Notochord Development Research Articles

Spinal scoliosis: insights into developmental mechanisms and animal models.

Spinal scoliosis, a prevalent spinal deformity impacting both physical and mental well-being, has a significant genetic component, though the exact pathogenic mechanisms remain elusive. This review offers a comprehensive exploration of current research on embryonic spinal development, focusing on the genetic and biological intricacies governing axial elongation and straightening. Zebrafish, a vital model in developmental biology, takes a prominent role in understanding spinal scoliosis. Insights from zebrafish studies illustrate genetic and physiological aspects, including notochord development and cerebrospinal fluid dynamics, revealing the anomalies contributing to scoliosis. In this review, we acknowledge existing challenges, such as deciphering the unique dynamics of human spinal development, variations in physiological curvature, and disparities in cerebrospinal fluid circulation. Further, we emphasize the need for caution when extrapolating findings to humans and for future research to bridge current knowledge gaps. We hope that this review will be a beneficial frame of reference for the guidance of future studies on animal models and genetic research for spinal scoliosis.

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.

Quantitative Phosphoproteomics Reveals the Requirement of DYRK1-Mediated Phosphorylation of Ion Transport- and Cell Junction-Related Proteins for Notochord Lumenogenesis in Ascidian.

The dual-specificity tyrosine phosphorylation-regulated kinase (DYRK1) phosphorylates diverse substrates involved in various cellular processes. Here, we found that blocking the kinase activity of DYRK1 inhibited notochord development and lumenogenesis in ascidian Ciona savignyi. By performing phosphoproteomics in conjunction with notochord-specific proteomics, we identified 1065 notochord-specific phosphoproteins that were present during lumen inflation, of which 428 differentially phosphorylated proteins (DPPs) were identified after inhibition of DYRK1 kinase activity. These DPPs were significantly enriched in metal ion transmembrane transporter activity, protein transport and localization, and tight junction. We next analyzed the downregulated phosphoproteins and focused on those belonging to the solute carrier (SLC), Ras-related protein (RAB), and tight junction protein (TJP) families. In vivo phospho-deficient study showed that alanine mutations on the phosphosites of these proteins resulted in defects of lumenogenesis during Ciona notochord development, demonstrating the crucial roles of phosphorylation of transmembrane transport-, vesicle trafficking-, and tight junction-related proteins in lumen formation. Overall, our study provides a valuable data resource for investigating notochord lumenogenesis and uncovers the molecular mechanisms of DYRK1-mediated notochord development and lumen inflation.

Proteomic identification of intracellular vesicle trafficking and protein glycosylation requirements for lumen inflation in Ciona notochord.

Lumen formation and inflation are crucial steps for tubular organ morphogenesis, yet the underling mechanism remains largely unrevealed. Here, we applied 4D proteomics to screen the lumenogenesis-related proteins and revealed the biological pathways potentially that are involved in lumen inflation during notochord lumen formation in the ascidian Ciona savignyi. In total, 910 differentiated expressed proteins (DEPs) were identified before and after notochord lumen formation utilizing Mfuzz analysis. Those DEPs were grouped into four upregulated clusters based on their quantitative expression patterns; the functions of these proteins were enriched in protein metabolic and biosynthetic process, the establishment of localization, and vesicle-mediated transport. We analyzed the vesicle trafficking cluster and focused on several vesicle transport hub proteins. In vivo function-deficient experiments showed that mutation of vesicle transport proteins resulted in an abnormal lumen in notochord development, demonstrating the crucial role of intracellular trafficking for lumen formation. Moreover, abundant extracellular matrix proteins were identified, the majority of which were predicted to be glycosylated proteins. Inhibition of glycosylation markedly reduced the lumen expansion rate in notochord cells, suggesting that protein glycosylation is essential for lumenogenesis. Overall, our study provides an invaluable resource and reveals the crucial mechanisms in lumen formation and expansion.

Diverse logics and grammar encode notochord enhancers.

The notochord is a defining feature of all chordates. The transcription factors Zic and ETS regulate enhancer activity within the notochord. We conduct high-throughput screens of genomic elements within developing Ciona embryos to understand how Zic and ETS sites encode notochord activity. Our screen discovers an enhancer located near Lama, a gene critical for notochord development. Reversing the orientation of an ETS site within this enhancer abolishes expression, indicating that enhancer grammar is critical for notochord activity. Similarly organized clusters of Zic and ETS sites occur within mouse and human Lama1 introns. Within a Brachyury (Bra) enhancer, FoxA and Bra, in combination with Zic and ETS binding sites, are necessary and sufficient for notochord expression. This binding site logic also occurs within other Ciona and vertebrate Bra enhancers. Collectively, this study uncovers the importance of grammar within notochord enhancers and discovers signatures of enhancer logic and grammar conserved across chordates.

Thiophanate-methyl induces notochord toxicity by activating the PI3K-mTOR pathway in zebrafish (Danio rerio) embryos

Thiophanate-methyl (TM), a typical pesticide widely used worldwide, was detected in rivers, soil, fruits, and vegetables. Thus, it is urgent to identify the potential harm of TM residual to non-target organisms and its molecular mechanisms. We used zebrafish (Danio rerio) in this study to evaluate TM toxicity. TM exposure induced developmental toxicity, including inhibited hatchability, reduced heart rates, restrained spontaneous locomotion, and decreased body length. Furthermore, we observed obvious toxicity in the notochord and detected increased expression levels of notochord-related genes (shha, col2a, and tbxta) by in situ hybridization in zebrafish larvae. In addition, calcein staining, alkaline phosphatase (ALP) activity analysis, and anatomic analysis indicated that TM induced notochord toxicity. We used rescue experiments to verify whether the PI3K-mTOR pathway involved in the notochord development was the cause of notochord abnormalities. Rapamycin and LY294002 (an inhibitor of PI3K) relieve notochord toxicity caused by TM, including morphological abnormalities. In summary, TM might induce notochord toxicity by activating the PI3K-mTOR pathway in zebrafish.

Oxamyl exerts developmental toxic effects in zebrafish by disrupting the mitochondrial electron transport chain and modulating PI3K/Akt and p38 Mapk signaling

Oxamyl, a carbamate insecticide, is mainly used to control nematodes in the agricultural field. Although oxamyl is a widely used insecticide that is associated with ecological concerns, limited studies have examined the toxic effects of oxamyl on the developmental stage and the underlying mechanisms. In this study, the developmental toxicity of oxamyl was demonstrated using zebrafish, which is a representative model as it is associated with rapid embryogenesis and a toxic response similar to that of other vertebrates. The morphological alteration of zebrafish larvae was analyzed to confirm the sub-lethal toxicity of oxamyl. Analysis of transgenic zebrafish (olig2:dsRED and flk1:eGFP line) and mRNA levels of genes associated with individual organ development revealed that oxamyl exerted toxic effects on the development of neuron, notochord, and vascular system. Next, the adverse effect of oxamyl on the mitochondrial electron transport chain was examined. Treatment with oxamyl altered the PI3K/Akt signaling and p38 Mapk signaling pathways in zebrafish. Thus, this study elucidated the mechanisms underlying the developmental toxicity of oxamyl and provided information on the parameters to assess the developmental toxicity of other environmental contaminants.

CD44 is highly expressed in stem/progenitor cells originating the intervertebral discs in the human notochord.

The notochord acts as a patterning structure, playing a key role in the formation of the vertebral column, both indirectly by inducing sclerotome cell differentiation and directly by forming the nucleus pulposus of intervertebral discs. The abnormal development of the notochord results in an easy equation with a variety of birth defects. Therefore, we focused our attention on the analysis of the early stages of human notochord development by highlighting the role of progenitor stem cells involved in the origin of intervertebral discs (IVDs). Eight human fetuses, ranging from 8 up to 21 weeks of gestational age, were obtained from spontaneous abortion or voluntary interruption of gestation. Samples were 10% formalin-fixed, routinely processed, and paraffin-embedded. Five micron-tick paraffin sections were obtained from each sample. Sections were stained with hematoxylin-eosin and PAS stain for a morphological examination. Tissue samples were immunostained with a commercial anti-human CD44 rabbit monoclonal antibody at 1:100 dilution. Immunoreactivity for CD44 was detected in six out of eight notochords examined in this study. Reactivity for CD44 was restricted to progenitor cells giving rise to the nucleus pulposus (NP) of the developing IVDs. Positive cells showed a membranous and/or cytoplasmic immunostaining, no reactivity was observed in the nuclear compartment. CD44 expression was always restricted to IVD precursor cells, whereas cartilage precursors were devoid of labelling. Our study shows, for the first time, that the stem cell marker CD44 selectively marks intervertebral disc progenitor cells, paralleling their differentiation toward a discogenic phenotype. Therefore, our results suggest that CD44 plays a key role in IVD development, allowing its differentiation from surrounding undifferentiated notochordal cells toward a IVD phenotype. Given the role of CD44 in IVD development, we may hypothesize that low CD44 levels might be associated with changes in IVD development and with susceptibility to develop back pain later in life.

Effect of monosultap on notochord development in zebrafish (Danio rerio) embryos

Monosultap (Mon) is a broad-spectrum insecticide used in agricultural production to control stem borers in rice fields. Currently, little evidence shows how Mon affects notochord development in zebrafish (Danio rerio). In our study, zebrafish embryos were exposed to 0.25, 0.5, and 0.75 mg/L Mon to determine the effects of different concentrations of Mon on notochord development. Mon exposure reduced the body length, decreased the heart rate and hatchability, and induced notochord deformity in zebrafish. The effects of Mon exposure on the internal organization of the notochord and the structural abnormalities were determined based on histological staining of paraffinized tissue sections. Quantitative polymerase chain reaction (qPCR) and in situ hybridization findings revealed that the expression levels of genes related to notochord development (shha, col2a, and ptch2) showed an increasing trend in a concentration-dependent manner. An abnormal increase of apoptosis and cell proliferation in some parts of the notochord suggested that Mon exposure could cause developmental abnormality of the notochord. This study revealed the toxicity of Mon in notochord development. Our findings provide information in assessing the risk of Mon to the ecological environment and human health.

Environmental Conditions along Tuna Larval Dispersion: Insights on the Spawning Habitat and Impact on Their Development Stages

Estimated larval backward trajectories of three Tuna species, namely, Atlantic Bluefin Tuna (Thunnus thynnus, Linnaeus, 1758), Bullet Tuna (Auxis Rochei, Risso, 1801) and Albacore Tuna (Thunnus alalunga, Bonnaterre, 1788) in the central Mediterranean Sea, were used to characterize their spawning habitats, and to assess the impact of changes due to the major environmental parameters (i.e., sea surface temperature and chlorophyll-a concentration) on larval development during their advection by surface currents. We assumed that the environmental variability experienced by larvae along their paths may have influenced their development, also affecting their survival. Our results showed that the Tuna larvae underwent an accelerated growth in favorable environmental conditions, impacting on the notochord development. In addition, further updated information on spawning and larval retention habitats of Atlantic Bluefin Tuna, Bullet and Albacore Tunas in the central Mediterranean Sea were delivered.

Bubble packing, eccentricity, and notochord development.

This paper develops a theoretical basis for the observed relationship between cell arrangements in notochords and analog physical models, and the eccentricity of their cross sections. Three models are developed and analyzed, of the mechanics of cell packing in sheaths. The key ratios governing the packing patterns and eccentricity are cells per unit length λ, tension ratio Γ, and eccentricity e. For flexible and semi-flexible sheaths, the optimal packing pattern shifts from "bamboo", with a symmetric cross section, to "staircase", with an eccentric cross section, at a critical value λ=1.13. In rigid tubes, this threshold is lowered as imposed eccentricity is increased. Patterns can be observed which are not optimal; pattern transitions may occur below or above the critical λ values. The eccentricity of staircase patterns in flexible and semi-flexible tubes is found to be dependent on the tension ratio Γ, increasing as sheath tension decreases relative to interior cell tension. A novel "serpentine" packing pattern appears for low Γ near the critical λ. The developmental utility of enforcing notochord eccentricity is discussed, as well as potential mechanisms for such control.

Functional Analysis of Zebrafish socs4a: Impacts on the Notochord and Sensory Function

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.

Effects of RTTN gene mutations and the need for complementary cDNA analysis for some transcripts: case report

Abstract RTTN is a gene coding for Rotatin protein which has been localized at ciliary body and centriole of human fibroblasts. Its exact function in human is not well identified. However, it is thought to play important role in maintenance of normal structure of primary cilia and hence in left to right organ specification, axial rotation, development of notochord as well as early cellular division in mice. Overall 28 cases have been reported with homozygous pathogenic mutations of RTTN gene till 2019 to our knowledge. They presented with primary microcephaly, short stature, polymicrogyria with or without seizures (MPPS). These symptoms can be summarized in microcephaly, generalized growth delay, malformation of the developing cortex (MDC) and hence neurodevelopmental delay with or without seizures. Meanwhile all of these signs usually appear in late prenatal or even postnatal life which emphasizes the need for other diagnostic tools in case of detection of RTTN variables of unknown significance or in case of accidental detection of multiple transcripts by NGS in prenatal genetic testing. A case with prenatal diagnosis of two different RTTN gene transcripts in fetal WES analysis. One of the transcripts showed RTTN homozygous gene mutation while the other transcript was normal. This article presents the multidisciplinary approach followed in prenatal as well as postnatal period. It also highlights the importance of gene expression analysis in prenatal genetics.

Origin of the Chordate Notochord

The phylum of Chordata is defined based on the discovery of a coelom-like dorsal notochord in ascidian and amphioxus embryos. Chordata can be classified into three subphylums, Cephalochordata, Urochordata, and Vertebrata, united by the presence of a notochord at some point during development. The origin of the notochord, the signature anatomical structure of chordates, has been under debate since the publication of Alexander Kovalevsky’s work in the mid-19th century that placed ascidians close to the vertebrates on the phylogenetic tree. During the late 20th century, the development of molecular and genetic tools in biology brought about a revival of studies on the evolutionary path of notochord development. Two main hypotheses for the origin of the notochord were proposed, the de novo theory and the axochord theory. The former states that notochord has developed de novo from the mid-dorsal archenteron of a chordate ancestor with simple morphology and no central nervous system nor notochord homolog. The putative notochord along the dorsal side of the animal is proposed to take on the signal functions later from the endoderm and ectoderm. An alternative hypothesis, the axochord theory, proposes that notochord has evolved from the mid-line muscle tissue, the so-called axochord, in annelids. Structural and molecular evidence point to the midline muscle of annelids as a distant homolog of the notochord. This hypothesis thus suggests a notochord-like structure in the urbilaterian ancestor, opposed to the consensus that notochord is a chordate-specific feature. In this review, we introduce the history of the formation of these views and summarize the current understandings of embryonic development, molecular profile, and gene regulatory networks of notochord and notochord-like structures.

Egfl6 expression in the pharyngeal pouch is dispensable for craniofacial development

ABSTRACT Epidermal growth factor-like domain multiple 6 (Egfl6) is a basement membrane protein and plays an important role in hair follicle morphogenesis, angiogenesis, notochord development in vertebrates. Although egfl6 expression in the developing head was observed in zebrafish, its role for craniofacial development and the determination of the pharyngeal region expressing egfl6, have not been reported yet. Here, we report the expression patterns and function of egfl6 in craniofacial development in zebrafish. egfl6 was expressed sequentially in the developing pharyngeal pouches that are key epithelial structures governing the development of the vertebrate head. However, loss-of-function mutations in egfl6 did not cause any craniofacial defects, including the pouches as well as the thymus and facial cartilages whose development is contingent upon appropriate pouch formation. egfl6 was unlikely redundant with egfl7 expressed in a distinct pharyngeal region from that of egfl6 in craniofacial development because reduction of egfl7 with a MO in egfl6 mutants did not affect craniofacial development. In addition, we found that egfl6 carried an endogenous start loss mutation in the wild-type Tübingen strain, implying egfl6 would be a non-functional gene. Taken all together, we suggest that egfl6 expression in the pharyngeal pouches is not required for craniofacial development in zebrafish.

Single-cell morphometrics reveals ancestral principles of notochord development.

ABSTRACTEmbryonic tissues are shaped by the dynamic behaviours of their constituent cells. To understand such cell behaviours and how they evolved, new approaches are needed to map out morphogenesis across different organisms. Here, we apply a quantitative approach to learn how the notochord forms during the development of amphioxus: a basally branching chordate. Using a single-cell morphometrics pipeline, we quantify the geometries of thousands of amphioxus notochord cells, and project them into a common mathematical space, termed morphospace. In morphospace, notochord cells disperse into branching trajectories of cell shape change, revealing a dynamic interplay between cell shape change and growth that collectively contributes to tissue elongation. By spatially mapping these trajectories, we identify conspicuous regional variation, both in developmental timing and trajectory topology. Finally, we show experimentally that, unlike ascidians but like vertebrates, posterior cell division is required in amphioxus to generate full notochord length, thereby suggesting this might be an ancestral chordate trait that is secondarily lost in ascidians. Altogether, our novel approach reveals that an unexpectedly complex scheme of notochord morphogenesis might have been present in the first chordates. This article has an associated ‘The people behind the papers’ interview.

Rare Germline Variants in Chordoma-Related Genes and Chordoma Susceptibility.

Simple SummaryChordoma is an extremely rare bone cancer that has not been fully characterized and few risk factors have been identified, highlighting the need for improving our understanding of the disease biology. Our study aims to identify chordoma susceptibility genes by investigating 265 genes involved in chordoma-related signaling pathways and other biological processes on germline DNA of 138 chordoma patients of European ancestry compared to internal control datasets and general population databases. Results were intersected with whole genome sequencing data from 80 skull-base chordoma patients of Chinese ancestry. Several rare loss-of-function and predicted deleterious missense variants were enriched in chordoma cases in both datasets, suggesting a complex model of pathways potentially involved in chordoma development and susceptibility, warranting further investigation in larger studies.Background: Chordoma is a rare bone cancer with an unknown etiology. TBXT is the only chordoma susceptibility gene identified to date; germline single nucleotide variants and copy number variants in TBXT have been associated with chordoma susceptibility in familial and sporadic chordoma. However, the genetic susceptibility of chordoma remains largely unknown. In this study, we investigated rare germline genetic variants in genes involved in TBXT/chordoma-related signaling pathways and other biological processes in chordoma patients from North America and China. Methods: We identified variants that were very rare in general population and internal control datasets and showed evidence for pathogenicity in 265 genes in a whole exome sequencing (WES) dataset of 138 chordoma patients of European ancestry and in a whole genome sequencing (WGS) dataset of 80 Chinese patients with skull base chordoma. Results: Rare and likely pathogenic variants were identified in 32 of 138 European ancestry patients (23%), including genes that are part of notochord development, PI3K/AKT/mTOR, Sonic Hedgehog, SWI/SNF complex and mesoderm development pathways. Rare pathogenic variants in COL2A1, EXT1, PDK1, LRP2, TBXT and TSC2, among others, were also observed in Chinese patients. Conclusion: We identified several rare loss-of-function and predicted deleterious missense variants in germline DNA from patients with chordoma, which may influence chordoma predisposition and reflect a complex susceptibility, warranting further investigation in large studies.

Potential role for the tumor suppressor CYLD in brain and notochord development

BackgroundThe 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.MethodsSemi‐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.ResultsThe 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.ConclusionsCYLD 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.

Pinhead antagonizes Admp to promote notochord formation

SummaryDorsoventral patterning of a vertebrate embryo critically depends on the activity of Smad1 that mediates signaling by BMP proteins, anti-dorsalizing morphogenetic protein (Admp), and their antagonists. Pinhead (Pnhd), a cystine-knot-containing secreted protein, is expressed in the ventrolateral mesoderm during Xenopus gastrulation; however, its molecular targets and signaling mechanisms have not been fully elucidated. Our mass spectrometry-based screen of the gastrula secretome identified Admp as Pnhd-associated protein. We show that Pnhd binds Admp and inhibits its ventralizing activity by reducing Smad1 phosphorylation and its transcriptional targets. Importantly, Pnhd depletion further increased phospho-Smad1 levels in the presence of Admp. Furthermore, Pnhd synergized with Chordin and a truncated BMP4 receptor in the induction of notochord markers in ectoderm cells, and Pnhd-depleted embryos displayed notochord defects. Our findings suggest that Pnhd binds and inactivates Admp to promote notochord development. We propose that the interaction between Admp and Pnhd refines Smad1 activity gradients during vertebrate gastrulation.

Multiple toxicity of propineb in developing zebrafish embryos: Neurotoxicity, vascular toxicity, and notochord defects in normal vertebrate development

A dithiocarbamate (DTC) fungicide, propineb, affects thyroid function and exerts immunotoxicity, cytotoxicity, and neurotoxicity in humans. Long-term exposure to propineb is associated with carcinogenicity, teratogenicity, malfunction of the reproductive system, and abnormalities in vital signs during organ development. However, there is no evidence of acute toxicity attributable to propineb in zebrafish. Therefore, in the present study, we assessed the toxicity of propineb in zebrafish by studying its adverse effects on embryo development, angiogenesis, and notochord development. Embryos with propineb exposure developed morphological and physiological defects and in larvae, apoptosis and notochord defects were induced in the early development stage. Transgenic fli1:eGFP zebrafish exposed to propineb showed abnormal larval development with defects in angiogenesis and deformed vasculature. Propineb induced irreversible damage to the neural development of embryos and neurogenic defects in developing zebrafish in transgenic olig2:dsRED zebrafish. These results show that exposure to propineb triggers abnormalities in different organ systems of zebrafish and suggests the physiological complexity of the response to propineb.