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.

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

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

Zebrafish embryos exposed to deltamethrin exhibit abnormalities despite induced expression of related genes ( you, you-too, momo and u-boot).

Evaluation of the toxic effects of a widely used synthetic pyrethroid, deltamethrin (DM), was carried out in this study. This pesticide is preferred for pest control because of its low environmental persistence and toxicity. We investigated the expression pattern of four genes, namely, you ( you), yot ( you-too), momo ( mom) and ubo ( u-boot) during early development of zebrafish, that is, from 12 hpf to 48 hpf stages. These stages are selected as most of the important developmental aspects take place during this period. All four genes are known to play a vital role in development of notochord and somites. To understand the effect of DM on development, embryos of 4 hpf stage were exposed to two concentrations (100 and 200 µg/L) of DM, and observations were made at 12, 24 and 48 hpf stages. Our earlier studies have shown phenotypic abnormalities such as notochord bending, tail deformation, yolk sac and pericardial edema, lightening of body and eye pigmentation and interfered in somite patterning, during these stages of development. Understanding the relationship of phenotypic abnormalities with these four genes has been our primary objective. These four genes were analyzed by Reverse transcription (RT)-polymerase chain reaction and intensity of the bands has shown induction in their expression after exposure to the toxicant. In spite of the expression of genes, it was noticed that DM caused abnormalities. It can be said from the results that translational pathway could have been affected.

Developmental neurotoxicity of maneb: Notochord defects, mitochondrial dysfunction and hypoactivity in zebrafish (Danio rerio) embryos and larvae.

Broad applications and exposure to the fungicide maneb can lead to toxicity in non-target organisms. Maneb is also associated with neurogenerative diseases such as Parkinson's disease (PD). The objectives of this study were to determine the acute toxicity of maneb to zebrafish by measuring mitochondrial bioenergetics, locomotor activity, and the expression of genes related to the oxidative damage response, as well as those related to dopamine signaling due to its association with PD. Zebrafish embryos at 6 h post-fertilization (hpf) were exposed to either solvent control (0.1% DMSO, v/v), or one dose of 0.1, 0.5, 1.0 and 10.0 µM maneb for 96 h. Maneb was moderately toxic to zebrafish embryos, and had a 96-h LC50 value of 4.29 µM (~ 1.14 mg/L). Maneb induced a dose-dependent increase in mortality, decreased hatching rate, and increased notochord deformity rate at both 1.0 and 10.0 µM after 72 and 96 h. Total body length was also significantly reduced with 1.0 µM maneb. A 50-60% decrease in mean basal oxygen consumption rate was also observed in embryos following a 24 hpf exposure to 10.0 µM maneb but oligomycin-induced ATP production and FCCP-induced maximum respiration remained unaffected. No change was detected in the expression levels of genes associated with oxidative stress (sod1 and sod2), nor those related to dopamine synthesis (th1), dopamine transporter (dat), dopamine receptors (drd1, drd2a, drd3, and drd4b). Thus, modifying the expression of these transcripts may not be a mechanism for maneb-induced developmental toxicity in zebrafish. To assess the potential for neurotoxicity, a dark photokinesis assay was conducted in larvae following 7 d exposure to 0.1, 0.5 and 1.0 µM maneb. Larvae exposed to 0.5 and 1.0 µM maneb showed signs related to hypoactivity, and this reduced activity is hypothesized to be associated with notochord defects as this deformity was prevalent at higher concentrations of maneb. Overall, these data demonstrate that maneb negatively affects embryonic development (i.e. notochord development), affects basal oxygen consumption rates of embryos, and induces hypoactivity in larval fish. This study improves understanding regarding the developmental neurotoxicity of the fungicide maneb to zebrafish.

The vacuolated morphology of chordoma cells is dependent on cytokeratin intermediate filaments.

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

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

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

Toxicity of malathion at early life stages of the Senegalese sole, Solea senegalensis (Kaup, 1858): notochord and somatic disruptions.

The toxicity of malathion to Solea senegalensis was studied in a static renewal bioassay for 24, 48 and 72 h, with toxicant concentrations ranging from 1.56 until 100 µgL⁻¹. The LC50 values of malathion for 48 and 72 h was 63.5 (95% C.I: 50.83-79.34) and 22.94 (95% C.I: 17.16-30.68) µgL⁻¹ respectively. The survival of larvae was non-affected by exposure to malathion at concentrations up to 25 µgL⁻¹ (24 h NOEC), 6.25 µgL⁻¹ (48 h NOEC) and <1.6 µg⁻¹ (72 h NOEC). At the end of the experiment, surviving larvae from concentrations smaller than the 72h-LC50 were chosen to study morphological changes during malathion exposure. Results revealed a strong disruption in the notochord and trunk musculature integrity as a result of toxicant exposure. Noticeable changes in the composition and reduction of collagen fibers from the perinotochordal connective sheath and perimysium were clearly detected. The trunk musculature was also altered, showing a general disorganization of fibers. Moreover, malathion exposure provoked pericardial and yolk-sac oedemas and histopathological alterations in some other organ- systems and tissues (i.e. liver, pancreas, intestine).

Butyl benzyl phthalate (BBP) induces caudal defects during embryonic development.

Butyl benzyl phthalate (BBP) is commonly added during the manufacturing of plastics to increase flexibility and elasticity. However, BBP leaches off of plastic and environment presence has been detected in soil, groundwater and sediment potentially effecting organisms in the environment. Given the widespread uses of BBP in household, consumer goods and the presence of BBP in the environment, studies on developmental toxicity are needed. Here, we use a zebrafish model to investigate the early developmental toxicity of BBP. We treated gastrula staged embryos with increasing concentrations of BBP and noted concentration-dependent defects in caudal tail development, but the effect was caudal specific with no other developmental defects noted. In situ hybridization studies using muscle and notochord markers show alterations in muscle development and non-linear, kinked notochord staining. A more detailed antibody staining using a myosin specific marker shows disorganized myofibrils and a loss of chevron shaped somites. Furthermore, vascular development in the tail was also disrupted in a concentration dependent manner. We conclude that BBP is toxic to caudal development in zebrafish. The sensitivity of zebrafish during development to environmental toxins and chemicals has been useful in assessing the health of the aquatic environment. The results presented here are a useful early warning system for contamination that could affect human health.

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

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

Zebrafish biosensor for toxicant induced muscle hyperactivity.

Robust and sensitive detection systems are a crucial asset for risk management of chemicals, which are produced in increasing number and diversity. To establish an in vivo biosensor system with quantitative readout for potential toxicant effects on motor function, we generated a transgenic zebrafish line TgBAC(hspb11:GFP) which expresses a GFP reporter under the control of regulatory elements of the small heat shock protein hspb11. Spatiotemporal hspb11 transgene expression in the musculature and the notochord matched closely that of endogenous hspb11 expression. Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression. Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency. The hspb11 transgene up-regulation induced by either chemicals or heat shock was eliminated after co-application of the anaesthetic MS-222. TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.

Reciprocal and dynamic polarization of planar cell polarity core components and myosin.

The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization.

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

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

Evaluation of the developmental toxicity of lead in the Danio rerio body.

Lead has been utilized throughout history and is widely distributed and mobilized globally. Although lead in the environment has been somewhat mitigated, the nature of lead and its extensive uses in the past prohibit it from being completely absent from our environment and exposure to lead is still a public health concern. Most studies regarding lead toxicity have focused on the brain. However, little is found in the literature on the effects of lead in other tissues. Here, we utilize the zebrafish model system to investigate effects of lead exposure during early developmental time windows at 24, 48 and 72 h post fertilization in the body. We analyze whole body, notochord and somatic muscle changes, vascular changes of the body, as well as motor neuron alterations. We find lead exposure induces a curved body phenotype with concomitant changes in somite length, decreased notochord staining and abnormal muscle staining using live and in situ approaches. Furthermore, altered vasculature within the somatic regions, loss and/or alternations of motor neuron extension both dorsally and ventrally from the spinal cord, loss of Rohon-Beard sensory neurons, and increased areas of apoptosis were found. We conclude that lead is developmentally toxic to other areas of the developing embryo, not just the brain.

Activation of intervertebral disc cells by co-culture with notochordal cells, conditioned medium and hypoxia.

BACKGROUND: Notochordal cells (NC) remain in the focus of research for regenerative therapy for the degenerated intervertebral disc (IVD) due to their progenitor status. Recent findings suggested their regenerative action on more mature disc cells, presumably by the secretion of specific factors, which has been described as notochordal cell conditioned medium (NCCM). The aim of this study was to determine NC culture conditions (2D/3D, fetal calf serum, oxygen level) that lead to significant IVD cell activation in an indirect co-culture system under normoxia and hypoxia (2% oxygen). METHODS: Porcine NC was kept in 2D monolayer and in 3D alginate bead culture to identify a suitable culture system for these cells. To test stimulating effects of NC, co-cultures of NC and bovine derived coccygeal IVD cells were conducted in a 1:1 ratio with no direct cell contact between NC and bovine nucleus pulposus cell (NPC) or annulus fibrosus cells (AFC) in 3D alginate beads under normoxia and hypoxia (2%) for 7 and 14 days. As a positive control, NPC and AFC were stimulated with NC-derived conditioned medium (NCCM). Cell activity, glycosaminoglycan (GAG) content, DNA content and relative gene expression was measured. Mass spectrometry analysis of the NCCM was conducted. RESULTS: We provide evidence by flow cytometry that monolayer culture is not favorable for NC culture with respect to maintaining NC phenotype. In 3D alginate culture, NC activated NPC either in indirect co-culture or by addition of NCCM as indicated by the gene expression ratio of aggrecan/collagen type 2. This effect was strongest with 10% fetal calf serum and under hypoxia. Conversely, AFC seemed unresponsive to co-culture with pNC or to the NCCM. Further, the results showed that hypoxia led to decelerated metabolic activity, but did not lead to a significant change in the GAG/DNA ratio. Mass spectrometry identified connective tissue growth factor (CTGF, syn. CCN2) in the NCCM. CONCLUSIONS: Our results confirm the requirement to culture NC in 3D to best maintain their phenotype, preferentially in hypoxia and with the supplementation of FCS in the culture media. Despite these advancements, the ideal culture condition remains to be identified.

FoxA4 favours notochord formation by inhibiting contiguous mesodermal fates and restricts anterior neural development in Xenopus embryos.

In vertebrates, the embryonic dorsal midline is a crucial signalling centre that patterns the surrounding tissues during development. Members of the FoxA subfamily of transcription factors are expressed in the structures that compose this centre. Foxa2 is essential for dorsal midline development in mammals, since knock-out mouse embryos lack a definitive node, notochord and floor plate. The related gene foxA4 is only present in amphibians. Expression begins in the blastula -chordin and -noggin expressing centre (BCNE) and is later restricted to the dorsal midline derivatives of the Spemann's organiser. It was suggested that the early functions of mammalian foxa2 are carried out by foxA4 in frogs, but functional experiments were needed to test this hypothesis. Here, we show that some important dorsal midline functions of mammalian foxa2 are exerted by foxA4 in Xenopus. We provide new evidence that the latter prevents the respecification of dorsal midline precursors towards contiguous fates, inhibiting prechordal and paraxial mesoderm development in favour of the notochord. In addition, we show that foxA4 is required for the correct regionalisation and maintenance of the central nervous system. FoxA4 participates in constraining the prospective rostral forebrain territory during neural specification and is necessary for the correct segregation of the most anterior ectodermal derivatives, such as the cement gland and the pituitary anlagen. Moreover, the early expression of foxA4 in the BCNE (which contains precursors of the whole forebrain and most of the midbrain and hindbrain) is directly required to restrict anterior neural development.

Zebrafish enpp1 mutants exhibit pathological mineralization, mimicking features of generalized arterial calcification of infancy (GACI) and pseudoxanthoma elasticum (PXE).

In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf), which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1), a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI) is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE) have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues - most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin). Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord mineralization phenotype. This indicates that enpp1 can exert its function in tissues that are remote from its site of expression.

The effects of the toxic cyanobacterium Limnothrix (strain AC0243) on Bufo marinus larvae.

Limnothrix (strain AC0243) is a cyanobacterium, which has only recently been identified as toxin producing. Under laboratory conditions, Bufo marinus larvae were exposed to 100,000 cells mL(-1) of Limnothrix (strain AC0243) live cultures for seven days. Histological examinations were conducted post mortem and revealed damage to the notochord, eyes, brain, liver, kidney, pancreas, gastrointestinal tract, and heart. The histopathological results highlight the toxicological impact of this strain, particularly during developmental stages. Toxicological similarities to ß-N-Methylamino-L-alanine are discussed.

A zebrafish model of chordoma initiated by notochord-driven expression of HRASV12.

Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer.

Multidimensional in vivo hazard assessment using zebrafish.

There are tens of thousands of man-made chemicals in the environment; the inherent safety of most of these chemicals is not known. Relevant biological platforms and new computational tools are needed to prioritize testing of chemicals with limited human health hazard information. We describe an experimental design for high-throughput characterization of multidimensional in vivo effects with the power to evaluate trends relating to commonly cited chemical predictors. We evaluated all 1060 unique U.S. EPA ToxCast phase 1 and 2 compounds using the embryonic zebrafish and found that 487 induced significant adverse biological responses. The utilization of 18 simultaneously measured endpoints means that the entire system serves as a robust biological sensor for chemical hazard. The experimental design enabled us to describe global patterns of variation across tested compounds, evaluate the concordance of the available in vitro and in vivo phase 1 data with this study, highlight specific mechanisms/value-added/novel biology related to notochord development, and demonstrate that the developmental zebrafish detects adverse responses that would be missed by less comprehensive testing strategies.

Emilin3 is required for notochord sheath integrity and interacts with Scube2 to regulate notochord-derived Hedgehog signals.

The notochord is a transient and essential structure that provides both mechanical and signaling cues to the developing vertebrate embryo. In teleosts, the notochord is composed of a core of large vacuolated cells and an outer layer of cells that secrete the notochord sheath. In this work, we have identified the extracellular matrix glycoprotein Emilin3 as a novel essential component of the zebrafish notochord sheath. The development of the notochord sheath is impaired in Emilin3 knockdown embryos. The patterning activity of the notochord is also affected by Emilin3, as revealed by the increase of Hedgehog (Hh) signaling in Emilin3-depleted embryos and the decreased Hh signaling in embryos overexpressing Emilin3 in the notochord. In vitro and in vivo experiments indicate that Emilin3 modulates the availability of Hh ligands by interacting with the permissive factor Scube2 in the notochord sheath. Overall, this study reveals a new role for an EMILIN protein and reinforces the concept that structure and function of the notochord are strictly linked.