Cis-regulatory interfaces reveal the molecular mechanisms underlying the notochord gene regulatory network of Ciona.

Tissue-specific gene expression is fundamental in development and evolution, and is mediated by transcription factors and by the cis-regulatory regions (enhancers) that they control. Transcription factors and their respective tissue-specific enhancers are essential components of gene regulatory networks responsible for the development of tissues and organs. Although numerous transcription factors have been characterized from different organisms, the knowledge of the enhancers responsible for their tissue-specific expression remains fragmentary. Here we use Ciona to study the enhancers associated with ten transcription factors expressed in the notochord, an evolutionary hallmark of the chordate phylum. Our results illustrate how two evolutionarily conserved transcription factors, Brachyury and Foxa2, coordinate the deployment of other notochord transcription factors. The results of these detailed cis-regulatory analyses delineate a high-resolution view of the essential notochord gene regulatory network of Ciona, and provide a reference for studies of transcription factors, enhancers, and their roles in development, disease, and evolution.

Snail Transcriptionally Represses Brachyury to Promote the Mesenchymal-Epithelial Transition in Ascidian Notochord Cells.

Mesenchymal-epithelial transition (MET) is a widely spread and evolutionarily conserved process across species during development. In Ciona embryogenesis, the notochord cells undergo the transition from the non-polarized mesenchymal state into the polarized endothelial-like state to initiate the lumen formation between adjacent cells. Based on previously screened MET-related transcription factors by ATAC-seq and Smart-Seq of notochord cells, Ciona robusta Snail (Ci-Snail) was selected for its high-level expression during this period. Our current knockout results demonstrated that Ci-Snail was required for notochord cell MET. Importantly, overexpression of the transcription factor Brachyury in notochord cells resulted in a similar phenotype with failure of lumen formation and MET. More interestingly, expression of Ci-Snail in the notochord cells at the late tailbud stage could partially rescue the MET defect caused by Brachyury-overexpression. These results indicated an inverse relationship between Ci-Snail and Brachyury during notochord cell MET, which was verified by RT-qPCR analysis. Moreover, the overexpression of Ci-Snail could significantly inhibit the transcription of Brachyury, and the CUT&Tag-qPCR analysis demonstrated that Ci-Snail is directly bound to the upstream region of Brachyury. In summary, we revealed that Ci-Snail promoted the notochord cell MET and was essential for lumen formation via transcriptionally repressing Brachyury.

Cell contacts and pericellular matrix in the Xenopus gastrula chordamesoderm.

Convergent extension of the chordamesoderm is the best-examined gastrulation movement in Xenopus. Here we study general features of cell-cell contacts in this tissue by combining depletion of adhesion factors C-cadherin, Syndecan-4, fibronectin, and hyaluronic acid, the analysis of respective contact width spectra and contact angles, and La3+ staining of the pericellular matrix. We provide evidence that like in other gastrula tissues, cell-cell adhesion in the chordamesoderm is largely mediated by different types of pericellular matrix. Specific glycocalyx structures previously identified in Xenopus gastrula tissues are absent in chordamesoderm but other contact types like 10-20 nm wide La3+ stained structures are present instead. Knockdown of any of the adhesion factors reduces the abundance of cell contacts but not the average relative adhesiveness of the remaining ones: a decrease of adhesiveness at low contact widths is compensated by an increase of contact widths and an increase of adhesiveness proportional to width. From the adhesiveness-width relationship, we derive a model of chordamesoderm cell adhesion that involves the interdigitation of distinct pericellular matrix units. Quantitative description of pericellular matrix deployment suggests that reduced contact abundance upon adhesion factor depletion is correlated with excessive accumulation of matrix material in non-adhesive gaps and the loss of some contact types.

scRNA-seq analysis of cells comprising the amphioxus notochord.

Cephalochordates occupy a key phylogenetic position for deciphering the origin and evolution of chordates, since they diverged earlier than urochordates and vertebrates. The notochord is the most prominent feature of chordates. The amphioxus notochord features coin-shaped cells bearing myofibrils. Notochord-derived hedgehog signaling contributes to patterning of the dorsal nerve cord, as in vertebrates. However, properties of constituent notochord cells remain unknown at the single-cell level. We examined these properties using Iso-seq analysis, single-cell RNA-seq analysis, and in situ hybridization (ISH). Gene expression profiles broadly categorize notochordal cells into myofibrillar cells and non-myofibrillar cells. Myofibrillar cells occupy most of the central portion of the notochord, and some cells extend the notochordal horn to both sides of the ventral nerve cord. Some notochord myofibrillar genes are not expressed in myotomes, suggesting an occurrence of myofibrillar genes that are preferentially expressed in notochord. On the other hand, non-myofibrillar cells contain dorsal, lateral, and ventral Müller cells, and all three express both hedgehog and Brachyury. This was confirmed by ISH, although expression of hedgehog in ventral Müller cells was minimal. In addition, dorsal Müller cells express neural transmission-related genes, suggesting an interaction with nerve cord. Lateral Müller cells express hedgehog and other signaling-related genes, suggesting an interaction with myotomes positioned lateral to the notochord. Ventral Müller cells also expressed genes for FGF- and EGF-related signaling, which may be associated with development of endoderm, ventral to the notochord. Lateral Müller cells were intermediate between dorsal/ventral Müller cells. Since vertebrate notochord contributes to patterning and differentiation of ectoderm (nerve cord), mesoderm (somite), and endoderm, this investigation provides evidence that an ancestral or original form of vertebrate notochord is present in extant cephalochordates.

Notochordal cells: A potential therapeutic option for intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is a prevalent musculoskeletal degenerative disorder worldwide, and ~40% of chronic low back pain cases are associated with IDD. Although the pathogenesis of IDD remains unclear, the reduction in nucleus pulposus cells (NPCs) and degradation of the extracellular matrix (ECM) are critical factors contributing to IDD. Notochordal cells (NCs), derived from the notochord, which rapidly degrades after birth and is eventually replaced by NPCs, play a crucial role in maintaining ECM homeostasis and preventing NPCs apoptosis. Current treatments for IDD only provide symptomatic relief, while lacking the ability to inhibit or reverse its progression. However, NCs and their secretions possess anti-inflammatory properties and promote NPCs proliferation, leading to ECM formation. Therefore, in recent years, NCs therapy targeting the underlying cause of IDD has emerged as a novel treatment strategy. This article provides a comprehensive review of the latest research progress on NCs for IDD, covering their biological characteristics, specific markers, possible mechanisms involved in IDD and therapeutic effects. It also highlights significant future directions in this field to facilitate further exploration of the pathogenesis of IDD and the development of new therapies based on NCs strategies.

Single cell RNA sequencing reveals emergent notochord-derived cell subpopulations in the postnatal nucleus pulposus.

Intervertebral disc degeneration is a leading cause of chronic low back pain. Cell-based strategies that seek to treat disc degeneration by regenerating the central nucleus pulposus (NP) hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native NP cells, which are unique amongst skeletal cell types in that they arise from the embryonic notochord. In this study, we use single cell RNA sequencing to demonstrate emergent heterogeneity amongst notochord-derived NP cells in the postnatal mouse disc. Specifically, we established the existence of progenitor and mature NP cells, corresponding to notochordal and chondrocyte-like cells, respectively. Mature NP cells exhibited significantly higher expression levels of extracellular matrix (ECM) genes including aggrecan, and collagens II and VI, along with elevated transforming growth factor-beta and phosphoinositide 3 kinase-protein kinase B signaling. Additionally, we identified Cd9 as a novel surface marker of mature NP cells, and demonstrated that these cells were localized to the NP periphery, increased in numbers with increasing postnatal age, and co-localized with emerging glycosaminoglycan-rich matrix. Finally, we used a goat model to show that Cd9+ NP cell numbers decrease with moderate severity disc degeneration, suggesting that these cells are associated with maintenance of the healthy NP ECM. Improved understanding of the developmental mechanisms underlying regulation of ECM deposition in the postnatal NP may inform improved regenerative strategies for disc degeneration and associated low back pain.

Embryo-scale reverse genetics at single-cell resolution.

The maturation of single-cell transcriptomic technologies has facilitated the generation of comprehensive cellular atlases from whole embryos1-4. A majority of these data, however, has been collected from wild-type embryos without an appreciation for the latent variation that is present in development. Here we present the 'zebrafish single-cell atlas of perturbed embryos': single-cell transcriptomic data from 1,812 individually resolved developing zebrafish embryos, encompassing 19 timepoints, 23 genetic perturbations and a total of 3.2 million cells. The high degree of replication in our study (eight or more embryos per condition) enables us to estimate the variance in cell type abundance organism-wide and to detect perturbation-dependent deviance in cell type composition relative to wild-type embryos. Our approach is sensitive to rare cell types, resolving developmental trajectories and genetic dependencies in the cranial ganglia neurons, a cell population that comprises less than 1% of the embryo. Additionally, time-series profiling of individual mutants identified a group of brachyury-independent cells with strikingly similar transcriptomes to notochord sheath cells, leading to new hypotheses about early origins of the skull. We anticipate that standardized collection of high-resolution, organism-scale single-cell data from large numbers of individual embryos will enable mapping of the genetic dependencies of zebrafish cell types, while also addressing longstanding challenges in developmental genetics, including the cellular and transcriptional plasticity underlying phenotypic diversity across individuals.

A collagen-rich arch in the urochordate notochord coordinates cell shaping and multi-tissue elongation.

Cell and tissue reshaping is crucial for coordinating three-dimensional pattern formation, in which the size and shape of the cells must be accurately regulated via signal transport and communication among tissues. However, the identity of signaling and transportation mechanisms in this process remains elusive. In our study, we identified an extracellular matrix (ECM) structure with a vertebra-like shape surrounding the central notochord tissue in the larval tail of the urochordate Ciona. Additionally, we verified that the ECM structure was formed de novo, mainly from collagens secreted by notochord cells. Fluorescence recovery after photobleaching and simulation results revealed that this structure was formed via diffusional collagen flow from a notochord that was restricted and molded in the spaces among tail tissues. We revealed that the collagen structure was essential for notochord cell arrangement and elongation. Furthermore, we observed that the central notochord connects with the epidermis through this ECM structure. The disruption of this structure by collagen knockdown and loss-of-collagen function caused the failure of notochord elongation. More importantly, the epidermis could not elongate proportionally with notochord, indicating that the collagen-rich structure serves as a scaffold to coordinate the concurrent elongation of the tail tissues. These findings provide insights into how the central tissue forms and molds its surrounding ECM structure, by not only regulating its own morphogenesis but also functioning as a scaffold for signal transmission to orchestrate the coordinated morphologic reshaping of the surrounding tissues.

Novel tissue thickening around the notochord sheath found in deformed Japanese eel Anguilla japonica leptocephali.

Even though reared leptocephalus larvae of the Japanese eel Anguilla japonica have a high incidence of notochord deformities (>60%), the cause is unknown. We performed histological examinations of the notochord and associated organs in reared larvae to better understand the process causing notochord deformation in eel larvae. In deformed larvae, unknown tissue thickening was discovered near the notochord sheath. Azan staining revealed that these tissue thickenings are most likely collagen fibers within fibrous connective tissue. This was almost identical to the connective tissue found in the primordium of the vertebral body around the notochord sheath in properly metamorphosing larvae. Furthermore, the amount of the thyroid hormone triiodothyronine (T3) was significantly higher in deformed larvae than in normal larvae, indicating that notochord deformity is probably linked to metamorphosis despite the immature stage of growth. We suggest that the aberrant growth of connective tissue surrounding the notochord sheath induced by incomplete metamorphosis causes deformities in eel larvae. The reason why deformed larvae have greater thyroid hormone levels is still unknown. It is important to assess how environmental and dietary factors affect the thyroid hormone levels of eel larvae raised in captivity.

Gene expression in notochord and nuclei pulposi: a study of gene families across the chordate phylum.

The transition from notochord to vertebral column is a crucial milestone in chordate evolution and in prenatal development of all vertebrates. As ossification of the vertebral bodies proceeds, involutions of residual notochord cells into the intervertebral discs form the nuclei pulposi, shock-absorbing structures that confer flexibility to the spine. Numerous studies have outlined the developmental and evolutionary relationship between notochord and nuclei pulposi. However, the knowledge of the similarities and differences in the genetic repertoires of these two structures remains limited, also because comparative studies of notochord and nuclei pulposi across chordates are complicated by the gene/genome duplication events that led to extant vertebrates. Here we show the results of a pilot study aimed at bridging the information on these two structures. We have followed in different vertebrates the evolutionary trajectory of notochord genes identified in the invertebrate chordate Ciona, and we have evaluated the extent of conservation of their expression in notochord cells. Our results have uncovered evolutionarily conserved markers of both notochord development and aging/degeneration of the nuclei pulposi.

Conserved enhancers control notochord expression of vertebrate Brachyury.

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development.

Genome-wide identification of notochord enhancers comprising the regulatory landscape of the brachyury locus in mouse.

The node and notochord are important signaling centers organizing the dorso-ventral patterning of cells arising from neuro-mesodermal progenitors forming the embryonic body anlage. Owing to the scarcity of notochord progenitors and notochord cells, a comprehensive identification of regulatory elements driving notochord-specific gene expression has been lacking. Here, we have used ATAC-seq analysis of FACS-purified notochord cells from Theiler stage 12-13 mouse embryos to identify 8921 putative notochord enhancers. In addition, we established a new model for generating notochord-like cells in culture, and found 3728 of these enhancers occupied by the essential notochord control factors brachyury (T) and/or Foxa2. We describe the regulatory landscape of the T locus, comprising ten putative enhancers occupied by these factors, and confirmed the regulatory activity of three of these elements. Moreover, we characterized seven new elements by knockout analysis in embryos and identified one new notochord enhancer, termed TNE2. TNE2 cooperates with TNE in the trunk notochord, and is essential for notochord differentiation in the tail. Our data reveal an essential role of Foxa2 in directing T-expressing cells towards the notochord lineage.

Case of split notochord syndrome: a neonate with thoracic neuroenteric cyst, abdominal duodenal duplication cyst, malrotation and vertebral anomalies.

The authors describe a case of a male neonate with split notochord syndrome presenting with cervico-thoracic deformity, thoracic neuroenteric cyst, separate abdominal duodenal duplication cyst and concurrent intestinal malrotation. This combination of abnormalities is very rare. When these lesions are suspected, patients must be investigated carefully.This case is presented not only to recount an infrequent combination of structural abnormalities but also to raise awareness of the signs that should point to clinical suspicion and prompt diagnosis.Following surgical excision of the thoracic neuroenteric cyst, the patient has made a good recovery.

Chordoma concurrent with benign notochordal cell tumor of the mobile spine. Should the chordoma part be biopsied? - A stepwise approach for management.

Some evidence suggests that benign notochordal tumors (BNCTs) could be a potential precursor of chordoma. We present an educational rare case of lumbar vertebral BNCTs concomitant with a destructive lesion not reachable on biopsy but thought to be chordoma. We present a stepwise approach for management of these difficult entities based on radiological features.

Ecchordosis Physaliphora: Does It Even Exist?

The term ecchordosis physaliphora (EP) has been used historically to describe a benign notochordal remnant with no growth potential, most commonly occuring in the central clivus. Unfortunately, the radiologic appearance of EP overlaps considerably with the appearance of low-grade chordomas, which do have the potential for growth. In this article, we review new pathologic terminology that better describes this family of diseases, and we propose new radiologic terms that better address the uncertainty of the radiologic diagnosis. The surgical importance of accurate terminology and the implications for patient care are discussed.

Loss of collagen gene expression in the notochord of the tailless tunicate Molgula occulta.

In tunicates, several species in the Molgulidae family have convergently lost the tailed, swimming larval body plan, including the morphogenesis of the notochord, a major chordate-defining trait. Through the comparison of tailless M. occulta and a close relative, the tailed species M. oculata, we show that notochord-specific expression of the Collagen Type I/II Alpha (Col1/2a) gene appears to have been lost specifically in the tailless species. Using CRISPR/Cas9-mediated mutagenesis in the tailed laboratory model tunicate Ciona robusta, we demonstrate that Col1/2a plays a crucial role in the convergent extension of notochord cells during tail elongation. Our results suggest that the expression of Col1/2a in the notochord, although necessary for its morphogenesis in tailed species, is dispensable for tailless species. This loss is likely a result of the accumulation of cis-regulatory mutations in the absence of purifying selective pressure. More importantly, the gene itself is not lost, likely due to its roles in other developmental processes, including during the adult stage. Our study further confirms the Molgulidae as an interesting family in which to study the evolutionary loss of tissue-specific expression of indispensable genes.

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

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

Dynamic BMP signaling mediates notochord segmentation in zebrafish.

The vertebrate spine is a metameric structure composed of alternating vertebral bodies (centra) and intervertebral discs.1 Recent studies in zebrafish have shown that the epithelial sheath surrounding the notochord differentiates into alternating cartilage-like (col2a1/col9a2+) and mineralizing (entpd5a+) segments which serve as a blueprint for centra formation.2,3,4,5 This process also defines the trajectories of migrating sclerotomal cells that form the mature vertebral bodies.4 Previous work demonstrated that notochord segmentation is typically sequential and involves the segmented activation of Notch signaling.2 However, it is unclear how Notch is activated in an alternating and sequential fashion. Furthermore, the molecular components that define segment size, regulate segment growth, and produce sharp segment boundaries have not been identified. In this study, we uncover that a BMP signaling wave acts upstream of Notch during zebrafish notochord segmentation. Using genetically encoded reporters of BMP activity and signaling pathway components, we show that BMP signaling is dynamic as axial patterning progresses, leading to the sequential formation of mineralizing domains in the notochord sheath. Genetic manipulations reveal that type I BMP receptor activation is sufficient to ectopically trigger Notch signaling. Moreover, loss of Bmpr1ba and Bmpr1aa or Bmp3 function disrupts ordered segment formation and growth, which is recapitulated by notochord-specific overexpression of the BMP antagonist, Noggin3. Our data suggest that BMP signaling in the notochord sheath precedes Notch activation and instructs segment growth, facilitating proper spine morphogenesis.