Somite Development Research Articles

Unravelling the Seasonal Dynamics of Limnological Parameters and Assessment of Ecological Health: A Case Study of Lingadheeranahalli Lake in Bangalore North, Karnataka, India

ABSTRACT: The present study investigates the seasonal dynamics of limnological variables and the developmental response of D. rerio in the wetland ecosystem of Lingadheeranahalli in the northern clutches of Bangalore in Karnataka. Once a vital water source for agriculture and replenishing groundwater, the lake faces degradation due to anthropogenic activities and the need for more awareness. A yearlong analysis was conducted with five sampling seasons and three sampling stations identified within the wetland. Parameters viz. physical, chemical, and biological were analyzed following American Public Health Association (APHA) guidelines. Somite development was studied using embryos while the heart rate was counted during the torpedo stage of D. rerio. The results of this comprehensive study revealed unique and significant seasonal variations over limnological parameters, providing novel insights into the dynamics of the wetland ecosystem. pH values are slightly acidic and influenced by precipitation and terrestrial vegetation. Turbidity spiked during winter due to colloidal dispersions, while conductivity peaked in spring due to sewage disposal. Reduced dissolved oxygen, high biochemical and chemical oxygen demand indicated organic pollution and microbial activity, particularly affecting the inlet station. Elevated phosphate and nitrate levels in spring indicated eutrophication potential. The influence of rains and mineral leaching during monsoons affected parameters such as alkalinity, total dissolved solids, total hardness, and Magnesium. Analysis of D. rerio demonstrated delayed development and decreased heart rates in the inlet and deposit stations, indicative of potent stressors in the wetland ecosystem. All-inclusive Lingadheeranahalli Lake exhibited poor water quality, focusing on the need for adaptive management strategies to mitigate pollution and safeguard wetland ecosystems. Continued wetland research gives more insights into water quality and seasonal dynamics, paving the way for its conservation.

Investigation of the potential teratogenic effects of fructose on the embryo using the rat whole embryo culture model

Excessive consumption of fructose-sweetened foods and beverages is a growing concern worldwide. Studies have demonstrated that fructose consumption before and during pregnancy can result in adverse outcomes such as decreased decidualization, increased fetal losses, and low birth weight. The study investigated the teratogenic effects of fructose on rat embryos during organogenesis using whole embryo culture. Within the scope of the study, 4 groups were formed as control, low, medium, and high-dose fructose (HDF) with 10 embryos in each group. The 9.5-day-old rat embryos were cultured with different concentrations of fructose (1, 5 and 10 mM) for 48 h and the possible effects of fructose were examined using morphological scoring, histochemistry, immunofluorescence, and TUNEL methods. According to the analyses, protein synthesis and proliferation were decreased, vascular formation was suppressed, and apoptosis was increased in embryos exposed to fructose, especially at concentrations of 5 mM and above. According to the morphological scoring results, it was determined that heart, hind limb, and somite development were retarded in all experimental groups compared to the control group, developmental retardation increased in direct proportion to fructose concentration, and also significant malformations were observed in all parameters examined in the HDF group. In addition, analysis of yolk sac diameter, head length, crown rump length and somite numbers showed that these parameters were significantly decreased in all experimental groups. End of the study, it was concluded that fructose at concentrations of 1 mM and above may induce embryonic development retardation and other anomalies by decreasing protein synthesis and cell proliferation, suppressing vascular formation, and increasing apoptosis in embryonic tissues.

Generation of musculoskeletal cells from human urine epithelium-derived presomitic mesoderm cells

BackgroundNumerous studies have shown that somite development is a necessary stage of myogenesis chondrogenesis and osteogenesis. Our previous study has established a stable presomitic mesoderm progenitor cell line (UiPSM) in vitro. Naturally, we wanted to explore whether UiPSM cell can develop bone and myogenic differentiation.ResultsSelective culture conditions yielded PAX3 and PAX7 positive skeletal muscle precursors from UiPSM cells. The skeletal muscle precursors undergo in vitro maturation resulting in myotube formation. MYOD effectively promoted the maturity of the skeletal myocytes in a short time. We found that UiPSM and MYOD mediated UiPSM cell-derived skeletal myocytes were viable after transplantation into the tibialis anterior muscle of MITRG mice, as assessed by bioluminescence imaging and scRNA-seq. Lack of teratoma formation and evidence of long-term myocytes engraftment suggests considerable potential for future therapeutic applications. Moreover, UiPSM cells can differentiate into osteoblast and chondroblast cells in vitro.ConclusionsUiPSM differentiation has potential as a developmental model for musculoskeletal development research and treatment of musculoskeletal disorders.

Core planar cell polarity genes VANGL1 and VANGL2 in predisposition to congenital vertebral malformations

Congenital scoliosis (CS), affecting approximately 0.5 to 1 in 1,000 live births, is commonly caused by congenital vertebral malformations (CVMs) arising from aberrant somitogenesis or somite differentiation. While Wnt/ß-catenin signaling has been implicated in somite development, the function of Wnt/planar cell polarity (Wnt/PCP) signaling in this process remains unclear. Here, we investigated the role of Vangl1 and Vangl2 in vertebral development and found that their deletion causes vertebral anomalies resembling human CVMs. Analysis of exome sequencing data from multiethnic CS patients revealed a number of rare and deleterious variants in VANGL1 and VANGL2, many of which exhibited loss-of-function and dominant-negative effects. Zebrafish models confirmed the pathogenicity of these variants. Furthermore, we found that Vangl1 knock-in (p.R258H) mice exhibited vertebral malformations in a Vangl gene dose- and environment-dependent manner. Our findings highlight critical roles for PCP signaling in vertebral development and predisposition to CVMs in CS patients, providing insights into the molecular mechanisms underlying this disorder.

Tracing the evolutionary origin of chordate somites in the hemichordate Ptychodera flava.

Metameric somites are a novel character of chordates with unclear evolutionary origins. In the early branching chordate amphioxus, anterior somites are derived from the paraxial mesodermal cells that bud off the archenteron (i.e., enterocoely) at the end of gastrulation. Development of the anterior somites requires FGF signaling, and distinct somite compartments express orthologs of vertebrate non-axial mesodermal markers. Thus, it has been proposed that the amphioxus anterior somites are homologous to the vertebrate head mesoderm, paraxial mesoderm and lateral plate mesoderm. To trace the evolutionary origin of somites, it is essential to study the chordates' closest sister group, Ambulacraria, which includes hemichordates and echinoderms. The anterior coeloms of hemichordate and sea urchin embryos (respectively called protocoel and coelomic pouches) are also formed by enterocoely and require FGF signals for specification and/or differentiation. In this study, we applied RNA-seq to comprehensively screen for regulatory genes associated with the mesoderm-derived protocoel of the hemichordate Ptychodera flava. We also used a candidate gene approach to identify P. flava orthologs of chordate somite markers. In situ hybridization results showed that many of these candidate genes are expressed in distinct or overlapping regions of the protocoel, which indicates that molecular compartments exist in the hemichordate anterior coelom. Given that the hemichordate protocoel and amphioxus anterior somites share a similar ontogenic process (enterocoely), induction signal (FGF), and characteristic expression of orthologous genes, we propose that these two anterior coeloms are indeed homologous. In the lineage leading to the emergence of chordates, somites likely evolved from enterocoelic, FGF-dependent, and molecularly compartmentalized anterior coeloms of the deuterostome last common ancestor.

Exploring Alternatives for Marine Toxicity Testing: Initial Evaluation of Fish Embryo and Mysid Tests.

Current regulations require that toxicity assessments be performed using standardized toxicity testing methods, often using fish. Recent legislation in both the European Union and United States has mandated that toxicity testing alternatives implement the 3Rs of animal research (replacement, reduction, and refinement) whenever possible. There have been advances in the development of alternatives for freshwater assessments, but there is a lack of analogous developments for marine assessments. One potential alternative testing method is the fish embryo toxicity (FET) test, which uses fish embryos rather than older fish. In the present study, FET methods were applied to two marine model organisms, the sheepshead minnow and the inland silverside. Another potential alternative is the mysid shrimp survival and growth test, which uses an invertebrate model. The primary objective of the present study was to compare the sensitivity of these three potential alternative testing methods to two standardized fish-based tests using 3,4-dichloroaniline (DCA), a common reference toxicant. A secondary objective was to characterize the ontogeny of sheepshead minnows and inland silversides. This provided a temporal and visual guide that can be used to identify appropriately staged embryos for inclusion in FET tests and delineate key developmental events (e.g., somite development, eyespot formation, etc.). Comparison of the testing strategies for assessing DCA indicated that: (1) the standardized fish tests possessed comparable sensitivity to each other; (2) the mysid shrimp tests possessed comparable sensitivity to the standardized fish tests; (3) the sheepshead minnow and inland silverside FET tests were the least sensitive testing strategies employed; and (4) inclusion of sublethal endpoints (i.e., hatchability and pericardial edema) in the marine FETs increased their sensitivity. Environ Toxicol Chem 2024;43:1285-1299. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Embryonic exposure to decitabine induces multiple neural tube defects in developing zebrafish.

Neural tube defects are severe congenital disorders of the central nervous system that originate during embryonic development when the neural tube fails to close completely. It affects one to two infants per 1000 births. The aetiology is multifactorial with contributions from both genetic and environmental factors. Dysregulated epigenetic mechanisms, in particular the abnormal genome-wide methylation during embryogenesis, have been linked to developmental abnormalities including neural tube defects. The current study investigated the influence of decitabine (DCT), a DNA methylation inhibitor, on embryonic development in zebrafish, with a focus on neural tube formation. The developing zebrafish embryos were exposed to graded concentrations of decitabine (from 13.69 μM to 1 mM) before the onset of neurulation. The developmental process was monitored at regular time intervals post fertilization. At 120 h post fertilization, the developing embryos were inspected individually to determine the incidence and severity of neural tube defects. Using alizarin red staining, the cranial and caudal neural tube morphology was examined in formaldehyde fixed larvae. Anomalies in neural tube and somite development, as well as a delay in hatching, were discovered at an early stage of development. As development continued, neural tube defects became increasingly evident, and there was a concentration-dependent rise in the prevalence and severity of various neural tube defects. 90% of growing embryos in the group exposed to decitabine 1 mM had multiple neural tube malformations, and 10% had isolated neural tube defects. With several abnormalities, the caudal region of the neural tube was seriously compromised. The histopathological studies supported the malformations in neural tube. Our study revealed the harmful impact of decitabine on the development of the neural tube in growing zebrafish. Moreover, these findings support the hypothesis that the hypomethylation during embryonic development causes neural tube defects.

The mucolipidosis III-causing mutation in GNPTAB, c.1760G>C, disrupts the development of somites in rats

The mucolipidosis III-causing mutation in GNPTAB, c.1760G>C, disrupts the development of somites in rats

Developmental toxicity in early chicken embryos on exposure to an organophosphorus flame retardant, tris(2-chloroisopropyl) phosphate

Tris(2-chloroisopropyl) phosphate (TCIPP) is an organophosphate flame retardant detected in the environment and eggs, feathers, and livers. Early-developmental-stage avian embryos are vulnerable to the toxic effects of chemicals. However, studies on the specific effects of TCIPP on avian embryonic development are limited. We aimed to investigate the toxicity of TCIPP in early chicken embryos using a previously developed shell-less incubation system. Fertilized chicken (Gallus gallus domesticus) eggs (n = 220) were exposed to 50 or 500 nmol TCIPP/(g egg) or dimethyl sulfoxide (DMSO) as a vehicle control on Day 0 of incubation. Development of 198 embryos was monitored from Days 3–9 of incubation, and 22 embryos on Day 4 and 74 embryos on Day 9 were dissected. Messenger RNA expression levels for several genes were measured in embryos on Day 4. Both TCIPP-exposed groups showed a significant reduction in survival rate. Imaging analyses revealed significant decreases in body length, head and bill length, eye diameter, and forelimb and hindlimb length in both TCIPP-treated groups. TCIPP exposure significantly impaired the development of extraembryonic blood vessels and the production of red blood cells. A TCIPP-dose-dependent decreasing trend in heart rate was observed on Days 4–7. The somitic angle increased significantly on Days 4–6, and embryos with curved somites showed cleavage in the back and gaps between somites, resulting in asymmetrical somite formation. A significant correlation was found between the somitic angle and FGF8 expression levels, suggesting that TCIPP exposure affects somite formation through an altered FGF-signaling pathway. Embryos with somitic deformities in TCIPP-exposed groups had significantly reduced survival rates, indicating that abnormal segment formation directly increased mortality. Finally, eye weight and ocular luminosity values were significantly reduced, suggesting that TCIPP may also affect eye development. Overall, these findings highlight severe toxic effects of TCIPP on avian embryonic development, including in vascularization, cardiac function, and somite and ocular development.

Downregulation of Zebrafish Cytosolic Sialidase Neu3.2 Affects Skeletal Muscle Development.

Sialidases remove terminal sialic acids residues from the non-reducing ends of glycoconjugates. They have been recognized as catabolic enzymes that work within different subcellular compartments and can ensure the proper turn-over of glycoconjugates. Four mammalian sialidases (NEU1-4) exist, with different subcellular localization, pH optimum and substrate specificity. In zebrafish, seven different sialidases, with high homology to mammalian counterparts, have been identified. Zebrafish Neu3.2 is similar to the human cytosolic sialidase NEU2, which is involved in skeletal muscle differentiation and exhibits a broad substrate specificity toward gangliosides and glycoproteins. In zebrafish neu3.2, mRNA is expressed during somite development, and its enzymatic activity has been detected in the skeletal muscle and heart of adult animals. In this paper, 1-4-cell-stage embryos injected with neu3.2 splice-blocking morpholino showed severe embryonic defects, mainly in somites, heart and anterior-posterior axis formation. Myog and myod1 expressions were altered in morphants, and impaired musculature formation was associated with a defective locomotor behavior. Finally, the co-injection of Neu2 mouse mRNA in morphants rescued the phenotype. These data are consistent with the involvement of cytosolic sialidase in pathologies related to muscle formation and support the validity of the model to investigate the pathogenesis of the diseases.

A transcriptional and regulatory map of mouse somite maturation

The mammalian body plan is shaped by rhythmic segmentation of mesoderm into somites, which are transient embryonic structures that form down each side of the neural tube. We have analyzed the genome-wide transcriptional and chromatin dynamics occurring within nascent somites, from early inception of somitogenesis to the latest stages of body plan establishment. We created matched gene expression andopen chromatin maps for the three leading pairs of somites at six time points during mouse embryonic development. We show that the rate of somite differentiation accelerates as development progresses. We identified a conserved maturation program followed by all somites, but somites from more developed embryos concomitantly switch on differentiation programs from derivative cell lineages soon after segmentation. Integrated analysis of the somitic transcriptional and chromatin activities identified opposing regulatory modules controlling the onset of differentiation. Our results provide a powerful, high-resolution view of the molecular genetics underlying somitic development in mammals.

Betaglycan promoter activity is differentially regulated during myogenesis in zebrafish embryo somites.

Betaglycan, also known as the TGFβ type III receptor (Tgfbr3), is a co-receptor that modulates TGFβ family signaling. Tgfbr3 is upregulated during C2C12 myoblast differentiation and expressed in mouse embryos myocytes. To investigate tgfbr3 transcriptional regulation during zebrafish embryonic myogenesis, we cloned a 3.2 kb promoter fragment that drives reporter transcription during C2C12 myoblasts differentiation and in the Tg(tgfbr3:mCherry) transgenic zebrafish. We detect tgfbr3 protein and mCherry expression in the adaxial cells concomitantly with the onset of their radial migration to become slow-twitch muscle fibers in the Tg(tgfbr3:mCherry). Remarkably, this expression displays a measurable antero-posterior somitic gradient expression. tgfbr3 is transcriptionally regulated during somitic muscle development in zebrafish with an antero-posterior gradient expression that preferentially marks the adaxial cells and their descendants.

MAP4K4 and cancer: ready for the main stage?

MAP4K4 is a serine/threonine kinase that belongs to the MAP kinase family and plays a critical role in embryogenesis and cellular migration. It contains approximately 1,200 amino acids and has a molecular mass of 140 kDa. MAP4K4 is expressed in most tissues where it has been examined and its knockout is embryonic lethal due to impaired somite development. Alterations in MAP4K4 function have a central role in the development of many metabolic diseases such as atherosclerosis and type 2 diabetes, but have recently been implicated in the initiation and progression of cancer. For example, it has been shown that MAP4K4 can stimulate the proliferation and invasion of tumor cells by activating pro-proliferative pathways (such as the c-Jun N-terminal kinase [JNK] and mixed-lineage protein kinase 3 [MLK3] pathways), attenuate anti-tumor cytotoxic immune responses, and stimulate cell invasion and migration by altering cytoskeleton and actin function. Recent in vitro experiments using RNA interference-based knockdown (miR) techniques have shown that inhibition of MAP4K4 function reduces tumor proliferation, migration, and invasion, and may represent a promising therapeutic approach in many types of cancer such as pancreatic cancer, glioblastoma, and medulloblastoma, among others. Over the last few years, specific MAP4K4 inhibitors such as GNE-495 have been developed but have not yet been tested in cancer patients. However, these novel agents may be useful for cancer treatment in the future.

Toxicological evaluation of industrial effluents using zebrafish: Efficacy of tertiary treatment of coking wastewater

The coking wastewater had a high chemical oxygen demand (COD) concentration (2 771 ± 397 mg/L) and was highly toxic to the zebrafish embryos. Although biological treatment removed over 80% of COD and almost all known toxic compound from the coking wastewater, the wastewater still caused nearly 90% mortality of zebrafish embryos, with severe cardiotoxicity and skeletal development toxicity, suggesting the presence of unknown toxic compounds in the biological effluent. Synchronized oxidation–adsorption (SOA) technology, i.e., Fenton-based oxidation coupled with hydroxyl radicals and adsorption through in-situ-formed hydrolyzed Fe3＋ particles, was employed as a tertiary treatment, resulting in further COD removal from 315 ± 36 to 129 ± 9 mg/L. More importantly, SOA tertiary treatment greatly reduced the toxicity of the biological effluent, effectively improving the survival rate of zebrafish from 10% to 95% at 80% of the wastewater concentration. Toxicological analysis showed that SOA treatment of wastewater alleviated its cardiovascular and somatic skeletal toxicity in zebrafish. Notably, nkx2.5, which affects circulatory system development, and her12, which regulates the development of skeletal somites, remained at low levels, which explain the malformations observed in the final effluent-exposed zebrafish. In addition, wastewater exposure induced apoptosis in zebrafish. This study is the first to report on the effects of coking wastewater on the cardiovascular system, somite bone development, and apoptosis in zebrafish embryos at the genetic level.

2,4-di-tert-butylphenol exposure impairs osteogenic differentiation

2,4-di-tert-butylphenol (2,4-DTBP) is a synthetic antioxidant used in polyethylene crosspolymer (PEX) water distribution pipes and food-related plastics. 2,4-DTBP can leach from plastic materials and has been found in breast milk, cord blood, and placental tissue, giving rise to the concern that this compound may interfere with fetal development. The objective of this study is to assess the impacts of 2,4-DTBP on cellular differentiation. Human induced pluripotent stem (HiPS) cells were differentiated into osteoblasts or myoblasts over 40 days, and analyzed for markers of somite, dermomyotome, sclerotome, myoblast, and osteoblast development. When cultured as stem cells, 2,4-DTBP did not alter cell viability and expression of markers (NANOG, OCT4). However, upon differentiation into somite-like cells, 2,4-DTBP had reduced levels of MEOX1 and TBX6 transcripts, while NANOG and OCT4 were in turn upregulated in a dose-dependent manner. At the sclerotome-like stage, PAX9 mRNA decreased by 2-fold in the 0.5 μM and 1.0 μM 2,4-DTBP exposure groups. After 40 days of differentiation into an osteoblast-like lineage, exposure to 2,4-DTBP significantly reduced expression of the osteogenesis transcripts RUNX2 and OSX in a dose-dependent manner. Further, Alizarin Red staining of calcium deposits was decreased in the 0.5 μM and 1.0 μM treatment groups. In contrast, myogenesis was not affected by 2,4-DTBP exposure. Interestingly, KEAP1 expression was significantly increased in the sclerotomal-like cells, but decreased in the dermomytomal-like cells, which may suggest a mechanism of action. Overall, this study shows that 2,4-DTBP can delay key processes during sclerotome and osteoblast development, leading to a potential for bone developmental issues in exposed individuals.

Short-term effects of various non-steroidal anti-inflammatory drugs (NSAIDs) on Danio rerio embryos

Due to the widespread use of non-steroidal anti-inflammatory drugs (NSAIDs) without a medical prescription and their frequent prevalence in aquatic habitats, there are major health and environmental issues. NSAIDs have been found in surface water and wastewater in concentrations ranging from ng/L to μg/L all over the world. The purpose of this study was to determine the relationship between NSAIDs (diclofenac, ketoprofen, paracetamol and ibuprofen) exposure and associated adverse effects in the assessment of indirect human health risks posed by Danio rerio (zebrafish) and Environmental Risk Assessment (ERA) of these NSAIDs in aquatic environments. Therefore, the objectives of this study were to (i) reveal abnormality endpoints of early developmental stages, after exposure of zebrafish and (ii) perform an ecological risk assessment of aquatic organisms upon exposure to NSAIDs detected in surface waters based on the risk quotients (RQs) method. According to the toxicity data collected, all of the malformations appeared after diclofenac exposure at all concentrations. The most notable malformations were the lack of pigmentation and an increase in yolk sac volume, with EC50 values of 0.6 and 1.03 mg/L, respectively. The results obtained for the ERA revealed RQs higher than 1 for all the four NSAIDs chosen, posing ecotoxicological pressure in aquatic environments. Overall, our findings provide a critical contribution to the formulation of high-priority actions, sustainable strategies and strict regulations that minimize the negative effects of NSAIDs on the aquatic ecosystem.•To determine the LC50, lethal conditions such as coagulation, absence of heartbeat and blood flow, absence of tail separation and development of somites were taken into account.•The EC50 was calculated using sublethal parameters such as blood coagulation, pericardial edema, yolk sac edema or hypertrophy.•The 4 compounds present a high risk individually and in mixture with a RQ >> 1.

Kinesin-5 Eg5 is essential for spindle assembly, chromosome stability and organogenesis in development

Chromosome stability relies on bipolar spindle assembly and faithful chromosome segregation during cell division. Kinesin-5 Eg5 is a plus-end-directed kinesin motor protein, which is essential for spindle pole separation and chromosome alignment in mitosis. Heterozygous Eg5 mutations cause autosomal-dominant microcephaly, primary lymphedema, and chorioretinal dysplasia syndrome in humans. However, the developmental roles and cellular mechanisms of Eg5 in organogenesis remain largely unknown. In this study, we have shown that Eg5 inhibition leads to the formation of the monopolar spindle, chromosome misalignment, polyploidy, and subsequent apoptosis. Strikingly, long-term inhibition of Eg5 stimulates the immune responses and the accumulation of lymphocytes in the mouse spleen through the innate and specific immunity pathways. Eg5 inhibition results in metaphase arrest and cell growth inhibition, and suppresses the formation of somite and retinal development in zebrafish embryos. Our data have revealed the essential roles of kinesin-5 Eg5 involved in cell proliferation, chromosome stability, and organogenesis during development. Our findings shed a light on the cellular basis and pathogenesis in microcephaly, primary lymphedema, and chorioretinal dysplasia syndrome of Eg5-mutation-positive patients.

Ethylene thiourea exposure induces neurobehavioral toxicity in zebrafish by disrupting axon growth and neuromuscular junctions

Ethylene thiourea (ETU) converted from ethylene bisdithiocarbamate (EBDC) fungicides has aroused great concern because of its prevalence and harmful effects. Although ETU-induced neurotoxicity has been reported, the potential mechanisms remain unclear. This study provided insights into its neurotoxic effects at environmentally relevant concentrations in zebrafish. Our findings showed that embryonic exposure to ETU decreased the hatch rate and delayed somite development. Furthermore, ETU treatment significantly reduced the dark velocity in the locomotion assay. The upregulated tendency of the mitogen-activated protein kinases (MAPK) pathway (mknk1, atf4, mapkapk3) screened by transcriptome analysis implied motor neuron degeneration, which was validated by subsequent morphological observation, as axon length and branches were truncated in the 62.5 µg/L ETU group. However, although the rescue experiment with a p38 MAPK inhibitor (SB203580) successfully ameliorated axon degeneration, it failed to reverse the locomotion behaviors. Further exploration of transcriptome data revealed the varied expression of presynaptic scaffold protein-related genes (pcloa, pclob, bsna), whose downregulation might impair the neuromuscular junction (NMJ). Therefore, we reasonably suspected that ETU-induced neurobehavioral deficits might result from the combined effects of the MAPK pathway and presynaptic proteins. Considering this, we highlighted the necessity to take precautions and early interventions for susceptible ETU-exposed populations.

Fish Embryo Acute Toxicity Testing and the RTgill-W1 Cell Line as In Vitro Models for Whole-Effluent Toxicity (WET) Testing: An In Vitro/In Vivo Comparison of Chemicals Relevant for WET Testing.

The fathead minnow (Pimephales promelas) fish embryo acute toxicity (FET) test was compared to the fish gill cells (RTgill-W1) in vitro assay and to the fish larvae acute toxicity test to evaluate their sensitivity for whole-effluent toxicity (WET) testing. The toxicity of 12 chemicals relevant for WET testing was compared as proof of principle. The concentrations lethal to 50% of a population (LC50) of embryos were compared to those in fish larvae and to the 50% effect concentration (EC50) in RTgill-W1 cells from previous literature. Along with traditional FET endpoints (coagulation, somite development, tail detachment, and heartbeat), cardiotoxicity was evaluated for WET applicability. Heart rate was measured at LC20 and LC50 values of six subselected chemicals (Cd, Cu, Ni, ammonia, 3,4-dichloraniline, and benzalkonium chloride). In addition, the toxicity of Cd and Ni was evaluated in RTgill-W1 cells exposed in a hypoosmotic medium to evaluate the effect that osmolarity may have on metal toxicity. A significant correlation was found between the FET and larvae LC50 values but not between the RTgill-W1 EC50 and FET LC50 values. Although sensitivity to Ni and Cd was found to increase with hypoosmotic conditions for FET and RTgill-W1 cells, a correlation was only found with removal of Ni from the analysis. Hypoosmotic conditions increased sensitivity with a significant correlation between RTgill-W1 cells and larvae. Cardiotoxicity was shown in three of the five subselected chemicals (Cd, Cu, and 3,4-dichloroaniline). Overall, both in vitro alternative models have shown good predictability of toxicity in fish in vivo for WET chemicals of interest. Environ Toxicol Chem 2022;41:2721-2731. © 2022 SETAC.

Cysteamine affects skeletal development and impairs motor behavior in zebrafish.

Cysteamine is a kind of feed additive commonly used in agricultural production. It is also the only targeted agent for the treatment of cystinosis, and there are some side effects in clinical applications. However, the potential skeletal toxicity remains to be further elucidated. In this study, a zebrafish model was for the first time utilized to synthetically appraise the skeletal developmental defects induced by cysteamine. The embryos were treated with 0.35, 0.70, and 1.05 mM cysteamine from 6 h post fertilization (hpf) to 72 hpf. Substantial skeletal alterations were manifested as shortened body length, chondropenia, and abnormal somite development. The results of spontaneous tail coiling at 24 hpf and locomotion at 120 hpf revealed that cysteamine decreased behavioral abilities. Moreover, the level of oxidative stress in the skeleton ascended after cysteamine exposure. Transcriptional examination showed that cysteamine upregulated the expression of osteoclast-related genes but did not affect osteoblast-related genes expression. Additionally, cysteamine exposure caused the downregulation of the Notch signaling and activating of Notch signaling partially attenuated skeletal defects. Collectively, our study suggests that cysteamine leads to skeletal developmental defects and reduces locomotion activity. This hazard may be associated with cysteamine-mediated inhibition of the Notch signaling and disorganization of notochordal cells due to oxidative stress and apoptosis.