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Mammals are generally poor at tissue regeneration, in contrast, fish maintain a high capacity for regenerating complex tissues after injury. Using larval zebrafish, we show that tail amputation triggers an metabolic shift to glycolysis in cells surrounding the notochord as they reposition to the amputation site. Blocking glycolysis prevents the fin from regenerating after amputation due to the failure to form a normal, pluripotent blastema. We performed a time series of scRNA-sequencing on regenerating tails under normal conditions or in the absence of glycolysis. Strikingly, we detected a transient cell population in the single cell analysis that represents notochord sheath cells undergoing a TGF–β dependent dedifferentiation and epithelium-to-mesenchyme transition to become pluripotent blastema cells. We further demonstrated that the metabolic switch to glycolysis is required for TGF–β signaling and blocking either glycolysis or TGF–β receptors results in aberrant blastema formation through the suppression of essential EMT mediators such assnai1.

scRNA-seq of an invariant cell lineage reveals insights into asymmetric cell division, FGF signaling, and notochord specification., Progressive unfolding of gene expression cascades underlies diverse embryonic lineage development. Here, we report a single-cell RNA sequencing analysis of the complete and invariant embryonic cell lineage of the tunicate Ciona savignyi from fertilization to the onset of gastrulation. We reconstructed a developmental landscape of 47 cell types over eight cell cycles in the wild-type embryo and identified eight fate transformations upon fibroblast growth factor (FGF) inhibition. For most FGF-dependent asymmetric cell divisions, the bipotent mother cell displays the gene signature of the default daughter fate. In convergent differentiation of the two notochord lineages, we identified additional gene pathways parallel to the master regulator T/Brachyury. Last, we showed that the defined Ciona cell types can be matched to E6.5-E8.5 stage mouse cell types and display conserved expression of limited number of transcription factors. This study provides a high-resolution single-cell dataset to understand chordate early embryogenesis and cell lineage differentiation.

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.

One of the most frequently applied techniques in zebrafish (Danio rerio) research is the visualisation or manipulation of specific cell populations using transgenic reporter lines. The generation of these transgenic zebrafish, displaying cell- or tissue-specific expression of frequently used fluorophores such as Green Fluorescent Protein (GFP) or mCherry, is relatively easy using modern techniques. Fluorophores with different emission wavelengths and driven by different promoters can be monitored simultaneously in the same animal. Photoconvertible fluorescent proteins (pcFPs) are different from these standard fluorophores because their emission spectrum is changed when exposed to UV light, a process called photoconversion. Here, the benefits and versatility of using pcFPs for both single and dual fluorochrome imaging in zebrafish skeletal research in a previously generated osx:Kaede transgenic line are illustrated. In this line, Kaede, which is expressed under control of the osterix, otherwise known as sp7, promoter thereby labelling immature osteoblasts, can switch from green to red fluorescence upon irradiation with UV light. First, this study demonstrates that osx:Kaede exhibits an expression pattern similar to a previously described osx:nuGFP transgenic line in both larval and adult stages, hereby validating the use of this line for the imaging of immature osteoblasts. More in-depth experiments highlight different applications for osx:Kaede, such as lineage tracing and its combined use with in vivo skeletal staining and other transgenic backgrounds. Mineral staining in combination with osx:Kaede confirms osteoblast-independent mineralisation of the notochord. Osteoblast lineage tracing reveals migration and dedifferentiation of scleroblasts during fin regeneration. Finally, this study shows that combining two transgenics, osx:Kaede and osc:GFP, with similar emission wavelengths is possible when using a pcFP such as Kaede.

This chapter discusses our current knowledge on the major segregation events that lead to the individualization of the building blocks of vertebrate organisms, starting with the segregation between "outer" and "inner" cells, the separation of the germ layers and the maintenance of their boundaries during gastrulation, and finally the emergence of the primary axial structure, the notochord. The amphibian embryo is used as the prototypical model, to which fish and mouse development are compared. This comparison highlights a striking conservation of the basic processes. It suggests that simple principles may account for the formation of divergent structures. One of them is based on the non-adhesive nature of the apical domain of epithelial cells, exploited to segregate superficial and deep cell populations as a result of asymmetric division. The other principle involves differential expression of contact cues, such as ephrins and protocadherins, to build up high tension along adhesive interfaces, which efficiently creates sharp boundaries.

Background The Oculo‐Auriculo‐Vertebral Spectrum (OAVS) or Goldenhar Syndrome is an embryonic developmental disorder characterized by hemifacial microsomia associated with auricular, ocular and vertebral malformations. The clinical heterogeneity of this spectrum and its incomplete penetrance limited the molecular diagnosis. In this study, we describe a novel causative gene, ZYG11B. Methods A sporadic case of OAVS was analyzed by whole exome sequencing in trio strategy. The identified candidate gene, ZYG11B, was screened in 143 patients by next generation sequencing. Overexpression and immunofluorescence of wild‐type and mutated ZYG11B forms were performed in Hela cells. Moreover, morpholinos were used for transient knockdown of its homologue in zebrafish embryo. Results A nonsense de novo heterozygous variant in ZYG11B, (NM_024646, c.1609G>T, p.Glu537*) was identified in a single OAVS patient. This variant leads in vitro to a truncated protein whose subcellular localization is altered. Transient knockdown of the zebrafish homologue gene confirmed its role in craniofacial cartilages architecture and in notochord development. Moreover, ZYG11B expression regulates a cartilage master regulator, SOX6, and is regulated by Retinoic Acid, a known developmental toxic molecule leading to clinical features of OAVS. Conclusion Based on genetic, cellular and animal model data, we proposed ZYG11B as a novel rare causative gene for OAVS., The Oculo‐Auriculo‐Vertebral Spectrum or Goldenhar Syndrome is an embryonic developmental disorder characterized by hemifacial microsomia associated with auricular, ocular and vertebral malformations. Its etiology is poorly characterized. In this study, we describe a novel causative gene, ZYG11B.

The segmented vertebral column comprises a repeat series of vertebrae, each consisting of two key components: the vertebral body (or centrum) and the vertebral arches. Despite being a defining feature of the vertebrates, much remains to be understood about vertebral development and evolution. Particular controversy surrounds whether vertebral component structures are homologous across vertebrates, how somite and vertebral patterning are connected, and the developmental origin of vertebral bone-mineralizing cells. Here, we assemble evidence from ichthyologists, palaeontologists and developmental biologists to consider these issues. Vertebral arch elements were present in early stem vertebrates, whereas centra arose later. We argue that centra are homologous among jawed vertebrates, and review evidence in teleosts that the notochord plays an instructive role in segmental patterning, alongside the somites, and contributes to mineralization. By clarifying the evolutionary relationship between centra and arches, and their varying modes of skeletal mineralization, we can better appreciate the detailed mechanisms that regulate and diversify vertebral patterning.

The founder cells of the Nucleus pulposus, the centre of the intervertebral disc, originate in the embryonic notochord. After birth, mature notochordal cells (NC) are identified as key regulators of disc homeostasis. Better understanding of their biology has great potential in delaying the onset of disc degeneration or as a regenerative-cell source for disc repair. Using human pluripotent stem cells, we developed a two-step method to generate a stable NC-like population with a distinct molecular signature. Time-course analysis of lineage-specific markers shows that WNT pathway activation and transfection of the notochord-related transcription factor NOTO are sucient to induce high levels of mesendoderm progenitors and favour their commitment toward the notochordal lineage instead of paraxial and lateral mesodermal or endodermal lineages. This study results in the identification of NOTO-regulated genes including some that are found expressed in human healthy disc tissue and highlights NOTO function in coordinating the gene network to human notochord di erentiation.

Chordomas are malignant, highly recurrent tumors of the midline skeleton that arise from the remnants of the notochord. The development of systemic therapy is critically important to ultimately managing this tumor. Several ongoing trials are attempting to use molecular targeted therapies for mutated pathways in recurrent and advanced chordomas and have shown promise. In addition, immunotherapies, including brachyury-directed vaccination and checkpoint inhibition, have also been attempted with encouraging results. This article discusses the major pathways that have been implicated in the pathogenesis of chordoma with an emphasis on molecular vulnerabilities that future therapies are attempting to exploit.

The notochord is a defining feature of chordates. During notochord formation in vertebrates and tunicates, notochord cells display dynamic morphogenetic movement, called convergent extension, in which cells intercalate and align at the dorsal midline. However, in cephalochordates, the most basal group of chordates, the notochord is formed without convergent extension. It is simply developed from mesodermal cells at the dorsal midline. This suggests that convergent extension movement of notochord cells is a secondarily acquired developmental attribute in the common ancestor of olfactores (vertebrates + tunicates), and that the chordate ancestor innovated the notochord upon a foundation of morphogenetic mechanisms independent of cell movement. Therefore, this review focuses on biological features specific to notochord cells, which have been well studied using clawed frogs, zebrafish, and tunicates. Attributes of notochord cells, such as vacuolation, membrane trafficking, extracellular matrix formation, and apoptosis, can be understood in terms of two properties: turgor pressure of vacuoles and strength of the notochord sheath. To maintain the straight rod-like structure of the notochord, these parameters must be counterbalanced. In the future, the turgor pressure-sheath strength model, proposed in this review, will be examined in light of quantitative molecular data and mathematical simulations, illuminating the evolutionary origin of the notochord.

Pathological changes in the vertebral column of farmed Atlantic salmon in Norway have been reported since the 1990s. Based on the characteristic radiographic findings, we here present a vertebral column deformity named "curved cross-stitch vertebrae" that mainly affects the middle aspect of the vertebral column. Sixty fish, from the west/northwest coast of mid-Norway, were sampled at slaughter and examined by radiography, computed tomography (CT), necropsy, macrophotography, and histology. The vertebral deformities were radiographically graded as mild, moderate, or marked. The main differences between these grades of changes were defined by increased curving of the peripheries of endplates, reduced intervertebral spaces, and vertical displacement of the vertebrae. The curved rims of endplates were located peripheral to a continuous and approximately circular borderline. The CT studies revealed small, multifocal, hypo-attenuating, round to crescent-shaped areas in the notochord, compatible with the presence of gas. Additionally, histology revealed that the axial parts of endplates had circular zones with perforations, through which either notochordal tissue prolapsed into the vertebrae or vascularized fibrochondroid proliferations extended from the vertebrae into the notochord. Inflammation was present in many vertebral bodies. To the best of our knowledge, this is the first report of gas in the notochord of fish.

The molecular expression profiles of zebrafish ep2aand ep4bhave not been defined to-date. Phylogenetic treesof EP2a and EP4b in zebrafish and other speciesrevealed that human EP4and zebrafish EP4b were more closely related than EP2a. ZebrafishEP2a is a 281amino acid protein with high identity to that of human (43%), mouse (44%), rat (43%),dog(44%), cattle(41%), and chicken (41%). ZebrafishEP4bencoded a precursor of 497amino acids with high amino acid identity to that of mammals, including human (57%), mouse (54%), rat (55%), dog (55%), cattle (56%), and chicken (54%). Whole-mount in situ hybridizationrevealed that ep2awas robustly expressed in the anterior four somitesat the 10-somites stages, but was absent in the somites at 19 hpf.Itwas observedagainin the pronephric duct at 24 hpf, in the intermediate cell masslocated in the trunk, and in the rostral blood island at 30 hpf. Ep2awas also expressed in the notochordat 48 hpf. During somitogenesis, ep4bwas highly expressed in the eyes, somites, and the trunk neural crest. From 30 to 48 hpf, ep4bcould be detected in the posterior cardinal vein and the neighboring ICM. From these data, we conclude that ep2aand ep4bare conserved in vertebrates and that the presence of ep2aand ep4btranscripts during developmental stages infers their role during early zebrafish larval development. In addition, the variable expression of the two receptor isoforms wasstrongly suggestive of divergent roles of molecular regulation.

The lymphatic vasculature develops primarily from pre-existing veins. A pool of lymphatic endothelial cells (LECs) first sprout from cardinal veins followed by migration and proliferation to colonise embryonic tissues. While much is known about the molecular regulation of LEC fate and sprouting during early lymphangiogenesis, we know far less about the instructive and permissive signals that support LEC migration through the embryo. Using a forward genetic screen, we identified mbtps1 and sec23a, components of the COP-II protein secretory pathway, as essential for developmental lymphangiogenesis. In both mutants, LECs initially depart the cardinal vein but then fail in their ongoing migration. A key cargo that failed to be secreted in both mutants was a type II collagen (Col2a1). Col2a1 is normally secreted by notochord sheath cells alongside which LECs migrate. col2a1a mutants displayed defects in the migratory behaviour of LECs and failed lymphangiogenesis. These studies thus identify Col2a1 as a key cargo secreted by notochord sheath cells and required for the migration of LECs. These findings combine with our current understanding to suggest that successive cell-to-cell and cell-matrix interactions regulate the migration of LECs through the embryonic environment during development.

The notochord drives longitudinal growth of the body axis by convergent extension, a highly conserved developmental process that depends on non-canonical Wnt/PCP (planar cell polarity) signaling. However, the role of cell-matrix interactions mediated by integrins in the development of the notochord is unclear. We developed transgenic Cre mice, in which β1 integrin gene is ablated at E8.0 in the notochord only or in the notochord and tail bud. These β1 integrin conditional mutants display misaligned, malformed vertebral bodies, hemi-vertebrae and truncated tails. From early somite stages, the notochord was interrupted and displaced in these mutants. Convergent extension of the notochord was impaired with defective cell movement. Treatment of E7.25 wild-type embryos with anti-β1 integrin blocking antibodies to target node pit cells, disrupted asymmetric localization of VANGL2. Our study implicates pivotal roles of β1 integrin for the establishment of PCP and convergent extension of the developing notochord, its structural integrity and positioning, thereby ensuring development of the nucleus pulposus and the proper alignment of vertebral bodies and intervertebral discs. Failure of this control may contribute to human congenital spine malformations.

The notochord defines the axial structure of all vertebrates during development. Notogenesis is a result of major cell reorganization in the mesoderm, the convergence and the extension of the axial cells. However, it is currently not fully understood how these processes act together in a coordinated way during notochord formation. The prechordal plate is an actively migrating cell population in the central mesoderm anterior to the trailing notochordal plate cells. We show that prechordal plate cells express Protocadherin 18a (Pcdh18a), a member of the cadherin superfamily. We find that Pcdh18a-mediated recycling of E-cadherin adhesion complexes transforms prechordal plate cells into a cohesive and fast migrating cell group. In turn, the prechordal plate cells subsequently instruct the trailing mesoderm. We simulated cell migration during early mesoderm formation using a lattice-based mathematical framework and predicted that the requirement for an anterior, local motile cell cluster could guide the intercalation and extension of the posterior, axial cells. Indeed, a grafting experiment validated the prediction and local Pcdh18a expression induced an ectopic prechordal plate-like cell group migrating towards the animal pole. Our findings indicate that the Pcdh18a is important for prechordal plate formation, which influences the trailing mesodermal cell sheet by orchestrating the morphogenesis of the notochord. Electronic supplementary material The online version of this article (10.1007/s00418-020-01887-5) contains supplementary material, which is available to authorized users.

While it is well known that the notochord of bony fishes changes over developmental time, less is known about how it varies across different body regions. In the development of the Atlantic salmon, Salmo salar L., cranial and caudal ends of the notochord are overlaid by the formation of the bony elements of the neurocranium and caudal fin, respectively. To investigate, we describe how the notochord of the cranium and caudal fin changes from embryo to spawning adult, using light microscopy, SEM, TEM, dissection, and CT scanning. The differences are dramatic. In contrast to the abdominal and caudal regions, at the ends of the notochord vertebrae never develop. While the cranial notochord builds a tapering, unsegmented cone of chordal bone, the urostylic notochordal sheath never ossifies: adjacent, irregular bony elements form from the endoskeleton of the caudal fin. As development progresses, two previously undescribed processes occur. First, the bony cone of the cranial notochord, and its internal chordocytes, are degraded by chordoclasts, an undescribed function of the clastic cell type. Second, the sheath of the urostylic notochord creates transverse septae that partly traverse the lumen in an irregular pattern. By the adult stage, the cranial notochord is gone. In contrast, the urostylic notochord in adults is robust, reinforced with septae, covered by irregularly shaped pieces of cellular bone, and capped with an opistural cartilage that develops from the sheath of the urostylic notochord. A previously undescribed muscle, with its origin on the opistural cartilage, inserts on the lepidotrich ventral to it. publishedVersion

The integration of clinical, radiographic and pathology-based information has long been intrinsic to practice of Orthopaedic Oncology, making it paradigm of multidisciplinary medicine. Extension of this multifactorial approach can lead to better understanding of heterogeneous group of entities. Historically, pathologists have approached the study of bone tumors from imaging to gross to histological findings. However, a reversal of this norm emphasizing a detailed understanding of the histopathology and growth parameters of each entity can allow a better understanding of the macroscopic / gross appearance of the orthopaedic tumors which in turn leads to a better appreciation of the imaging features. The chapter emphasizes those tumors most unique to bone: cartilage, osseous, small cell, fibrous, vascular, notochord, adamantinoma and giant cell lesions.

The notochord is a structure required for support and patterning of all chordate embryos, from sea squirts to humans. An increasing amount of information on notochord development and on the molecular strategies that ensure its proper morphogenesis has been gleaned through studies in the sea squirt Ciona. This invertebrate chordate offers a fortunate combination of experimental advantages, ranging from translucent, fast-developing embryos to a compact genome and impressive biomolecular resources. These assets have enabled the rapid identification of numerous notochord genes and cis-regulatory regions, and provide a rather unique opportunity to reconstruct the gene regulatory network that controls the formation of this developmental and evolutionary chordate landmark. This chapter summarizes the morphogenetic milestones that punctuate notochord formation in Ciona, their molecular effectors, and the current knowledge of the gene regulatory network that ensures the accurate spatial and temporal orchestration of these processes.

Summary The notochord is an evolutionary novelty in vertebrates that functions as an important signaling center during development. Notochord ablation in chicken has demonstrated that it is crucial for pancreas development; however, the molecular mechanism has not been fully described. Here, we show that in zebrafish, the loss of function of nog2, a Bmp antagonist expressed in the notochord, impairs β cell differentiation, compatible with the antagonistic role of Bmp in β cell differentiation. In addition, we show that nog2 expression in the notochord is induced by at least one notochord enhancer and its loss of function reduces the number of pancreatic progenitors and impairs β cell differentiation. Tracing Nog2 diffusion, we show that Nog2 emanates from the notochord to the pancreas progenitor domain. Finally, we find a notochord enhancer in human and mice Nog genomic landscapes, suggesting that the acquisition of a Nog notochord enhancer occurred early in the vertebrate phylogeny and contributes to the development of complex organs like the pancreas., Graphical Abstract, Highlights • nog2 loss of function leads to a reduced number of pancreatic progenitor and β cells • A tissue-specific enhancer controls the expression of nog2 in the notochord • The nog2 notochord enhancer is required for establishing a correctly sized pancreas • Regulation of notochord expression of Nog might be similar in many vertebrate lineages, Amorim et al. find that Nog2 is expressed in the zebrafish notochord by the action of a tissue-specific enhancer, and it diffuses to the pancreatic domain and controls its size. The identification of Nog enhancers in other vertebrate lineages suggests a conserved mechanism for pancreas development in vertebrates.

The zebrafish is a vertebrate model suited to the exploration of cell biology within a whole organism. Hypotheses in cell mechanics can be tested by using the zebrafish notochord as a manipulable experimental system. Here, the methodologies to prepare, label, and simultaneously induce and image mechanical loading on live zebrafish notochord cells via electrical stimulation are described. This approach investigates membrane mechanics in a live, physiological setting and is thus suited for caveola research where observations within the tissues of an intact organism are increasingly relevant. This chapter also aims to introduce fundamental methodologies for the use of zebrafish in "in vivo cell biology."

Polymelia is a rare congenital disease in humans characterized by extra limbs and a poorly understood etiology. To understand the mechanisms responsible for polymelia, DNA was isolated from two polymelia children with congenital malformations and their healthy parents for whole-exome sequencing. Using three hypothetical inheritance patterns and Ingenuity Pathway Analysis (IPA) the disease network analysis identified a network of Cancer, Connective Tissue Disorders, Organismal Injury and Abnormalities which included eleven mutated genes that might play roles in the proband multi-malformations. Variants in three collagen-associated genes, including rs146977141 c.5168 A > G and rs372335521 c.5207 C > T in COL27A1, c.1786_1794del and c.2106_2114del in COL18A1 and rs754836509 c.3940 C > T in COL4A5, were further validated. A significant defect of notochord was observedin both COL18A1 and COL4A5 morphants in zebrafish. Our results identified variants in collagen-containing extracellular matrix genes might be critical in the development of polymelia.

The development of the craniocervical junction is a complex sequence that requires perfect arrangement between cranial and cervical components. The neural crest cells of the upper somites and the notochord are both important in the development of this region. Of particular significance is the proatlas. Recent molecular studies have increased our knowledge of the processes involved in the formation of the base of the skull and upper cervical spine. The orchestration of the developing rhombencephalon and craniocervical junction is critical in the proper adult neuro-osseous relationships. Derailment of these processes may result in hindbrain herniation.

The suppressor of cytokine signaling (SOCS) proteins play important roles in cytokine and growth factor signaling, where they act principally as negative feedback regulators, particularly of the downstream signal transducer and activator of transcription (STAT) transcription factors. This critical mode of regulation impacts on both development and homeostasis. However, understanding of the function of SOCS4 remains limited. To address this, we investigated one of the zebrafish SOCS4 paralogues, socs4a, analyzing its expression and the consequences of its ablation. The socs4a gene had a dynamic expression profile during zebrafish embryogenesis, with initial ubiquitous expression becoming restricted to sensory ganglion within the developing nervous system. The knockdown of zebrafish socs4a revealed novel roles in notochord development, as well as the formation of a functional sensory system.

Context: Spine duplication is a rare condition, with various extents and severe additional anomalies. The goal of this study was to describe a unique case of a boy with split notochord syndrome who was followed up from birth until maturity. Findings: Physical examination at birth showed defects of the abdominal wall and cloacal exstrophy with visible urether outlets. A transposed anus was present in the perineal region. Split bony elements of the spine with nonpalpable sacral bone were noted. A soft, skin-covered lump, with the consistency of a lipoma, was present in the sacral area. There was asymmetry of the lower limbs: the left was hypoplastic, with a deformed foot and hip. Computed tomography revealed a normal shape of the Th12 and L1 vertebrae, whereas the L2 was split. Downward from L3, there were two vertebrae at each level, with two spinal canals. The spinal cord divided into two “semicords” at the level of L1. Neurologic status and the shape of the spine remained unchanged during puberty. The last follow-up was performed at the age of 18 years. He managed to walk independently in prosthesis with visible limping. Conclusion: Spine deformities are always suspected in neonates with lipoma in the sacral region, which may sometimes be serious. Walking ability and quality of life depend on neurologic deficits; even with long duplication and double sacrum, walking can be a feasible option.

Reactions of embryonic tissues to a distributed and concentrated stretching are described and compared with the mechanics of the normal gastrulation movements. A role of mechanically stressed dynamic cell structures in the gastrulation, demarcation of notochord borders and in providing proportionality of the axial rudiments is demonstrated. A morphomechanical scheme of amphibian gastrulation is presented.

AimsTo study the clinicopathological and molecular features of benign notochordal cell tumours (BNCTs) and their differential diagnosis from chordoma.Methods13 cases of BNCT were investigated. The genome-wide copy number imbalances were performed using Oncoscan CNV array in three cases and fluorescence in situ hybridisation (FISH) detection of epidermal growth factor receptor (EGFR)/chromosome 7 enumeration probe (CEP7), LSI1p36/1q21, LSI19p13/19q13, CEP3/CEP12 and Telvysion 6 P was performed in 13 cases.ResultsAll 13 BNCTs were symptomatic and eight cases showed a close relationship with the bones of the skull base. The important histological character for differential diagnosis with chordoma was the absence of extracellular matrix and eosinophil cells and the presence of vacuoles in most tumour cells. Immunohistochemical staining of AE1/AE3, vimentin, epithelial membrane antigen, S-100 and brachyury (100% each) were positive in BNCTs. Gain of chromosome 7 occurred in 10 cases (76.9%), gain of 1p in four (30.8%), gain of 1q in five (38.5%), gain of 19p and 19q in five (38.5%), gain of chromosome 12 in 11 cases (84.6%), gain of 6p in eight (61.5%) and gain of chromosome 3 in four cases (30.8%).ConclusionsIn contrast to chordoma, chromosome gain or normal copy number was more common while chromosome loss was infrequent in BNCTs. This may be a differential diagnosis clue for chordoma and may be an important characteristic in the progression of notochordal cell tumours.

Notochordal cells (NCs), characterized by their vacuolated morphology and coexpression of cytokeratin and vimentin intermediate filaments (IFs), form the immature nucleus pulposus (NP) of the intervertebral disc. As humans age, NCs give way to mature NP cells, which do not possess a vacuolated morphology and typically only express vimentin IFs. In light of their concomitant loss, we investigated the relationship between cytosolic vacuoles and cytokeratin IFs, specifically those containing cytokeratin-8 proteins, using a human chordoma cell line as a model for NCs. We demonstrate that the chemical disruption of IFs with acrylamide, F-actin with cytochalasin-D, and microtubules with nocodazole all result in a significant (p < 0.001) decrease in vacuolation. However, vacuole loss was the greatest in acrylamide-treated cells. Examination of the individual roles of vimentin and cytokeratin-8 IFs in the existence of vacuoles was accomplished using small interfering RNA-mediated RNA interference to knock down either vimentin or cytokeratin-8 expression. Reduction of cytokeratin-8 expression was associated with a less-vacuolated cell morphology. These data demonstrate that cytokeratin-8 IFs are involved in stabilizing vacuoles and that their diminished expression could play a role in the loss of vacuolation in NCs during aging. A better understanding of the NCs may assist in preservation of this cell type for NP maintenance and regeneration.

Background The blood filtering organ in zebrafish embryos is the pronephros, which consists of two functional nephrons. Segmentation of a nephron into different domains is essential for its function and is well conserved among vertebrates. Zebrafish has been extensively used as a model to understand nephron segmentation during development. Here, we have identified EF-hand domain containing 2 (Efhc2) as a novel component of genetic programme regulating nephron segmentation in zebrafish. Human EFHC2 is a protein with one predicted calcium-binding EF-hand motif and three DM10 domains, whose function is unknown. EFHC2 has been implicated in several brain-related genetic diseases like Turner syndrome and juvenile myoclonic epilepsy. However, there is limited information on its normal physiological function. Results efhc2 mRNA is primarily expressed in the pronephros of zebrafish embryos. Other sites of expression include olfactory placode, notochord, otic vesicle, epiphysis and neuromast cells. Morpholino antisense oligonucleotide-mediated knock-down of Efhc2 resulted in defects in pronephros development and function in zebrafish embryos. Efhc2 knock-down leads to expansion of distal early segment of pronephros, whereas, the corpuscle of stannius and distal late segments were reduced. The number of multi-ciliated cells (MCC) that are present in a salt-and-pepper fashion throughout the middle of each nephron and vital for fluid flow were also reduced. It is known that retinoic acid (RA) signaling regulates pronephros segmentation in vertebrates and we show that Efhc2 function is crucial for nephron segmentation in zebrafish. Our data suggests that RA and Efhc2 function independent of each other in pronephros segmentation. However, Efhc2 and RA synergistically regulate MCC development. Conclusion In this study, we have identified Efhc2 as a regulator of segmentation of the distal part of nephron and pronephros function during zebrafish development. Electronic supplementary material The online version of this article (10.1186/s13578-018-0253-z) contains supplementary material, which is available to authorized users.

The adult nucleus pulposus (NP) and articular cartilage are similar in terms of their histocytological components and biomechanical functionalities, requiring a deep understanding of NP-specific markers to better evaluate stem-cell-based NP regeneration. Here, we seek to distinguish NP cells from articular chondrocytes (ACs), focusing on differences in their embryonic formation and evolutionary origin. Embryonically, NP cells are conservatively derived from the axial notochord, whereas ACs originate in a diversified manner from paraxial mesoderm and neural crest cells. Evolutionarily, although the origins of vertebrate NP and AC cells can be traced to similar structures within protostomia-like bilaterian ancestors, the distant phylogenetic relationship between the two groups of animals and the differences in the bodily origins of the tissues suggest that the tissues may in fact have undergone parallel evolution within the protostomia and deuterostomia. The numbers of supposedly NP-specific markers are increasing gradually as microarray studies proceed, but no final consensus has been attained on the specificity and physiology of "exclusive" NP markers because of innate variations among species; intrinsic expression of genes that destabilize the circadian clock; and cooperation by, and crosstalk among, different genes in terms of physiology-related phenotypes. We highlight the embryonic and evolutionary boundaries between NP and AC cells, to aid in recognition of the challenges associated with evaluation of the role played by nucleopulpogenic differentiation during stem-cell-based intervertebral disc regeneration.

Chordomas are rare malignant tumors that develop from the residual of embryonic notochord. These tumors may be seen along the spine and have a local aggressive progression. Skull base chordomas often originate from the clivus as localization. These tumors are usually found to be overgrown when they are diagnosed. They are locally invasive and rarely develop distant metastasis. These chordomas cannot usually be completely removed due to their localization. Because these tumors are advanced at the time of diagnosis and are adjacent to important structures, they are among the tumors with high rates of mortality and morbidity. Surgery and/or radiotherapy is administered for the treatment.

Background Bilaterally symmetric animals have evolved highly reproducible asymmetries between left and right. In teleosts, Kupffer's vesicle, the structure necessary for the determination of left-right asymmetry, is derived from a group of cells in the gastrula termed the dorsal forerunners. Results Wilson cells are a ring of marginal enveloping layer cells that are cytoplasmically connected to the yolk cell and thus the last blastomeres to inherit yolk cell cytoplasm. Afterward, they collapse into the yolk to form the yolk syncytial layer. Without exception, forerunner cells are the progeny of dorsal Wilson cells. At the beginning of gastrulation, these Wilson cell progeny ingress beneath the enveloping layer, transform into Kupffer's vesicle, and eventually become tail notochord and muscle. Before ingressing, the forerunner precursor cells express endodermal promoting genes and require high-levels of Nodal signaling. Conclusions Despite a derived function of the enveloping layer as an epithelium covering the entire embryo, its dorsal margin retains many behaviors of what might be expected of the dorsal superficial layers of the ancestral fish embryo, including an early program of endodermal development, cell ingression, and an eventual contribution of cells to caudal notochord and muscle, as well as the control of laterality. Developmental Dynamics 247:1057-1069, 2018. © 2018 Wiley Periodicals, Inc.

Golenberg et al. report a citrullination-deficient zebrafish and demonstrate a role for Padi2 in fin wound responses and regeneration. This work identifies a distinct population of cells within the regenerative notochord bead that exhibits wound-induced histone citrullination., Calcium is an important early signal in wound healing, yet how these early signals promote regeneration remains unclear. Peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes, catalyze citrullination, a post-translational modification that alters protein function and has been implicated in autoimmune diseases. We generated a mutation in the single zebrafish ancestral pad gene, padi2, that results in a loss of detectable calcium-dependent citrullination. The mutants exhibit impaired resolution of inflammation and regeneration after caudal fin transection. We identified a new subpopulation of cells displaying citrullinated histones within the notochord bead following tissue injury. Citrullination of histones in this region was absent, and wound-induced proliferation was perturbed in Padi2-deficient larvae. Taken together, our results show that Padi2 is required for the citrullination of histones within a group of cells in the notochord bead and for promoting wound-induced proliferation required for efficient regeneration. These findings identify Padi2 as a potential intermediary between early calcium signaling and subsequent tissue regeneration.

BackgroundEcchordosis physaliphora is a congenital, benign lesion originating from notochordal remnants along the craniospinal axis, most frequently located at the level of the clivus and sacrum. Sometimes ecchordosis physaliphora is difficult to recognise and treat, with a total of twenty-six cases described in the literature.MethodsThis study reports on three cases of previously undiagnosed ecchordosis physaliphora presenting with cerebrospinal fluid rhinorrhoea and meningitis.ConclusionEndoscopic transclival or transsphenoid surgery including three-layer (fat, fascia and nasoseptal flap) reconstruction was used in all cases with complete resolution of the symptoms.

The fertilized egg (or first embryonic cell) is described as totipotent because it is capable of giving birth to an entire embryo. Embryonic development is the consequence of the gene activity within the cells and the interactions received by this cell from its environment. The result is changes in shape, movement, proliferation and death, differentiation, and specialization of cells.

The Ciona notochord has emerged as a simple and tractable in vivo model for tubulogenesis. Here, using a chemical genetics approach, we identified UTKO1 as a selective small molecule inhibitor of notochord tubulogenesis. We identified 14-3-3εa protein as a direct binding partner of UTKO1 and showed that 14-3-3εa knockdown leads to failure of notochord tubulogenesis. We found that UTKO1 prevents 14-3-3εa from interacting with ezrin/radixin/moesin (ERM), which is required for notochord tubulogenesis, suggesting that interactions between 14-3-3εa and ERM play a key role in regulating the early steps of tubulogenesis. Using live imaging, we found that, as lumens begin to open between neighboring cells, 14-3-3εa and ERM are highly colocalized at the basal cortex where they undergo cycles of accumulation and disappearance. Interestingly, the disappearance of 14-3-3εa and ERM during each cycle is tightly correlated with a transient flow of 14-3-3εa, ERM, myosin II, and other cytoplasmic elements from the basal surface toward the lumen-facing apical domain, which is often accompanied by visible changes in lumen architecture. Both pulsatile flow and lumen formation are abolished in larvae treated with UTKO1, in larvae depleted of either 14-3-3εa or ERM, or in larvae expressing a truncated form of 14-3-3εa that lacks the ability to interact with ERM. These results suggest that 14-3-3εa and ERM interact at the basal cortex to direct pulsatile basal accumulation and basal–apical transport of factors that are essential for lumen formation. We propose that similar mechanisms may underlie or may contribute to lumen formation in tubulogenesis in other systems.

Fossa navicularis magna was detected in multislice spiral computed tomography in two patients who turned to the medical centers with pathology of the paranasal sinuses. Its appearance is determined during the development of the basilar part of the occipital bone and the body of the sphenoid bone in embryogenesis. This fossa has the appearance of an edge defect on the ventral surface of the clivus in CBCT scans. Practical radiologist should interpret such a finding as a congenital anomaly of development, but not as an invasive lesion.

The clivus is a bony structure formed by the fusion of the basioccipital and basispheniod bone at the sphenooccipital synchondrosis. This downward sloping structure from the dorsum sellae to the foramen magnum is derived from mesoderm and ectoderm properties. This comprehensive review of the clivus will discuss its basic anatomy, embryology, pathological findings, and surgical implications. The clivus is an endochondral bone, formed under two processes; first, a cartilaginous base is developed, and it is secondly reabsorbed and replaced with bone. Knowledge of its embryological structure and growth of development will clarify the pathogenesis of anatomical variants and pathological findings of the clivus. Understanding the anatomy including proximity to anatomical structures, adjacent neurovasculature properties, and anatomical variants will aid neurosurgeons in their surgical management when treating pathological findings around the clivus.

MicroRNAs (miRNAs) are highly conserved small non-coding RNA molecules that post-transcriptionally regulate gene expression in multicellular organisms. Within the set of muscle-specific miRNAs, miR-206 expression is largely restricted to skeletal muscle and is found exclusively within the bony fish lineage. Although many studies have implicated miR-206 in muscle maintenance and disease, its role in skeletal muscle development remains largely unknown. Here, we examine the role of miR-206 during Xenopus laevis somitogenesis. In Xenopus laevis, miR-206 expression coincides with the onset of somitogenesis. We show that both knockdown and over-expression of miR-206 result in abnormal somite formation affecting muscle cell rotation, attachment, and elongation. In particular, our data suggests that miR-206 regulates changes in cell adhesion that affect the ability of newly formed somites to adhere to the notochord as well as to the intersomitic boundaries. Additionally, we show that β-dystroglycan and F-actin expression levels are significantly reduced, suggesting that knockdown of miR-206 levels affects cellular mechanics necessary for cell shape changes and attachments that are required for proper muscle formation.

Objective The nucleus pulposus of the human intervertebral disc contains 2 cell types: notochordal (NC) and mature nucleus pulposus (MNP) cells. NC cell loss is associated with disc degeneration and this process may be initiated by mechanical stress and/or nutrient deprivation. This study aimed to investigate the functional responses of NC and MNP cells to hydrostatic pressures and glucose restriction. Design Bovine MNP and NC cells were cultured in 3-dimensional alginate beads under low (0.4-0.8 MPa) and high (1.6-2.4 MPa) dynamic pressure for 24 hours. Cells were cultured in either physiological (5.5 mM) glucose media or glucose-restriction (0.55 mM) media. Finally, the combined effect of glucose restriction and high pressure was examined. Results Cell viability and notochordal phenotypic markers were not significantly altered in response to pressure or glucose restriction. MNP cells responded to low pressure with an increase in glycosaminoglycan (GAG) production while high pressure significantly decreased ACAN gene expression compared with atmospheric controls. NC cells showed no response in matrix gene expression or GAG production with either loading regime. Glucose restriction decreased NC cell TIMP-1 expression but had no effect on MNP cells. The combination of glucose restriction and high pressure only affected MNP cell gene expression, with decreased ACAN, Col2α1, and ADAMTS-5 expression. Conclusion This study shows that NC cells are more resistant to acute mechanical stresses than MNP cells and provides a strong rationale for future studies to further our understanding the role of NC cells within the disc, and the effects of long-term exposure to physical stresses.