Early Zebrafish Development Research Articles

Genetic and Epigenetic Evidence for Nonestrogenic Disruption of Otolith Development by Bisphenol A in Zebrafish.

Bisphenol A (BPA) is a well-known endocrine-disrupting chemical (EDC) that has estrogenic activities. In addition to disrupting reproductive development and function via estrogenic signaling pathways, BPA can also interfere with nonreproductive functions through nonestrogenic pathways; however, the mechanisms underlying such nonestrogenic activities are not well understood. In this study, we demonstrated that BPA could disrupt otolith formation during the early development of zebrafish with long-lasting ethological effects. Using multiple mutants of estrogen receptors, we provided strong genetic evidence that the BPA-induced otolith malformation was independent of estrogen signaling. Transcriptome analysis revealed that two genes related to otolith development, otopetrin 1 (otop1) and starmaker (stm), decreased their expression significantly after BPA exposure. Knockout of both otop1 and stm genes could phenocopy the BPA-induced otolith malformation, while microinjection of their mRNAs could rescue the BPA-induced abnormalities of otolith formation. Further experiments showed that BPA inhibited the expression of otop1 and stm by activating the MEK/ERK-EZH2-H3K27me3 signaling pathway. Taken together, our study provided comprehensive genetic and molecular evidence that BPA induced the otolith malformation through nonestrogenic pathway during zebrafish early development and its activities involved epigenetic control of key genes (e.g., otop1 and stm) participating in otolith formation.

An improved Erk biosensor detects oscillatory Erk dynamics driven by mitotic erasure during early development

Extracellular signal-regulated kinase (Erk) signaling dynamics elicit distinct cellular responses in a variety of contexts. The early zebrafish embryo is an ideal model to explore the role of Erk signaling dynamics invivo, as a gradient of activated diphosphorylated Erk (P-Erk) is induced by fibroblast growth factor (Fgf) signaling at the blastula margin. Here, we describe an improved Erk-specific biosensor, which we term modified Erk kinase translocation reporter (modErk-KTR). We demonstrate the utility of this biosensor invitro and in developing zebrafish and Drosophila embryos. Moreover, we show that Fgf/Erk signaling is dynamic and coupled to tissue growth during both early zebrafish and Drosophila development. Erk activity is rapidly extinguished just prior to mitosis, which we refer to as mitotic erasure, inducing periods of inactivity, thus providing a source of heterogeneity in an asynchronously dividing tissue. Our modified reporter and transgenic lines represent an important resource for interrogating the role of Erk signaling dynamics invivo.

Deletion of the chd7 Hinders Oligodendrocyte Progenitor Cell Development and Myelination in Zebrafish.

CHD7, an encoding ATP-dependent chromodomain helicase DNA-binding protein 7, has been identified as the causative gene involved in CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia choanae, Retardation of growth and/or development, Genital abnormalities and Ear abnormalities). Although studies in rodent models have expanded our understanding of CHD7, its role in oligodendrocyte (OL) differentiation and myelination in zebrafish is still unclear. In this study, we generated a chd7-knockout strain with CRISPR/Cas9 in zebrafish. We observed that knockout (KO) of chd7 intensely impeded the oligodendrocyte progenitor cells' (OPCs) migration and myelin formation due to massive expression of chd7 in oilg2+ cells, which might provoke upregulation of the MAPK signal pathway. Thus, our study demonstrates that chd7 is critical to oligodendrocyte migration and myelination during early development in zebrafish and describes a mechanism potentially associated with CHARGE syndrome.

Systematic Proteomics Study on the Embryonic Development of Danio rerio.

The early development of zebrafish (Danio rerio) is a complex and dynamic physiological process involving cell division, differentiation, and movement. Currently, the genome and transcriptome techniques have been widely used to study the embryonic development of zebrafish. However, the research of proteomics based on proteins that directly execute functions is relatively vacant. In this work, we apply label-free quantitative proteomics to explore protein profiling during zebrafish's embryogenesis, and a total of 5961 proteins were identified at 10 stages of zebrafish's early development. The identified proteins were divided into 11 modules according to weighted gene coexpression network analysis (WGCNA), and the characteristics between modules were significantly different. For example, mitochondria-related functions enriched the early development of zebrafish. Primordial germ cell-related proteins were identified at the 4-cell stage, while the eye development event is dominated at 5 days post fertilization (dpf). By combining with published transcriptomics data, we discovered some proteins that may be involved in activating zygotic genes. Meanwhile, 137 novel proteins were identified. This study comprehensively analyzed the dynamic processes in the embryonic development of zebrafish from the perspective of proteomics. It provided solid data support for further understanding of the molecular mechanism of its development.

Avermectin reduces bone mineralization via the TGF-β signaling pathway in zebrafish

Avermectin, a widely used insecticide, is primarily effective against animal parasites and insects. Given its extensive application in agriculture, a large amount of avermectin accumulates in natural water bodies. Studies have shown that avermectin has significant toxic effects on various organisms and on the nervous system, spine, and several other organs in humans. However, the effects of avermectin on bone development have not been reported yet. In this study, zebrafish embryos were treated with different concentrations of avermectin to explore the effects of avermectin on early bone development. The results showed that avermectin disturbed early bone development in zebrafish, caused abnormal craniofacial chondrogenesis, and reduced bone mineralization. Avermectin treatment significantly reduced mineralization in zebrafish scales and increased osteoclast activity. Real-time quantitative PCR results showed that avermectin decreased the expression of genes related to osteogenesis and transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling pathways. The TGF-β inhibitor SB431542 rescued avermectin-induced bone mineralization and osteogenesis related gene expression in zebrafish during early development. Thus, this study provides insight into the mechanism of damage caused by avermectin on bone development, thus helping demonstrate its toxicity.

Deiodinase inhibition impairs the formation of the three posterior swim bladder tissue layers during early embryonic development in zebrafish

Thyroid hormone system disruption (THSD) negatively affects multiple developmental processes and organs. In fish, inhibition of deiodinases, which are enzymes crucial for (in)activating thyroid hormones (THs), leads to impaired swim bladder inflation. Until now, the underlying mechanism has remained largely unknown. Therefore, the objective of this study was to identify the process during swim bladder development that is impacted by deiodinase inhibition. Zebrafish embryos were exposed to 6 mg/L iopanoic acid (IOP), a model deiodinase inhibitor, during 8 different exposure windows (0–60, 60–120, 24–48, 48–72, 72–96, 96–120, 72–120 and 0–120 h post fertilization (hpf)). Exposure windows were chosen based on the three stages of swim bladder development: budding (24–48 hpf), pre-inflation, i.e., the formation of the swim bladder tissue layers (48–72 hpf), and inflation phase (72–120 hpf). Exposures prior to 72 hpf, during either the budding or pre-inflation phase (or both), impaired swim bladder inflation, while exposure during the inflation phase did not. Based on our results, we hypothesize that DIO inhibition before 72 hpf leads to a local decrease in T3 levels in the developing swim bladder. Gene transcript analysis showed that these TH level alterations disturb both Wnt and hedgehog signaling, known to be essential for swim bladder formation, eventually resulting in impaired development of the swim bladder tissue layers. Improper development of the swim bladder impairs swim bladder inflation, leading to reduced swimming performance. This study demonstrates that deiodinase inhibition impacts processes underlying the formation of the swim bladder and not the inflation process, suggesting that these processes primarily rely on maternal rather than endogenously synthetized THs since TH measurements showed that THs were not endogenously synthetized during the sensitive period.

The Importance of Assessing Water Quality in Tributaries: A Case Study in an Urban Waterway Using Zebrafish (Danio rerio)

Tributaries are important for fish recruitment and diversity. Here, we examine the biological impact of inorganic and organic contaminants in Paint Branch stream (PBS), a tributary of the Anacostia river in Washington D.C. The Anacostia has suffered severe ecological damage because of decades of pollution and deposited wastewater runoff; however, PBS, which connects to the northern part of the river, is forested and less urbanized, suggesting higher water quality. However, the impact of PBS water on early fish development has not been studied. To address this question, we examined if chronic (28 day) exposure to water collected from PBS can support the proper early development of zebrafish (Danio rerio), a vertebrate model in toxicological studies. We assessed their overall growth and swimming behaviors and correlated these results with a water quality analysis. The water chemistry identified high levels of calcium, sodium, and nitrate in PBS water samples. A gas chromatography–mass spectroscopy analysis of extracted non-polar compounds in the water column revealed siloxanes (congeners D6–D10) were the only component identified with >90% certainty. In our fish experiments, we observed age-dependent increases in growth and eye development, consistent with normal development. In contrast, general swimming behaviors showed an early increase in angular velocity at 7 days postfertilization (dpf; p = 0.001) and a decreased total distance traveled at 14 dpf (p = 0.015) for PBS-treated larvae. Using the open field test, we observed that the PBS-treated fish made fewer visits to the edge at 7 (p = 0.01), 14 (p < 0.001), and 21 dpf (p = 0.038) and spent significantly more time at the edge at 21 dpf (p < 0.001). Fewer visits to the center were also noted at 14 and 21 dpf, suggesting reduced overall movement at these two ages in response to chronic PBS water exposure. Interestingly, by 28 dpf, no differences were noted in any parameter measured. Overall, these results indicate zebrafish larvae grew well in PBS water; however, their reduced movement and anxiogenic behavior suggested subtle behavioral abnormalities. The identified chemicals likely originated from runoff or sewage and have potentially deleterious consequences for fish living in PBS or migrating to/from upstream spawning/nursery locations.

Sulfonamides (SAs) exposure causes neurobehavioral toxicity at environmentally relevant concentrations (ERCs) in early development of zebrafish

Antibiotics, due to their stability and persistence in the environment, can have chronic impacts on various ecosystems and organisms. However, the molecular mechanisms underlying antibiotic toxicity at environmental concentrations, particularly the neurotoxic effects of sulfonamides (SAs), remain poorly understood. In this study, we assessed the neurotoxicity of six SAs including the sulfadiazine (SD), sulfathiazole (ST), sulfamethoxazole (SMX), sulfisoxazole (SIZ), sulfapyridine (SPD), and sulfadimethoxine (SDM) by exposing zebrafish to environmentally relevant concentrations (ERCs). The SAs exhibited concentration-dependent effects on zebrafish behavior, including spontaneous movement, heartbeat, survival rate, and body metrics, ultimately leading to depressive-like symptoms and sublethal toxicity during early life stages. Notably, even the lowest SA concentration (0.05 μg/L) induced neurotoxicity and behavioral impairment in zebrafish. We observed a dose-dependent increase in melancholy behavior as indicated by increased resting time and decreased motor activity in zebrafish larvae. Following exposure to SAs from 4 to 120 h post-fertilization (hpf), key genes involved in folate synthesis [sepiapterin reductase a (spra), phenylalanine hydroxylase (pah), tyrosine hydroxylase (th), and tryptophan hydroxylase 1 (tryptophan 5-monooxygenase) a tryptophan hydroxylase (tph1a)] and carbonic anhydrase (CA) metabolism [carbonic anhydrase II (ca2), carbonic anhydrase IV a (ca4a), carbonic anhydrase VII (ca7), and carbonic anhydrase XIV (ca14)] were significantly downregulated or inhibited at different concentrations. Our findings demonstrate that acute exposure to six SAs at environmentally relevant concentrations induces developmental and neurotoxic effects in zebrafish, impacting folate synthesis pathways and CA metabolism. These results provide valuable insights into the potential role of antibiotics in depressive disorders and neuroregulatory pathways.

Transcriptome analysis reveals an Atoh1b-dependent gene set downstream of Dlx3b/4b during early inner ear development in zebrafish.

The vertebrate inner ear is the sensory organ mediating hearing and balance. The entire organ develops from the otic placode, which itself originates from the otic-epibranchial progenitor domain (OEPD). Multiple studies in various species have shown the importance of the forkhead-box and distal-less homeodomain transcription factor families for OEPD and subsequent otic placode formation. However, the transcriptional networks downstream of these factors are only beginning to be understood. Using transcriptome analysis, we here reveal numerous genes regulated by the distal-less homeodomain transcription factors Dlx3b and Dlx4b (Dlx3b/4b). We identify known and novel transcripts displaying widespread OEPD expression in a Dlx3b/4b-dependent manner. Some genes, with a known OEPD expression in other vertebrate species, might be members of a presumptive vertebrate core module required for proper otic development. Moreover, we identify genes controlling early-born sensory hair cell formation as well as regulating biomineral tissue development, both consistent with defective sensory hair cell and otolith formation observed in dlx3b/4b mutants. Finally, we show that ectopic Atoh1b expression can rescue early sensorigenesis even in the absence of Dlx3b/4b. Taken together, our data will help to unravel the gene regulatory network underlying early inner ear development and provide insights into the molecular control of vertebrate inner ear formation to restore hearing loss in humans ultimately.

Environmentally relevant levels of BPA and NOR disturb early skeletal development in zebrafish

Bisphenol A (BPA) and fluoroquinolone antibiotics (FQs) have been detected frequently in aquatic environments. Studies have shown that high levels of BPA and FQs exposure have adverse effects on chondrogenesis in young terrestrial vertebrates. However, little is known about their combined toxicity to bone metabolism. Here, we evaluated the single and combined effects of BPA and norfloxacin (NOR, a typical species of FQs) at an environmentally relevant level (1 μg/L) on early skeletal development in zebrafish. We found that both individual and combined BPA and NOR exposure resulted in poor embryo quality and decreased calcium-phosphorus ratio. The malformation increased after exposure to BPA and NOR, and craniofacial cartilage ossification was delayed. At the molecular level, the transcriptions of genes related to ossification were down-regulated significantly, and the lysine oxidase activity decreased. Hence, we infer that an environmentally relevant concentration of BPA and NOR have adverse effects on early skeletal development in fish. In addition, combined exposure to BPA and NOR seems to have an antagonistic effect on early skeletal development.

Environmentally relevant concentrations of triclosan induce lethality and disrupt thyroid hormone activity in zebrafish (Danio rerio)

Triclosan is an antimicrobial agent that has been used in common household products and can be detected in water environment. In this study, therefore, I aimed at clarifying the effects of environmentally relevant concentrations of triclosan on the early life stage development in zebrafish. A lethal effect was observed: the lowest effect and the no effect concentrations were 70.6 and 48.4 μg/L, respectively. These concentrations are very close to the environmentally detected residual concentrations. In 10.9, 19.8, 48.4, and 70.6 μg/L of triclosan, the iodothyronine deiodinase 1 gene expression was found to be significantly increased when compared with that of the control group. These findings indicate that triclosan can potentially disrupt the thyroid hormone activity in zebrafish. The exposure to triclosan (at 149.2 μg/L) was also found to inhibit the gene expression of insulin-like growth factor-1. My findings suggest that triclosan can exert a thyroid hormone-disrupting effect on fish.

CRISPR/Cas9-Mediated Knockout of tnfaip1 in Zebrafish Plays a Role in Early Development.

TNF α-induced protein 1 (TNFAIP1) was first identified in human umbilical vein endothelial cells and can be induced by tumor necrosis factor α (TNFα). Early studies have found that TNFAIP1 is involved in the development of many tumors and is closely associated with the neurological disorder Alzheimer's disease. However, little is known about the expression pattern of TNFAIP1 under physiological conditions and its function during embryonic development. In this study, we used zebrafish as a model to illustrate the early developmental expression pattern of tnfaip1 and its role in early development. First, we examined the expression pattern of tnfaip1 during early zebrafish development using quantitative real-time PCR and whole mount in situ hybridization and found that tnfaip1 was highly expressed in early embryonic development and, subsequently, expression became localized to anterior embryonic structures. To investigate the function of tnfaip1 during early development, we constructed a model of a stably inherited tnfaip1 mutant using the CRISPR/Cas9 system. Tnfaip1 mutant embryos showed significant developmental delays as well as microcephaly and microphthalmia. At the same time, we found decreased expression of the neuronal marker genes tuba1b, neurod1, and ccnd1 in tnfaip1 mutants. Analysis of transcriptome sequencing data revealed altered expression of the embryonic development related genes dhx40, hspa13, tnfrsf19, nppa, lrp2b, hspb9, clul1, zbtb47a, cryba1a, and adgrg4a in the tnfaip1 mutants. These findings suggest an important role for tnfaip1 in the early development of zebrafish.

First In Vivo Insights on the Effects of Tempol-Methoxycinnamate, a New UV Filter, as Alternative to Octyl Methoxycinnamate, on Zebrafish Early Development

The demand for organic UV filters as active components in sunscreen products has rapidly risen over the last century, as people have gradually realized the hazards of overexposure to UV radiation. Their extensive usage has resulted in their ubiquitous presence in different aquatic matrices, representing a potential threat to living organisms. In this context, the need to replace classic UV filters such as octyl methoxycinnamate (OMC), one of the most popular UV filters reported to be a potential pollutant of aquatic ecosystems, with more environmentally friendly ones has emerged. In this study, using zebrafish, the first in vivo results regarding the effect of exposure to tempol-methoxycinnamate (TMC), a derivative of OMC, are reported. A comparative study between TMC and OMC was performed, analyzing embryos exposed to similar TMC and OMC concentrations, focusing on morphological and molecular changes. While both compounds seemed not to affect hatching and embryogenesis, OMC exposure caused an increase in endoplasmic reticulum (ER) stress response genes, according to increased eif2ak3, ddit3, nrf2, and nkap mRNA levels and in oxidative stress genes, as observed from modulation of the sod1, sod2, gpr, and trx mRNA levels. On the contrary, exposure to TMC led to reduced toxicity, probably due to the presence of the nitroxide group in the compound's molecular structure responsible for antioxidant activity. In addition, both UV filters were docked with estrogen and androgen receptors where they acted differently, in agreement with the molecular analysis that showed a hormone-like activity for OMC but not for TMC. Overall, the results indicate the suitability of TMC as an alternative, environmentally safer UV filter.

Abstract 3525: VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis

Abstract Clinical sequencing efforts are rapidly identifying sarcoma gene fusions that have not been functionally validated for their transformation capacity and biological activity. An example is the new fusion of transcriptional coactivators, VGLL2-NCOA2, found in infantile rhabdomyosarcoma. To delineate VGLL2-NCOA2 tumorigenic mechanisms and identify therapeutic vulnerabilities, we implemented a cross-species comparative oncology approach with zebrafish, mouse allograft, and patient samples. We found that in our transgenic zebrafish and mouse allograft models, VGLL2-NCOA2 is sufficient to generate mesenchymal tumors that display features of immature skeletal muscle. Zebrafish and mouse VGLL2-NCOA2 tumors are consistent with the human disease histologically and transcriptionally, and express diagnostic rhabdomyosarcoma markers. We found a shared molecular feature of these tumors was a suppression of myogenic differentiation pathways and reactivation of developmental programs. To identify impacted developmental programs, we transcriptionally clustered zebrafish VGLL2-NCOA2 tumors with the trajectory of early zebrafish development. A subset of VGLL2-NCOA2 zebrafish tumors cluster with embryonic somitogenesis and pinpoint VGLL2-NCOA2 developmental targets, including a RAS family GTPase, ARF6. In VGLL2-NCOA2 zebrafish, mouse allograft, and patient tumors, ARF6 is highly expressed and is absent from mature skeletal muscle. ARF6 knockout in our C2C12-VGLL2-NCOA2 cell culture model suppresses VGLL2-NCOA2 soft agar colony formation capacity, suggesting a genetic cooperating event in the disease. Our data indicate that VGLL2-NCOA2 is an oncogene which leverages developmental programs for tumorigenesis, and that the reactivation or persistence of ARF6 could represent a therapeutic opportunity. Citation Format: Sarah Watson, Collette A. LaVigne, Lin Xu, Didier Surdez, Joanna Cyrta, Delia Calderon, Matthew V. Cannon, Matthew R. Kent, Katherine M. Silvius, Jack P. Kucinski, Emma N. Harrison, Whitney Murchison, Dinesh Rakheja, Franck Tirode, Olivier Delattre, James F. Amatruda, Genevieve C. Kendall. VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3525.

To the fin and back again: expanding our understanding of congenital limb malformation

Fins are not limbs. However, findings from comparative developmental and genetic analyses show that many of the processes essential in establishing a limb are similar for fin formation. Nevertheless, due to their structural differences, fins are not often used as models to understand the causes or potential treatment of limb disorders.Split hand/foot malformation (SHFM) presents with loss or mis-patterning of elements of hands or feet leading to a ‘clefting-like’ phenotype, often with joining of the digits. This rare disorder is genetically and phenotypically heterogeneous, and knowledge of its etiology remains largely incomplete. Recent work by Truong et al. provides new insight into SHFM by identifying new genetic variants within three different families, predicted to cause changes in function of the PRDM1/Blimp1 gene. PRDM1 is a transcription factor known to be essential for early appendage development in both zebrafish and mice. However, PRDM1 action is nuanced, in part due to its multifaceted roles in integrating signaling, transcription and chromatin state. This complicates the functional characterization of variants in this gene that have unknown significance in SHFM patients. Truong et al. demonstrate the utility of the zebrafish fin as an assay to directly address the function of domain-specific mutations in prdm1a by using the ability of the mutant alleles to rescue fin phenotypes caused by loss of Prdm1a function or, in the case of putative dominant variants, to suppress wildtype zebrafish fin development.A fin may not, or more likely will not, have the complete genetic networks underlying limb development and patterning. However, core mechanisms remain active and, at times, latent in fin development, allowing experimental dissection of mechanisms and gene function. Truong et al. harnessed the experimental malleability of zebrafish to isolate pectoral fin cells in early development and detail Prdm1a-dependent regulation of genes. These data establish aspects of the downstream regulatory network of fin development regulated by Prdm1a, providing insight into the broader array of changes involved in the etiology of this disorder.Core mechanisms and common evolutionary foundations allow for the establishment of defined genetic models of limb disorders in zebrafish and, importantly, accessible experimental models to formulate and test hypotheses on how these disease states are attained. Conversely, identification of disease-associating alleles provides insight into molecular action of genes in development and reveals details of the specificity of their regulation. Fin-in-hand, leading towards new discoveries for congenital limb malformations.

Lethal and Sublethal Toxicity of Un-Ionized Ammonia to Early-Life Stages of Danio rerio.

Un-ionized ammonia (NH3) is a prevalent contaminant found in aquatic ecosystems, frequently associated with complex mixtures of other toxic contaminants. Early embryo-larval stages of zebrafish Danio rerio became an important model for water quality monitoring, and it is necessary to characterize its susceptibility to NH3 exposure. Fertilized eggs were exposed to NH3 concentrations ranging from 0.02 to 5.23 mg NH3 L-1 until 168 h postfertilization (hpf). The lethal concentration to 50% of exposed zebrafish during 96 h was 2.07 mg NH3 L-1, 25% above the median value reported values for early developmental stages of fishes. Sublethal toxicity endpoints indicated the lowest observed effect concentrations for slow blood circulation at 24 hpf, decreased heart ventricular contractions at 72 hpf, incomplete yolk sac absorption at 96 hpf, failure in swim bladder inflation at 96 hpf, developmental retardation at 96 hpf, decreased total length, decreased swimming speed, altered trajectories, and acetylcholinesterase inhibition at 168 hpf of 0.85, 0.06, 0.15, 0.06, 0.15, 0.61, 1.35, 0.35, and 0.85 mg NH3 L-1, respectively. Environmentally relevant NH3 concentrations can affect zebrafish's early development and larval viability, and our results help discriminate NH3 contribution to the toxicity of complex environmental mixtures when zebrafish is used in water quality monitoring.

Immunotoxicity induced by triclocarban exposure in zebrafish triggering the risk of pancreatic cancer

Owing to frequent application as a broad-spectrum bactericide, triclocarban (TCC) exposure has raised great concern for aquatic organisms and human health. Herein, based on transcriptome sequencing data analysis of zebrafish, we confirmed that TCC induced oxidative stress and dysimmunity through transcriptional regulation of the related genes. With aid of the Cancer Genome Atlas (TCGA) assembler database, 52 common differentially expressed genes, whose functions were related to immunity, were screened out by virtue of the meta-analysis of pancreatic cancer sample data and differential transcription profiles from TCC-exposed larvae. Acute TCC exposure affected formation of the innate immune cells, delayed mature thymic T-cell development, reduced immunoglobulin M (IgM) levels and promoted excessive release of the pro-inflammatory factors (IL-6, IL-1β and tnfα). Under TCC exposure, the expressions of the genes associated with immune cell abundance in pancreatic cancer were significantly down-regulated, while the levels of ROS were prominently increased in concomitant with suppressed antioxidant activity. Moreover, a series of marker genes (pi3k, nrf2, keap1, ho-1 and nqo1) in the PI3K/Nrf2 antioxidant-stress pathway were abnormally expressed under TCC exposure. Interestingly, vitamin C decreased the malformation and increased the survival rate of 120-hpf larvae and effectively alleviated TCC-induced oxidative stress and immune responses. Overall, TCC exposure induced immunotoxicity and increased the risk of pancreatic cancer by inhibiting the antioxidant capacity of the PI3K/Nrf2 signal pathway. These observations enrich our in-depth understanding of the effects of TCC on early embryonic-larval development and immune damage in zebrafish.

The infection of murine gammaherpesvirus-68 delays the early embryonic development of zebrafish

Zebrafish is a model animal for aquaculture as well as bio-medicine. Murine gamma-herpesvirus 68 (MHV68) is widely regarded as a model for the study of human gamma herpesviruses such as Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, the infection of MHV68 on zebrafish was investigated through incubating fertilized zebrafish eggs with MHV68. The development of zebrafish delayed or stopped at early stage separately when the zebrafish eggs were incubated with MHV68 at different dose for 2 h. The lytic gene of MHV68, including ORF65, RTA and ORF57, were detected in the genome of larvae hatched from the infected eggs. The transcription of the latent gene LANA but not of these lytic genes was detected by RT-PCR. The mRNA level of growth factor TGF-β1, but not of FGF3, was obviously decreased in the larvae from the MHV68 treated eggs. The mRNA levels of cytokines, such as IFNφ3, ISG15 and TNFα, varied post MHV68 infection; however, the transcription of the cytosolic DNA sensors, including DDX41, DHX9 and cGAS, did not change significantly. Moreover, the data generated in zebrafish cell line demonstrated that MHV68 could infect ZF4 cells. All the data support the conclusion that MHV68 could infect zebrafish embryo in the egg envelope and go into latent infection, which will delay the development of the embryos through some unknown mechanism.

Cpeb1b-mediated cytoplasmic polyadenylation of shha mRNA modulates zebrafish definitive hematopoiesis

During vertebrate embryogenesis, hematopoietic stem and progenitor cell (HSPC) production through endothelial-to-hematopoietic transition requires suitable developmental signals, but how these signals are accurately regulated remains incompletely understood. Cytoplasmic polyadenylation, which is one of the posttranscriptional regulations, plays a crucial role in RNA metabolism. Here, we report that Cpeb1b-mediated cytoplasmic polyadenylation is important for HSPC specification by translational control of Hedgehog (Hh) signaling during zebrafish early development. Cpeb1b is highly expressed in notochord and its deficiency results in defective HSPC production. Mechanistically, Cpeb1b regulates hemogenic endothelium specification by the Hedgehog-Vegf-Notch axis. We demonstrate that the cytoplasmic polyadenylation element motif-dependent interaction between Cpeb1b and shha messenger RNA (mRNA) in the liquid-like condensates, which are induced by Pabpc1b phase separation, is required for cytoplasmic polyadenylation of shha mRNA. Intriguingly, the cytoplasmic polyadenylation regulates translation but not stability of shha mRNA, which further enhances the Shha protein level and Hh signal transduction. Taken together, our findings uncover the role of Cpeb1b-mediated cytoplasmic polyadenylation in HSPC development and provide insights into how posttranscriptional regulation can direct developmental signals with high fidelity to translate them into cell fate transition.

The toxicity of 4-tert-butylphenol in early development of zebrafish: morphological abnormality, cardiotoxicity, and hypopigmentation.

Endocrine disrupting effects of 4-tert-butylphenol (4-t-BP) are well described in literature. However, the evidence regarding developmental toxic effect of 4-t-BP is still vague. The present study used zebrafish as a model organism to investigate the toxic effect of 4-t-BP. The results showed that 4-t-BP exposure at 3, 6, and 12 μM induced developmental toxicity in zebrafish, such as reduced embryo hatchability and abnormality morphological. Flow cytometry analysis showed that 4-t-BP also induced intracellular ROS production. 4-t-BP induced changes in the expression of genes related to cardiac development and melanin synthesis, resulting in cardiotoxicity and hypopigmentation. 4-t-BP also caused oxidative stress, and initiated apoptosis through p53-bcl-2/bax-capase3 pathway. Integrative biomarker response analysis showed time- and dose-dependent effects of 4-t-BP on oxidative damage and developmental toxicity in zebrafish embryos. Overall, this study contributed to a comprehensive evaluation of the toxicity of 4-t-BP, and the findings provided new evidence for early warning of residues in aquatic environments.