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BACKGROUND Defects in secondary neurulation play an important role in neural tube defects. Researchers have investigated the processes of secondary neurulation and caudal body formation mainly by microscopic observations and molecular experiments. Although conventional histology is a powerful tool for observing the details of morphology, it has limitations in the presentation of gross three-dimensional (3D) configurations of small embryos. The goal of this study was to visualize secondary neurulation and related structures in chick embryos in Hamburger and Hamilton (HH) stages 10-22 using microCT. RESULTS The gross morphology of the chick embryo of various developmental stages was well visualized using microCT. Also, the detailed structures of the caudal cell mass (CCM) were presented starting from HH stage 12 to stage 16. The spatiotemporal relationship of CCM with the floor plate of the neural tube and notochord was shown. The dynamic changes of the chordoneural hinge, the cavitation of the secondary neural tube, and the primitive streak were described throughout the early stages of secondary neurulation. CONCLUSIONS By utilizing the advantages of the microCT technique, our study shed light on the secondary neurulation in early-stage chick embryos and this can be the 3D reference for related structures.

We modeled swimming kinematics and body mechanics of several fish species of varying habitat and body shape based on measurements of internal vertebral morphology. SYNOPSIS One key evolutionary innovation that separates vertebrates from invertebrates is the notochord, a central element that provides the stiffness needed for powerful movements. Later, the notochord was further stiffened by the vertebrae, cartilaginous, and bony elements, surrounding the notochord. The ancestral notochord is retained in modern vertebrates as intervertebral material, but we know little about its mechanical interactions with surrounding vertebrae. In this study, the internal shape of the vertebrae-where this material is found-was quantified in 16 species of fishes with various body shapes, swimming modes, and habitats. We used micro-computed tomography to measure the internal shape. We then created and mechanically tested physical models of intervertebral joints. We also mechanically tested actual vertebrae of five species. Material testing shows that internal morphology of the centrum significantly affects bending and torsional stiffness. Finally, we performed swimming trials to gather kinematic data. Combining these data, we created a model that uses internal vertebral morphology to make predictions about swimming kinematics and mechanics. We used linear discriminant analysis (LDA) to assess the relationship between vertebral shape and our categorical traits. The analysis revealed that internal vertebral morphology is sufficient to predict habitat, body shape, and swimming mode in our fishes. This model can also be used to make predictions about swimming in fishes not easily studied in the laboratory, such as deep sea and extinct species, allowing the development of hypotheses about their natural behavior.

Chordomas are centrally located, expansile soft tissue neoplasms that arise from the remnants of the embryological notochord. Hemorrhagic presentation is exceedingly rare and can resemble pituitary apoplexy. Moreover, a purely intrasellar location of a chordoma is extremely uncommon. We report a case of a hemorrhagic intrasellar chordoma in an adult male, which presented similarly to pituitary apoplexy and was resolved with surgical resection.A 69-year-old male presented with a 4 week history of acute onset headache and concurrent diplopia, with significantly reduced testosterone and slightly reduced cortisol. His left eye demonstrated a sixth cranial nerve palsy. Magnetic resonance imaging of the brain showed a large hemorrhagic mass in the pituitary region with significant compression of the left cavernous sinus and superior displacement of the pituitary gland. The patient underwent an endoscopic endonasal transsphenoidal approach for the resection of the lesion. Near total resection was achieved. Final pathology revealed chordoma with evidence of intratumoral hemorrhage, further confirmed by immunopositive stain for brachyury. Post-operatively, the patient had improved diplopia and was discharged home on low dose hydrocortisone. At 3-month follow-up, his diplopia was resolved and new MRI showed stable small residual disease.Apoplectic chordomas are uncommon given chordoma's characteristic lack of intralesional vascularity and represent a diagnostic challenge in the sellar region. Our unique case demonstrates that despite our initial impression of pituitary apoplexy, this was ultimately a case of apoplectic chordoma that responded well to endoscopic endonasal surgery.

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.

Background The cylindromatosis (CYLD) tumor suppressor is a microtubule‐associated deubiquitinase that plays a critical role in the regulation of cell signaling and contributes to a variety of physiological and pathological processes. However, the functions of CYLD in zebrafish are less well known, particularly with regard to their development and physiology. In this context, we investigated the loss of function of CYLD in zebrafish via transcription activator‐like effector nuclease (TALEN)‐based gene deletion. Methods Semi‐quantitative RT‐PCR was used to quantify CYLD mRNA expression in zebrafish embryos at various developmental stages. We also performed whole‐mount in situ hybridization to further assess the dynamic expression and distribution of CYLD in the entire zebrafish embryos at different stages. In addition, we deleted CYLD in zebrafish with TALENs to investigate its potential impact on embryonic development. Results The expression of CYLD mRNA varied during early embryonic development. The CYLD mRNA localized to the brain and notochord of developing zebrafish embryos. Homozygous deletion of CYLD resulted in embryonic death before 8 h post‐fertilization. Conclusions CYLD appears to play an important role in central nervous system development in zebrafish. Although severe embryonic death restricted analysis of homozygous mutants, further research into the role of CYLD in central nervous system development is warranted., Cylindromatosis (CYLD) has important pathophysiological roles in humans. More than just a tumor suppressor, CYLD has many potential functions. We exploited the zebrafish model to investigate dynamic changes in CYLD expression during embryonic development. In doing so, we discovered a potential role for CYLD in brain and notochord development, providing experimental evidence for a novel pathophysiological role in central nervous system development.

Objectives Ecchordosis physaliphora (EP) is a benign notochord lesion of the clivus arising from the same cell line as chordoma, its malignant counterpart. Although usually asymptomatic, it can cause spontaneous cerebrospinal fluid (CSF) rhinorrhea. Benign notochordal cell tumor (BNCT) is considered another indolent, benign variant of chordoma. Although aggressive forms of chordoma require maximal safe resection followed by proton beam radiotherapy, BNCT and EP can be managed with close imaging surveillance without resection or radiotherapy. However, while BNCT and EP can be distinguished from more aggressive forms of chordoma, differentiating the two is challenging as they are radiologically and histopathologically identical. This case series aims to characterize the clinicopathological features of EP and to propose classifying EP and BNCT together for the purposes of clinical management. Design Case series. Setting Tertiary referral center, United Kingdom. Participants Patients with suspected EP from 2015 to 2019. Main Outcome Measures Diagnosis of EP. Results Seven patients with radiological suspicion of EP were identified. Five presented with CSF rhinorrhea and two were asymptomatic. Magnetic resonance imaging features consistently showed T1-hypointense, T2-hyperintense nonenhancing lesions. Diagnosis was made on biopsy for patients requiring repair and radiologically where no surgery was indicated. The histological features of EP included physaliphorous cells of notochordal origin (positive epithelial membrane antigen, S100, CD10, and/or MNF116) without mitotic activity. Conclusion EP is indistinguishable from BNCT. Both demonstrate markers of notochord cell lines without malignant features. Their management is also identical. We therefore propose grouping EP with BNCT. Close imaging surveillance is required for both as progression to chordoma remains an unquantified risk.

Ecchordosis physaliphora is a rare congenital benign hamartomatous lesion originating from nodal cord remnants. This is histopathologically indistinguishable from chordoma, and hence imaging plays a key role in diagnosis. These lesions are hypointense on T1-weighted and hyperintense on T2-weighted images, and follow CSF signal. In contrast to chordoma, Ecchordosis Physaliphora does not demonstrate contrast enhancement. Here, we present a case of 32-year-old male with no prior medical history, who presented to an outside facility for chronic headache workup and incidentally detected indeterminate lytic defect in the bony clivus with a well demarcated smoothly corticated margin. Further assessment with MRI brain showed findings characteristic of Ecchordosis physaliphora, a benign congenital hamartomatous lesion originating from nodal cord remnants requiring no additional follow-up imaging or intervention.

Study Design: Retrospective cohort study. Objective: Spinal chordomas are rare primary malignant neoplasms of the primitive notochord. They are slow growing but locally aggressive lesions that have high rates of recurrence and metastasis after treatment. Gold standard treatment remains en-bloc surgical resection with questionable efficacy of adjuvant therapies such as radiation and chemotherapy. Here we provide a comprehensive analysis of prognostic factors, treatment modalities, and survival outcomes in patients with spinal chordoma. Methods: Patients with diagnosis codes specific for chordoma of spine, sacrum, and coccyx were queried from the National Cancer Database (NCDB) during the years 2004-2016. Outcomes were investigated using Cox univariate and multivariate regression analyses, and survival curves were generated for comparative visualization. Results: 1,548 individuals were identified with a diagnosis of chordoma, 60.9% of which were at the sacrum or coccyx and 39.1% at the spine. The mean overall survival of patients in our cohort was 8.2 years. Increased age, larger tumor size, and presence of metastases were associated with worsened overall survival. 71.2% of patients received surgical intervention and both partial and radical resection were associated with significantly improved overall survival ( P < 0.001). Neither radiotherapy nor chemotherapy administration improved overall survival; however, amongst patients who received radiation, those who received proton-based radiation had significantly improved overall survival compared to traditional radiation. Conclusion: Surgical resection significantly improves overall survival in patients with spinal chordoma. In those patients receiving radiation, those who receive proton-based modalities have improved overall survival. Further studies into proton radiotherapy doses are required.

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.

Objectives: To compare results obtained since 1970 from craniofacial embryological studies on human fetal tissues with results from similar studies performed on animal tissues and focus on how the different tissue types and methods can enrich the human postnatal craniofacial research. There are three sections: i) research on normal and pathological human fetal material, ii) animal experimental research performed on different species, and iii) comparisons of the results obtained. Human Material: On human prenatal material, normal and pathological developmental processes in the mandible, maxilla, nasal cavity, body axis, cranial base, vomeronasal organs, pituitary gland and nervous system were related to postnatal findings. Specific focus was given to pre/postnatal bridging of observations on holoprosencephaly, cleft lip/palate, Down syndrome, myelomeningocele/spina bifida, Kallmann syndrome, Turner syndrome and Fragile X syndrome. Demarcations of pathological regions in Cri du chat, Velo-cardio-Facial syndrome and Crouzon syndrome were mapped. Animal Material: Animal experimental results from studies on the notochord, gastrulation, neural crest, hindbrain, rhombomere, homeobox genes and experimentally induced malformation were presented. Comparing materials: The educational background of the scientists performing human and animal research, their postnatal clinical experience, the research materials used and the methods available for research are compared. Furthermore, the obtained findings result in a “pro et contra list” indicating what is positive and what is negative for improving postnatal human diagnostics. Conclusion: Human and animal studies in craniofacial embryology enrich human postnatal craniofacial insight differently. Future cooperation between human and animal research cultures is recommended.

In recent years, the development and implementation of animal-free approaches to chemical and pharmaceutical hazard and risk assessment has taken off. Alternative approaches are being developed starting from the perspective of human biology and physiology. Neural tube closure is a vital step that occurs early in human development. Correct closure of the neural tube depends on a complex interplay between proteins along a number of protein concentration gradients. The sensitivity of neural tube closure to chemical disturbance of signalling pathways such as the retinoid pathway, is well known. To map the pathways underlying neural tube closure, literature data on the molecular regulation of neural tube closure were collected. As the process of neural tube closure is highly conserved in vertebrates, the extensive literature available for the mouse was used whilst considering its relevance for humans. Thus, important cell compartments, regulatory pathways, and protein interactions essential for neural tube closure under physiological circumstances were identified and mapped. An understanding of aberrant processes leading to neural tube defects (NTDs) requires detailed maps of neural tube embryology, including the complex genetic signals and responses underlying critical cellular dynamical and biomechanical processes. The retinoid signaling pathway serves as a case study for this ontology because of well-defined crosstalk with the genetic control of neural tube patterning and morphogenesis. It is a known target for mechanistically-diverse chemical structures that disrupt neural tube closure The data presented in this manuscript will set the stage for constructing mathematical models and computer simulation of neural tube closure for human-relevant AOPs and predictive toxicology.

SOX9 plays an important role in chondrocyte differentiation and, in the developing axial skeleton, maintains the notochord and the demarcation of intervertebral disc compartments. Diminished expression is linked to campomelic dysplasia, resulting in severe scoliosis and progressive disc degeneration. However, the specific functions of SOX9 in the adult spinal column and disc are largely unknown. Accordingly, employing a strategy to conditionally delete Sox9 in Acan-expressing cells (Acan(CreERT2)Sox9(fl/lf)), we delineated these functions in the adult intervertebral disc. Acan(CreERT2)Sox9(fl/lf) mice (Sox9(cKO)) showed extensive and progressive remodeling of the extracellular matrix in nucleus pulposus (NP) and annulus fibrosus (AF), consistent with human disc degeneration. Progressive degeneration of the cartilaginous endplates (EP) was also evident in Sox9(cKO) mice, and it preceded morphological changes seen in the NP and AF compartments. Fate mapping using tdTomato reporter, EdU chase, and quantitative immunohistological studies demonstrated that SOX9 is crucial for disc cell survival and phenotype maintenance. Microarray analysis showed that Sox9 regulated distinct compartment-specific transcriptomic landscapes, with prominent contributions to the ECM, cytoskeleton-related, and metabolic pathways in the NP and ion transport, the cell cycle, and signaling pathways in the AF. In summary, our work provides new insights into disc degeneration in Sox9(cKO) mice at the cellular, molecular, and transcriptional levels, underscoring tissue-specific roles of this transcription factor. Our findings may direct future cell therapies targeting SOX9 to mitigate disc degeneration.

No abstract available Keywords: Brachyury; Notochord; Physaliferous cells; Proton therapy; Rare diseases.

The loss of nucleus pulposus (NP) notochordal cells is one of the key initial hallmarks of age-related intervertebral disc degeneration. Although the transmembrane mechanoreceptor integrin α5β1 is important in the process of disc degeneration, the relationship between integrin α5β1 and notochordal cell disappearance remains unclear. The purpose of this study was to elucidate the role of integrin α5β1 in the homeostasis of notochordal cells using an ex-vivo dynamic loading culture system that we developed. Rat tail functional spinal units (n = 80 from 40 rats) were cultured under unloading or 1.3-MPa, 1.0-Hz dynamic compressive loading for 48 or 144 h with or without an integrin α5β1 inhibitor. Disc histomorphology, cell viability, apoptosis, senescence, and phenotypic expression were investigated. Consequently, histological degenerative disc changes with decreased cell viability and increased cell apoptosis and senescence were observed with an extended loading duration. Immunofluorescence revealed that the expression of notochordal cell markers, CD24 and brachyury, and chondrocyte markers, collagen type II and SRY-box 9, declined with loading. In particular, reduction in notochordal cell marker expression was more dramatic than that in chondrocyte marker expression. Apoptotic terminal deoxynucleotidyl transferase dUTP nick-end labeling positivity was also higher in brachyury-positive notochordal cells. Furthermore, all these changes were delayed by inhibiting integrin α5β1. Findings of our dynamic loading regimen with a relatively high pressure suggest reproducibility of the cellularity and phenotypic disappearance of NP notochordal cells during adolescence, the susceptibility of notochordal cells to mechanical stimuli partially through the integrin α5β1 pathway, and future potential treatment of integrin regulation for intervertebral disc disease.

Protein phosphatases regulate a wide array of proteins through post-translational modification and are required for a plethora of intracellular events in eukaryotes. While some core components of the protein phosphatase complexes are well characterized, many subunits of these large complexes remain unstudied. Here we characterize a loss-of-function allele of the protein phosphatase 1 regulatory subunit 35 (Ppp1r35) gene. Homozygous mouse embryos lacking Ppp1r35 are developmental delayed beginning at embryonic day (E) 7.5 and have obvious morphological defects at later stages. Mutants fail to initiate turning and do not progress beyond the size or staging of normal E8.5 embryos. Consistent with recent in vitro studies linking PPP1R35 with the microcephaly protein Rotatin and with a role in centrosome formation, we show that Ppp1r35 mutant embryos lack primary cilia. Histological and molecular analysis of Ppp1r35 mutants revealed that notochord development is irregular and discontinuous and consistent with a role in primary cilia, that the floor plate of the neural tube is not specified. Similar to other mutant embryos with defects in centriole function, Ppp1r35 mutants displayed increased cell death that is prevalent in the neural tube and an increased number of proliferative cells in prometaphase. We hypothesize that loss of Ppp1r35 function abrogates centriole homeostasis, resulting in a failure to produce functional primary cilia, cell death and cell cycle delay/stalling that leads to developmental failure. Taken together, these results highlight the essential function of Ppp1r35 during early mammalian development and implicate this gene as a candidate for human microcephaly.

Chordomas are rare primary bone malignancies derived from notochord remnants. The tumors often are slow-growing and often present with indolent, nonspecific symptoms. Nevertheless, chordomas are locally aggressive and highly prone to local recurrence, necessitating precise planning before biopsy and/or surgical resection. Familiarity with the imaging features of chordomas is, therefore, essential. This case highlights the typical imaging and pathologic features of a spinal chordoma as well as the surgical approach and the patient’s subsequent outcome.

Valproic acid (VPA) is a branched short-chain fatty acid primarily used in epilepsy, but is also used in bipolar disorder, migraine, and psychotic disorders. Despite its wide range of use, it is a teratogen resulting in various congenital abnormalities. Although a large number of scientific studies evidenced the teratogenic effects, there are limited data on embryonic exposure to VPA at specific or different stages of early embryogenesis. Based on this, the present study was planned to investigate the embryonic exposure to VPA at specific and different hours post fertilization (hpf) in zebrafish embryonic model. In first set of experiments, embryos from spawning groups of adult zebrafish were exposed to different molar concentrations of VPA at 2.5 hpf, and in the second set of experiments, embryos were exposed to VPA 100 μM at 24 hpf, 36 hpf, 48 hpf, 72 hpf, and 96 hpf. The parameters examined were hatching rate, mortality, morphology, body length, pericardial sac size, heartrate, anatomical changes in heart, skeletal and notochord till 120 hpf. It was observed that the embryos exposed to VPA at 2.5 hpf suffered from cardiac abnormalities including heart malformation, bradycardia, circulatory failure, and pericardial sac enlargement which was more apparent in embryos exposed to 100 μM VPA. In the second set of experiments, embryos exposed to VPA 100 μM at 24 hpf and 36 hpf suffered from heart malformations, but there was no incidence of cardiac malformation in embryos exposed to VPA at 48 hpf, 72 hpf, and 96 hpf. From the results, it was evident that exposure to VPA at early developmental stage of embryogenesis produced congenital cardiac abnormalities. Since VPA is a selective HDAC inhibitor, histone acetylation with aberrant gene expression during cardiogenesis might be the underlying cause of cardiac malformation.

Here we describe the histology and microanatomy of vertebral centra of the iconic pelycosaur-grade synapsids Edaphosaurus boanerges and Dimetrodon spp. Vertebrae from different axial positions and, in the case of Dimetrodon, from different ontogenetic stages were selected. For the histological description, we produced histological petrographic thin sections ground to a thickness of 50-80 μm of the vertebrae in the sagittal and transversal cutting planes. After the preparation process, the thin sections were examined under transmitted and cross-polarized light in a polarized microscope. The analyzed vertebrae reveal similar bone tissues, where both taxa have cortical parallel-fibered bone (PFB). PFB and lamellar bone (LB) forms in the cancellous part. However, in juvenile Dimetrodon, woven-fibered bone (WFB) is also deposited and shows a high degree of vascularity. This suggests that Dimetrodon had slightly faster bone growth than Edaphosaurus, which is mainly made of PFB and LB and shows poorly developed vascular canals. In addition, one specimen of Dimetrodon displays the preservation of an ossified notochord, which can be assumed to be indicative of how the intervertebral tissues were developed. Historically, evidence of how the joint between Dimetrodon vertebral centra was built was lacking until this specimen appeared. If the notochord ran persistently through the vertebrae, it would have possibly increased the stiffness of the vertebral column and would have affected the limbs and locomotion. Furthermore, the organization of trabeculae and relative thickness of the vertebral cortex gives insights into how the animals were adapted to their habitat.

Chordomas are rare, malignant and locally aggressive tumors that are derived from the remnants of primitive notochord, out of which chondroid chordomas are even rarer. Very few case reports have described this variant, which is difficult to pick up on cytopathology alone and has a number of other differentials too. We report here a case of chondroid chordoma at the sacrococcygeal region that was diagnosed on FNAC in an elderly male patient emphasizing on its cytomorphology and how to differentiate from its cytological mimickers. Keywords: FNAC, notochordal tumors, Cartilaginous, Physaliphorous.

Background The Weberian apparatus enhances hearing in otophysan fishes, including Zebrafish (Danio rerio). Several studies have examined aspects of morphological development of the Weberian apparatus and hearing ability in Zebrafish. A comprehensive developmental description including both hard and soft tissues is lacking. This information is critical for both interpretation of genetic developmental analyses and to better understand the role of morphogenesis and integration on changes in hearing ability. Results Histological development of hard and soft tissues of the Weberian apparatus, including ossicles, ear, swim bladder, and ligaments are described from early larval stages (3.8 mm notochord length) through adult. Results show a strong relationship in developmental timing and maturation across all regions. All required auditory elements are present and morphologically integrated early, by 6.5 mm SL. Dynamic ossification patterns and changes in shape continue throughout the examined developmental period. Conclusions This study provides the first comprehensive histological description of Weberian apparatus development in Zebrafish. Morphological integration was found early, before increases in hearing ability were detected in functional studies (>10 mm total length), suggesting morphological integration precedes functional integration. Further research is needed to examine the nature of the functional delay, and how maturation of the Weberian apparatus influences functionality.

The tailbud stage is part of the organogenesis period—an evolutionarily conserved developmental period among chordates that is essential for determining the characteristics of the chordate body plan. When the volume of the egg is artificially decreased by cutting, ascidians produce a normal-looking but miniature (dwarf) tailbud embryo. Although cell lineages during ascidian embryogenesis are invariant, the number of cell divisions in the dwarf embryo is altered by a different mechanism in each tissue (Yamada and Nishida, 1999). Here, to elucidate the size-regulation strategies of the Ciona robusta dwarf tailbud embryo, we compared anatomical structure, developmental speed, and cell number/volume in each tissue between dwarf and wild type (WT) embryos. To do this, we constructed a 3D virtual mid-tailbud embryo (Nakamura et al., 2012). We could make a Ciona dwarf tailbud embryo from eggs with a diameter over 108 ​μm (correspond to ​> ​40% of the wild type egg volume). The timings of cleavage (~St. 12) and subsequent morphogenesis were nearly the same but blastomeres of animal hemisphere slightly delayed the timing of mitosis in the early cleavage period. Intriguingly, the tissue-to-tissue volume ratios of dwarf tailbud embryos were similar to those of wild type embryos suggesting that the ratio of tissue volumes is essential for maintaining the proper shape of the tailbud embryo. The number of cells in the epidermis, nervous system, and mesenchyme was significantly reduced in the dwarf embryos whereas the cell volume distribution of these tissues was similar in the dwarf and wild type. In contrast, the number of cells in the notochord, muscle, heart, and endoderm were maintained in the dwarf embryos; cell volumes were significantly reduced. Neither parameter changed in germline precursors. These results indicate that each tissue uses different scaling strategies to coordinate cell number and cell volume in accordance with the embryo size.

Background/objective: Intervertebral disc degeneration (IDD) remains to be an intractable clinical challenge. Although IDD is characterised by loss of notochordal cells (NCs) and dysfunction of nucleus pulposus (NP) cells, little is known about the origin, heterogeneity, fate and maintenance of NCs and NP cells, which further stunts the therapeutic development. Thus, effective tools to spatially and temporally trace specific cell lineage and clarify cell functions in intervertebral disc (IVD) development and homoeostasis are urgently required. Methods: In this study, NP specimens were obtained from 20 patients with degenerative disc disease or scoliosis. LepR-Cre mice was crossed with R26R-Tdtomato mice to generate LepR-Cre; R26R-Tdtomato mice, which enabled fate-mapping of NPs from embryo stage to late adult. LMNA G609G/G609G mice was used to determine the effect of premature-aging induced IDD on LepR NPs. X-ray imaging was used to measure lumber disc height of mice. Results: Here, we provide the first evidence that the leptin receptor (LepR) is preferentially expressed in NCs at embryonic stages and notochord-derived cells in the postnatal IVD. By using R26R-Tdtomato fluorescent reporter mice, we systematically analysed the specificity of activity and targeting efficiency of leptin receptor-Cre (LepR-Cre) in IVD tissues from the embryonic stage E15.5 to 6-month-old LepR-Cre; Rosa26-Tdtomato (R26R-Tdtomato) mice. Specifically, LepR-Cre targets a distinct subpopulation of notochord-derived cells closely associated with disc homoeostasis. The percentage of LepR-expressing NP cells markedly decreases in the postnatal mouse IVD and, more importantly, in the human IVD with the progression of IDD. Moreover, both spine instability–induced and premature ageing–induced IDD mouse models display the phenotype of IDD with decreased percentage of LepR-expressing NP cells. These findings uncover a potential role of LepR-expressing notochord-derived cells in disc homoeostasis and open the gate for therapeutically targeting the NP cell subpopulation. Conclusion: In conclusion, our data prove LepR-Cre mice useful for mapping the fate of specific subpopulations of IVD cells and uncovering the underlying mechanisms of IDD. The translational potential of this article: The translation potential of article is that we first identified LepR as a candidate marker of subpopulation of nucleus pulposus (NP) cells and provided LepR as a potential target for the treatment of intervertebral disc degeneration (IDD), which have certain profound significance. Keywords: LepR, Lineage tracing, Notochordal cells, Nucleus pulposus cells, Subpopulation

Notochord formation begins between the third and fourth weeks of embryonic development. As the axial skeleton develops, the notochord regresses and eventually contributes to the formation of the nucleus pulposus of the intervertebral disks. Notochord regression can be variable, especially at the 2 poles of the skeleton, which results in the persistence of ectopic notochordal remnants. These remnants include benign entities (eg, ecchordosis physaliphora, benign notochordal cell tumor, Tornwaldt cyst, canalis basilaris medianus, fossa navicularis) or malignant lesions (eg, chordoma). We present a review of these lesions, their clinical presentations, and their typical radiographic appearances. Notochord remnants may have overlapping features in terms of their location and appearance; however, differentiation is crucial because the proper treatment and management may differ drastically. Familiarity with notochord development aids in understanding the characteristic locations of notochordal remnants. Noting the unique imaging findings of notochordal remnants can often help narrow the differential diagnoses.Learning Objective: To describe notochord development, normal regression during fetal life, and notochord remnants that give rise to anatomic variants, including benign and malignant lesions.

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., Congenital scoliosis is a complex genetic disorder characterized by vertebral malformation. Here, the authors demonstrate that loss of dstyk leads to scoliosis in zebrafish due to dysregulated biogenesis of notochord vacuoles and that DSTYK is required for lysosome biogenesis through mTORC1 regulation.

Highlights • Several evolutionary steps led to the emergence of vertebrate movement needed for social behaviour. • Descending systems rely on diverse spinal cord neurons to produce a rich repertoire of movement syllables. • The notochord and ventricular zone led to this diversity of neuronal types. • The sympathetic nervous system provided the substrate for homeothermic endothermy. • Homeothermic endothermy is a necessary building block for power, speed, and endurance., Rich repertoires of movements underlie the complex social interactions of mammals. The building blocks, or syllables, of these movements are produced by spinal cord circuits that are comprised of diverse neuronal types that control musculoskeletal systems comprised of multi-segmented limbs. Together, these systems provide mammals with the evolutionary advantages of power, speed, and endurance. Here, I propose that the key steps in chordate evolution that led to these traits began with the development of the notochord and a proliferative ventricular zone (with associated Notch signalling). This step led to the production of diverse neuronal types that included the development of a sympathetic nervous system that could regulate the evolving cardiovascular system. And the sympathetic nervous system in turn led to the development of homeothermic endothermy, a requirement for motor systems to produce a combination of power, speed, and endurance. Furthermore, the evolution of the continuous structure of the spinal cord led not only to a structure fit for cartesian signalling molecules, but also to one with high processing power in which circuits for effecting movement syllables formed. These syllables are harnessed by higher regions of nervous systems to produce the complex movements required for interactions with others and with the surrounding environment.

Neurotrophic signaling regulates neural cell behaviors in development and physiology, although its role in regeneration has not been fully investigated. Here, we examined the role of neurotrophic signaling in Xenopus laevis tadpole tail regeneration. After the tadpole tails were amputated, the expression of neurotrophin ligand family genes, especially ngf and bdnf, was up-regulated as regeneration proceeded. Moreover, notochordal expression of the NGF receptor gene TrkA, but not that of other neurotrophin receptor genes TrkB and TrkC, became prominent in the regeneration bud, a structure arising from the tail stump after tail amputation. The regenerated tail length was significantly shortened by the pan-Trk inhibitor K252a or the TrkA inhibitor GW-441756, but not by the TrkB inhibitor ANA-12, suggesting that TrkA signaling is involved in elongation of regenerating tails. Furthermore, during Xenopus laevis embryonic development, TrkA expression was detected in the dorsal mesoderm at the gastrula stage and in the notochord at the neurula stage, and its knockdown led to gastrulation defects with subsequent shortening of the body axis length. These results suggest that Xenopus laevis TrkA signaling, which can act in the mesoderm/notochord, plays a key role in body axis elongation during embryogenesis as well as tail elongation during tadpole regeneration.

Intervertebral disc degeneration (IDD) is a public health dilemma as it is associated with low back and neck pain, a frequent reason for patients to visit the physician. During IDD, nucleus pulposus (NP), the central compartment of intervertebral disc (IVD) undergo degeneration. Stem cells have been adopted as a promising biological source to regenerate the IVD and restore its function. Here, we describe a simple, two-step differentiation strategy using a cocktail of four factors (LDN, AGN, FGF, and CHIR) for efficient derivation of notochordal cells from human embryonic stem cells (hESCs). We employed a CRISPR/Cas9 based genome-editing approach to knock-in the mCherry reporter vector upstream of the 3' untranslated region of the Noto gene in H9-hESCs and monitored notochordal cell differentiation. Our data show that treatment of H9-hESCs with the above-mentioned four factors for 6 days successfully resulted in notochordal cells. These cells were characterized by morphology, immunostaining, and gene and protein expression analyses for established notochordal cell markers including FoxA2, SHH, and Brachyury. Additionally, pan-genomic high-throughput single cell RNA-sequencing revealed an efficient and robust notochordal differentiation. We further identified a key regulatory network consisting of eight candidate genes encoding transcription factors including PAX6, GDF3, FOXD3, TDGF1, and SOX5, which are considered as potential drivers of notochordal differentiation. This is the first single cell transcriptomic analysis of notochordal cells derived from hESCs. The ability to efficiently obtain notochordal cells from pluripotent stem cells provides an additional tool to develop new cell-based therapies for the treatment of IDD.

The node-streak border region comprising notochord progenitor cells (NPCs) at the posterior node and neuro-mesodermal progenitor cells (NMPs) in the adjacent epiblast is the prime organizing center for axial elongation in mouse embryos. The T-box transcription factor brachyury (T) is essential for both formation of the notochord and maintenance of NMPs, and thus is a key regulator of trunk and tail development. The T promoter controlling T expression in NMPs and nascent mesoderm has been characterized in detail; however, control elements for T expression in the notochord have not been identified yet. We have generated a series of deletion alleles by CRISPR/Cas9 genome editing in mESCs, and analyzed their effects in mutant mouse embryos. We identified a 37 kb region upstream of T that is essential for notochord function and tailbud outgrowth. Within that region, we discovered a T-binding enhancer required for notochord cell specification and differentiation. Our data reveal a complex regulatory landscape controlling cell type-specific expression and function of T in NMP/nascent mesoderm and node/notochord, allowing proper trunk and tail development., Summary: Genetic dissection of the mouse brachyury locus identifies a notochord enhancer and predicts additional control elements essential for trunk and tail development of the mouse embryo.

Chordomas are rare neoplasms arising from remnants of the notochord. We report a case of a 61-year-old female who underwent work-up for bariatric surgery and was found to have bilateral pulmonary nodules on computed tomography scan. Video-assisted thoracoscopic wedge resections were performed, and immunohistochemical stains confirmed the diagnosis of chordoma in all lesions. Much of our current knowledge of chordomas comes from axial and extra axial, non pulmonary presentations. Surgery appears to be the best treatment option as these tumors are generally chemo resistant. However, there are limited numbers of case reports of this entity.

Back and neck pain are significant healthcare burdens that are commonly associated with pathologies of the intervertebral disc (IVD). The poor understanding of the cellular heterogeneity within the IVD makes it difficult to develop regenerative IVD therapies. To address this gap, we developed an atlas of bovine (Bos taurus) caudal IVDs using single-cell RNA-sequencing (scRNA-seq). Unsupervised clustering resolved 15 unique clusters, which we grouped into the following annotated partitions: nucleus pulposus (NP), outer annulus fibrosus (oAF), inner AF (iAF), notochord, muscle, endothelial, and immune cells. Analyzing the pooled gene expression profiles of the NP, oAF, and iAF partitions allowed us to identify novel markers for NP (CP, S100B, H2AC18, SNORC, CRELD2, PDIA4, DNAJC3, CHCHD7, and RCN2), oAF (IGFBP6, CTSK, LGALS1, and CCN3), and iAF (MGP, COMP, SPP1, GSN, SOD2, DCN, FN1, TIMP3, WDR73, and GAL) cells. Network analysis on subpopulations of NP and oAF cells determined that clusters NP1, NP2, NP4, and oAF1 displayed gene expression profiles consistent with cell survival, suggesting these clusters may uniquely support viability under the physiological stresses of the IVD. Clusters NP3, NP5, oAF2, and oAF3 expressed various extracellular matrix (ECM)-associated genes, suggesting their role in maintaining IVD structure. Lastly, transcriptional entropy and pseudotime analyses found that clusters NP3 and NP1 had the most stem-like gene expression signatures of the NP partition, implying these clusters may contain IVD progenitor cells. Overall, results highlight cell type diversity within the IVD, and these novel cell phenotypes may enhance our understanding of IVD development, homeostasis, degeneration, and regeneration.

Neurulation is the process in early vertebrate embryonic development during which the neural plate folds to form the neural tube. Spinal neural tube folding in the posterior neuropore changes over time, first showing a median hingepoint, then both the median hingepoint and dorsolateral hingepoints, followed by dorsolateral hingepoints only. The biomechanical mechanism of hingepoint formation in the mammalian neural tube is poorly understood. Here, we employ a mechanical finite element model to study neural tube formation. The computational model mimics the mammalian neural tube using microscopy data from mouse and human embryos. While intrinsic curvature at the neural plate midline has been hypothesized to drive neural tube folding, intrinsic curvature was not sufficient for tube closure in our simulations. We achieved neural tube closure with an alternative model combining mesoderm expansion, non-neural ectoderm expansion and neural plate adhesion to the notochord. Dorsolateral hingepoints emerged in simulations with low mesoderm expansion and zippering. We propose that zippering provides the biomechanical force for dorsolateral hingepoint formation in settings where the neural plate lateral sides extend above the mesoderm. Together, these results provide a new perspective on the biomechanical and molecular mechanism of mammalian spinal neurulation.

Evolutionary novelties entail the origin of morphologies that enable new functions. These features can arise through changes to gene function and regulation. One important novelty is the fused rod at the end of the vertebral column in anurans, the urostyle. This feature is composed of a coccyx and an ossifying hypochord, and both structures ossify during metamorphosis. We used Laser Capture Micro-dissection of these identified tissues and subjected them to RNA-seq and ATAC-seq analyses at three developmental stages in tadpoles of Xenopus tropicalis. These experiments reveal that the coccyx and hypochord have two different molecular signatures. ATAC-seq data reveals potential regulatory regions that are observed in proximity to candidate genes identified from RNA-seq. Neuronal (TUBB3) and muscle markers (MYH3) are upregulated in coccygeal tissues, whereas T-box genes (TBXT, TBXT.2), corticosteroid stress hormones (CRCH.1), and matrix metallopeptidases (MMP1, MMP8, MMP13) are upregulated in the hypochord. Even though an ossifying hypochord is only present in anurans, this ossification between the vertebral column and the notochord appears to resemble a congenital vertebral anomaly seen prenatally in humans, caused by an ectopic expression of the TBXT/TBXT.2 gene. This work opens the way to functional studies that help us better elucidate anuran bauplan evolution.

The transient but pronounced ventral tail bending is found in many chordate embryos and constitutes an interesting model of how tissue interactions control embryo shape (Lu et al., 2020). Here, we identify one key upstream regulator of ventral tail bending in the ascidian Ciona embryo. We show that during early tailbud stage, ventral epidermal cells exhibit a boat-shaped morphology (boat cell) with a narrow apical surface where phosphorylated myosin (pMLC) accumulated. We further show that interfering with the function of the BMP ligand Admp leads to pMLC localizing to the basal instead of the apical side of ventral epidermal cells and a reduced number of boat cells. Finally, we show that cutting ventral epidermal midline cells at their apex using a ultraviolet laser relaxes ventral tail bending. Based on these results, we propose a novel function for Admp in localizing pMLC to the apical side of ventral epidermal cells, which causes the tail to bend ventrally by resisting antero-posterior notochord extension at the ventral side of the tail.Summary StatementAdmp is an upstream regulator of tail bending in the chordate Ciona tailbud embryo, determining tissue polarity of the ventral midline epidermis by localizing phosphorylated myosin.

Gene regulatory networks coordinate the formation of organs and structures that compose the evolving body plans of different organisms. We are using a simple chordate model, the Ciona embryo, to investigate the essential gene regulatory network that orchestrates morphogenesis of the notochord, a structure necessary for the proper development of all chordate embryos. Although numerous transcription factors expressed in the notochord have been identified in different chordates, several of them remain to be positioned within a regulatory framework. Here, we focus on Xbp1, a transcription factor expressed during notochord formation in Ciona and other chordates. Through the identification of Xbp1-downstream notochord genes in Ciona, we found evidence of the early co-option of genes involved in the unfolded protein response to the notochord developmental program. We report the regulatory interplay between Xbp1 and Brachyury, and by extending these results to Xenopus, we show that Brachyury and Xbp1 form a cross-regulatory subcircuit of the notochord gene regulatory network that has been consolidated during chordate evolution.