral column malformations. Furthermore, we provide evidence that a transient loss of col8a1a function or inhibition of Lysyl oxidases with drugs during embryogenesis was sufficient to generate vertebral fusions and scoliosis in the adult spine. Using periodic imaging of individual zebrafish, we correlate focal notochord defects of the embryo with vertebral malformations (VM) in the adult. Finally, we show that bends and kinks in the notochord can lead to aberrant apposition of osteoblasts normally confined to well-segmented areas of the developing vertebral bodies. Our results afford a novel mechanism for the formation of VM, independent of defects of somitogenesis, resulting from aberrant bone deposition at regions of misshapen notochord tissue.

Mutations in the T (brachyury) gene cause a novel syndrome consisting of sacral agenesis, abnormal ossification of the vertebral bodies and a persistent notochordal canal.

BACKGROUND: The T gene (brachyury gene) is the founding member of the T-box family of transcription factors and is vital for the formation and differentiation of the mesoderm and the axial development of all vertebrates. RESULTS: We report here on four patients from three consanguineous families exhibiting sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies, and the identification and characterisation of their underlying genetic defect. Given the consanguineous nature and the similarity of the phenotypes between the three families, we performed homozygosity mapping and identified a common 4.1 Mb homozygous region on chromosome 6q27, containing T, brachyury homologue (mouse) or T. Sequencing of T in the affected individuals led to the identification of a homozygous missense mutation, p.H171R, in the highly conserved T-box. The homozygous mutation results in diminished DNA binding, increased cell growth, and interferes with the normal expression of genes involved in ossification, notochord maintenance and axial mesoderm development. CONCLUSIONS: We have identified a shared homozygous mutation in three families in T and linked it to a novel syndrome consisting of sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies. We suggest that screening for the ossification of the vertebrae is warranted in patients with sacral agenesis to evaluate the possible causal involvement of T.