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Polyhalogenated carbazoles (PHCZs) are emerging environmental pollutants, yet limited information is available on their embryotoxicity and neurotoxicity. Therefore, the current work was performed to investigate the adverse effects of 3,6-dibromocarbazole (3,6-DBCZ), a typical PHCZs homolog, on the early life stages of zebrafish larvae. It revealed that the 96-hour post-fertilization (hpf) median lethal concentration (LC50) value of 3,6-DBCZ in zebrafish larvae was determined to be 0.7988 mg/L. Besides, 3,6-DBCZ reduced survival rates at concentrations ≥ 1 mg/L and decreased hatching rates at ≥ 0.25 mg/L at 48 hpf. In behavior tests, it inhibited locomotor activities and reduced the frequency of recorded acceleration states in response to optesthesia (a sudden bright light stimulus) at concentrations ≥ 160 μg/L. Meanwhile, 3,6-DBCZ exposure decreased the frequency of recorded acceleration states in the startle response (tapping mode) at concentrations ≥ 6.4 μg/L. Pathologically, with the transgenic zebrafish model (hb9-eGFP), we observed a strikingly decreased axon length and number in motor neurons after 3,6-DBCZ treatment, which may be ascribed to the activation of the AhR signaling pathway, as evidenced by the molecular docking analysis and Microscale thermophoresis (MST) assay suggested that 3,6-DBCZ binding to AhR-ARNT2 compound proteins. Through interaction with AhR-ARNT, a striking reduction of the anti-oxidative stress (sod1/2, nqo1, nrf2) and neurodevelopment-related genes (elavl3, gfap, mbp, syn2a) were observed after 3,6-DBCZ challenge, accompanied by a marked increased inflammatory genes (TNFβ, IL1β, IL6). Collectively, our findings reveal a previously unrecognized adverse effect of 3,6-DBCZ on zebrafish neurodevelopment and locomotor behaviors, potentially mediated through the activation of the AhR pathway. Furthermore, it provides direct evidence for the toxic concentrations of 3,6-DBCZ and the potential target signaling in zebrafish larvae, which may be beneficial for the risk assessment of the aquatic ecosystems.

CYP1A is the most commonly used biomarker and transgenic fish which carrying a cyp1a promoter to drive a reporter gene can be used as reliable way to monitor dioxin/dioxin-like compounds (DLCs) in the environment. Here, we cloned the cyp1a promoter of Gambusia affinis and this promoter showed stronger transcriptional activity than that of zebrafish. Then, a Tg(GAcyp1a:eGFP/Luc) transgenic zebrafish line was first constructed with the G. affinis cyp1a promoter driving eGFP expression using meganuclease I-SceI mediated transgenesis technology. The Tg(GAcyp1a:eGFP/Luc) larvae at 72 h post-fertilization (hpf) were tested by exposing to TCDD for 72 h, and induced GFP was mainly expressed in the liver with low background. The Tg(GAcyp1a:eGFP/Luc) zebrafish showed high sensitivity (limit of detection of 0.322 ng/L TCDD and 0.7 TEQ-ng/L PCDD/Fs) and specificity (insensitive to responses to PAHs and PCBs). In addition, the transgenic line showed a low detection concentration of the DLCs contaminated environmental samples (as low as 1.8 TEQ-ng/L), and the eGFP fluorescence intensity and the chemical-TEQ values were closely correlated. In conclusion, a sensitively and specifically transgenic zebrafish line was established to convenient and effective to detect DLCs in the environment.

Piperlongumine (PL) and piperine (PP) are alkaloids presented in long pepper (Piper longum), and they exhibit various biological activities, especially anti-cancer properties. With these regards, they are considered as future medicines with high potential. Even they are exposed to humans such a long time, their potential toxicities in the environment have not been studied. Therefore, their ecological toxicities were assessed using zebrafish embryos. PP showed low mortality and no abnormal phenotype up to 10 µM. However, PL exhibited strong acute toxicity at the concentration of 5–10 µM ranges, and abnormal development were frequently found in the range of 1–2.5 µM with pericardial and yolk sac edemas. In transgenic zebrafish embryos, PL induced an increase in the number of intersegmental vessels and delayed the early-stage development. PL treatment affected heart formation and heart rate. The presence of PL induced the expression of cytokines, inflammatory markers, and inflammasome in the embryos. The PL treatment changed the mRNA levels of the ER stress and apoptosis-related genes. In addition, ROS production was observed during early-stage development of PL-treated zebrafish embryos. These results indicate that developing PL as a medicine would require extremely meticulous strategies to prevent potential toxicity.

Compared with other species, freshwater fish are more capable of synthesizing DHA via same biosynthetic pathways. Freshwater fish have a “Sprecher” pathway to biosynthesize DHA in a peroxisome-dependent manner. Enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) is involved in the hydration and dehydrogenation reactions of fatty acid β-oxidation in peroxisomes. However, the role of Ehhadh in the synthesis of DHA in freshwater fish remains largely unclear. In this study, the knockout of Ehhadh significantly inhibited DHA synthesis in zebrafish. Liver transcriptome analysis showed that Ehhadh deletion significantly inhibited SREBF and PPAR signaling pathways and decreased the expression of PUFA synthesis-related genes. Our results from the analysis of transgenic zebrafish (Tg:Ehhadh) showed that Ehhadh overexpression significantly increased the DHA content in the liver and significantly upregulated the expression of genes related to PUFA synthesis. In addition, the DHA content in the liver of Tg:Ehhadh fed with linseed oil was significantly higher than that of wildtype, but the expression of PUFA synthesis-related genes fads2 and elovl2 were significantly lower, indicating that Ehhadh had a direct effect on DHA synthesis. In conclusion, our results showed that Ehhadh was essential for DHA synthesis in the “Sprecher” pathway, and Ehhadh overexpression could promote DHA synthesis. This study provides insight into the role of Ehhadh in freshwater fish.

Microcystin-LR (MC-LR) is widely distributed in the natural environment and causes hepatotoxicity. However, whether MC-LR promotes liver tumor progression remains controversial. krasV12 transgenic zebrafish were used as an inducible liver tumor model to evaluate the potential tumor-promoting effect of MC-LR. First, krasV12 transgenic larvae were exposed to 0, 0.1 and 1 mg/L MC-LR with 20 mg/L doxycycline (Dox) for 4 d. The gray values and histopathological examinations of the liver demonstrated that MC-LR aggravated liver tumor progression, which could be inhibited by the Protein arginine methyltransferase 5 (Prmt5) inhibitor compound 5 (CMP5). Second, 1-month-old juvenile transgenic zebrafish were exposed to 0, 20 mg/L Dox, 1 μg/L MC-LR, and 20 mg/L Dox with 0.1 or 1 μg/L MC-LR for 15 d to determine whether the exposure to environmental concentrations of MC-LR promoted hepatocellular carcinoma (HCC) progression. We found that environmental concentrations of MC-LR increased the hepatosomatic index (HSI) and gray value (intensity/area) and promoted HCC progression. The results indicate that environmental concentrations of MC-LR have the potential to promote liver tumor progression. Taken together, the present study demonstrates that MC-LR can promote tumor in krasV12 transgenic zebrafish and that the upregulation of prmt5 expression might contribute to MC-LR-mediated promotion of liver tumorigenesis.

Pyrethroid and organochlorine insecticides are enormously used to control agricultural and indoor insect pests. The metabolites of pyrethroid and endosulfan were used to evaluate environmental toxicities using a representative animal model, zebrafish (Danio rerio) embryos in this study. The LC50 values in 3-phenoxy benzoic acid (3-PBA) and endosulfan sulfate (ES) were 1461 μg/L and 1459 μg/L, respectively. At the concentration of 2000 μg/L, spine curvature was observed in the ES-treated embryos. ES showed seizure-like events with an EC50 value of 354 μg/L. At the concentration of 1000 μg/L, the pericardial edema was observed in 3-PBA-treated embryos. The inhibition of heart development and the reduction of beating rates were observed in Tg(cmlc2:EGFP) embryos after the exposure to 3-PBA. Down-regulation of the vmhc gene coding ventricular myosin during heart development was significantly found in 3-PBA-treated embryos at 48 hpf, but recovered afterward. It indicates that ventricular malformation occurred at the initial stage of 3-PBA exposure. Considered together, both 3-PBA and ES need public concerns with periodic monitoring of these metabolites in households and agricultural areas to prevent humans and environmental organisms from their unexpected attacks.

Ferulic acid (FA) derivatives have applied in American clinical trials for preventing and treating auditory dysfunctions. However, the effects of FA on neomycin-mediated sensorineural hearing loss are still unknown. We developed a transgenic zebrafish (pvalb3b:TagGFP) expressing green-colored hair cells in the inner ear and lateral line neuromasts by Tol2 system. Then, we determined the effects of FA on neomycin-induced ototoxicity in 4 dpf transgenic larvae. FA conferred significant protection against hair cell loss across a wide range of neomycin concentrations. In addition, FA significantly decreased intracellular ROS production and TUNAL reactions in larvae pretreated with FA prior to neomycin exposure. These findings suggest that FA has antioxidant effects and can attenuate neomycin-induced hair cell death in neuromasts. The green hair cells of transgenic zebrafish lateral line may provide a useful basis on which to investigate the cellular pathways related to the hair cell death and survival imparted by FA.

Polyhalogenated carbazoles (PHCZs) are emerging environmental pollutants, yet limited information is available on their embryotoxicity and neurotoxicity. Therefore, the current work was performed to investigate the adverse effects of 3,6-dibromocarbazole (3,6-DBCZ), a typical PHCZs homolog, on the early life stages of zebrafish larvae. It revealed that the 96-hour post-fertilization (hpf) median lethal concentration (LC 50 ) value of 3,6-DBCZ in zebrafish larvae was determined to be 0.7988 mg/L. Besides, 3,6-DBCZ reduced survival rates at concentrations ≥ 1 mg/L and decreased hatching rates at ≥ 0.25 mg/L at 48 hpf. In behavior tests, it inhibited locomotor activities and reduced the frequency of recorded acceleration states in response to optesthesia (a sudden bright light stimulus) at concentrations ≥ 160 μg/L. Meanwhile, 3,6-DBCZ exposure decreased the frequency of recorded acceleration states in the startle response (tapping mode) at concentrations ≥ 6.4 μg/L. Pathologically, with the transgenic zebrafish model (hb9-eGFP), we observed a strikingly decreased axon length and number in motor neurons after 3,6-DBCZ treatment, which may be ascribed to the activation of the AhR signaling pathway, as evidenced by the molecular docking analysis and Microscale thermophoresis (MST) assay suggested that 3,6-DBCZ binding to AhR-ARNT2 compound proteins. Through interaction with AhR-ARNT, a striking reduction of the anti-oxidative stress (sod1/2, nqo1, nrf2) and neurodevelopment-related genes (elavl3, gfap, mbp, syn2a) were observed after 3,6-DBCZ challenge, accompanied by a marked increased inflammatory genes (TNFβ, IL1β, IL6). Collectively, our findings reveal a previously unrecognized adverse effect of 3,6-DBCZ on zebrafish neurodevelopment and locomotor behaviors, potentially mediated through the activation of the AhR pathway. Furthermore, it provides direct evidence for the toxic concentrations of 3,6-DBCZ and the potential target signaling in zebrafish larvae, which may be beneficial for the risk assessment of the aquatic ecosystems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Although nanoplastics (NPs) can penetrate the blood-brain barrier and accumulate in the brain, the neurotoxicity of these particles and the mechanisms associated with their unique physio-chemical properties have yet to be sufficiently ascertained. In this study, we assessed the neuroexcitatory symptoms of zebrafish (Danio rerio) larvae treated with polystyrene (PS) NPs based on an examination of locomotory behaviour, dopamine levels, and acetylcholinesterase activity. We found that PS NPs caused oxidative stress and inhibited atoh1a expression in the cerebellum of Tg(atoh1a:dTomato) transgenic zebrafish larvae, thereby indicating damage to the central nervous system. In contrast to the Parkinson's disease (PD) like effects induced by most types of nanoparticles, such as graphene oxide, we established that PS NPs influenced the neuronal proteomic profiles of zebrafish larvae in a manner contrary to the molecular pathways characteristic of PD-like effects, which could be explained by the molecular dynamic simulation. Unlike graphene oxide nanoparticles that promote significant change in the internal structure of neuroproteins, the complex macromolecular polymers of PS NPs promoted the coalescence and increased expression of neuroproteins, thereby plausibly contributing to the neuroexcitatory symptoms observed in treated zebrafish larvae. Consequently, compared with traditional nanoparticles, we believe that the unique physio-chemical properties of NPs could be a potential factor contributing to their toxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

CYP1A is the most commonly used biomarker and transgenic fish which carrying a cyp1a promoter to drive a reporter gene can be used as reliable way to monitor dioxin/dioxin-like compounds (DLCs) in the environment. Here, we cloned the cyp1a promoter of Gambusia affinis and this promoter showed stronger transcriptional activity than that of zebrafish. Then, a Tg(GAcyp1a:eGFP/Luc) transgenic zebrafish line was first constructed with the G. affinis cyp1a promoter driving eGFP expression using meganuclease I-SceI mediated transgenesis technology. The Tg(GAcyp1a:eGFP/Luc) larvae at 72 h post-fertilization (hpf) were tested by exposing to TCDD for 72 h, and induced GFP was mainly expressed in the liver with low background. The Tg(GAcyp1a:eGFP/Luc) zebrafish showed high sensitivity (limit of detection of 0.322 ng/L TCDD and 0.7 TEQ-ng/L PCDD/Fs) and specificity (insensitive to responses to PAHs and PCBs). In addition, the transgenic line showed a low detection concentration of the DLCs contaminated environmental samples (as low as 1.8 TEQ-ng/L), and the eGFP fluorescence intensity and the chemical-TEQ values were closely correlated. In conclusion, a sensitively and specifically transgenic zebrafish line was established to convenient and effective to detect DLCs in the environment., Competing Interests: Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Piperlongumine (PL) and piperine (PP) are alkaloids presented in long pepper (Piper longum), and they exhibit various biological activities, especially anti-cancer properties. With these regards, they are considered as future medicines with high potential. Even they are exposed to humans such a long time, their potential toxicities in the environment have not been studied. Therefore, their ecological toxicities were assessed using zebrafish embryos. PP showed low mortality and no abnormal phenotype up to 10 µM. However, PL exhibited strong acute toxicity at the concentration of 5-10 µM ranges, and abnormal development were frequently found in the range of 1-2.5 µM with pericardial and yolk sac edemas. In transgenic zebrafish embryos, PL induced an increase in the number of intersegmental vessels and delayed the early-stage development. PL treatment affected heart formation and heart rate. The presence of PL induced the expression of cytokines, inflammatory markers, and inflammasome in the embryos. The PL treatment changed the mRNA levels of the ER stress and apoptosis-related genes. In addition, ROS production was observed during early-stage development of PL-treated zebrafish embryos. These results indicate that developing PL as a medicine would require extremely meticulous strategies to prevent potential toxicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Nanoplastics (NPs) have received global attention due to their wide application and detection in various environmental or biological media. NPs can penetrate physical barriers and accumulate in organisms after being ingested, producing a variety of toxic effects and possessing particle size-dependent effects, distinguishing them from traditional contaminants. This paper explored the neurotoxicity of polystyrene (PS)-NPs of different particle sizes on zebrafish (Danio rerio) embryos at environmental concentrations at the tissue and molecular levels using visualized transgenic zebrafish. Results showed that all particle sizes of PS-NPs produced developmental toxicity in zebrafish embryos and induced neuronal loss, axonal deletion/shortening/hybridization, and developmental and apoptotic-related genetic alterations, ultimately leading to behavioral abnormalities. PS-NPs with smaller sizes may have more severe neurotoxicity due to their entry into the embryo and brain through the chorionic pore before hatching. In addition, PS-NPs at 100 nm and 1000 nm can specifically interfere with GABAergic, cholinergic or serotonergic system and affect neuronal signaling. Our results reveal the neurotoxic risk of NPs, and smaller particle-size NPs may have a greater ecological risk. We anticipate that our study can provide a basis for exploring the toxicity mechanisms of NPs and the environmental risk assessment of NPs., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Compared with other species, freshwater fish are more capable of synthesizing DHA via same biosynthetic pathways. Freshwater fish have a "Sprecher" pathway to biosynthesize DHA in a peroxisome-dependent manner. Enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) is involved in the hydration and dehydrogenation reactions of fatty acid β-oxidation in peroxisomes. However, the role of Ehhadh in the synthesis of DHA in freshwater fish remains largely unclear. In this study, the knockout of Ehhadh significantly inhibited DHA synthesis in zebrafish. Liver transcriptome analysis showed that Ehhadh deletion significantly inhibited SREBF and PPAR signaling pathways and decreased the expression of PUFA synthesis-related genes. Our results from the analysis of transgenic zebrafish (Tg:Ehhadh) showed that Ehhadh overexpression significantly increased the DHA content in the liver and significantly upregulated the expression of genes related to PUFA synthesis. In addition, the DHA content in the liver of Tg:Ehhadh fed with linseed oil was significantly higher than that of wildtype, but the expression of PUFA synthesis-related genes fads2 and elovl2 were significantly lower, indicating that Ehhadh had a direct effect on DHA synthesis. In conclusion, our results showed that Ehhadh was essential for DHA synthesis in the "Sprecher" pathway, and Ehhadh overexpression could promote DHA synthesis. This study provides insight into the role of Ehhadh in freshwater fish., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Pyrethroid and organochlorine insecticides are enormously used to control agricultural and indoor insect pests. The metabolites of pyrethroid and endosulfan were used to evaluate environmental toxicities using a representative animal model, zebrafish (Danio rerio) embryos in this study. The LC 50 values in 3-phenoxy benzoic acid (3-PBA) and endosulfan sulfate (ES) were 1461 μg/L and 1459 μg/L, respectively. At the concentration of 2000 μg/L, spine curvature was observed in the ES-treated embryos. ES showed seizure-like events with an EC 50 value of 354 μg/L. At the concentration of 1000 μg/L, the pericardial edema was observed in 3-PBA-treated embryos. The inhibition of heart development and the reduction of beating rates were observed in Tg(cmlc2:EGFP) embryos after the exposure to 3-PBA. Down-regulation of the vmhc gene coding ventricular myosin during heart development was significantly found in 3-PBA-treated embryos at 48 hpf, but recovered afterward. It indicates that ventricular malformation occurred at the initial stage of 3-PBA exposure. Considered together, both 3-PBA and ES need public concerns with periodic monitoring of these metabolites in households and agricultural areas to prevent humans and environmental organisms from their unexpected attacks., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Microcystin-LR (MC-LR) is widely distributed in the natural environment and causes hepatotoxicity. However, whether MC-LR promotes liver tumor progression remains controversial. kras V12 transgenic zebrafish were used as an inducible liver tumor model to evaluate the potential tumor-promoting effect of MC-LR. First, kras V12 transgenic larvae were exposed to 0, 0.1 and 1 mg/L MC-LR with 20 mg/L doxycycline (Dox) for 4 d. The gray values and histopathological examinations of the liver demonstrated that MC-LR aggravated liver tumor progression, which could be inhibited by the Protein arginine methyltransferase 5 (Prmt5) inhibitor compound 5 (CMP5). Second, 1-month-old juvenile transgenic zebrafish were exposed to 0, 20 mg/L Dox, 1 μg/L MC-LR, and 20 mg/L Dox with 0.1 or 1 μg/L MC-LR for 15 d to determine whether the exposure to environmental concentrations of MC-LR promoted hepatocellular carcinoma (HCC) progression. We found that environmental concentrations of MC-LR increased the hepatosomatic index (HSI) and gray value (intensity/area) and promoted HCC progression. The results indicate that environmental concentrations of MC-LR have the potential to promote liver tumor progression. Taken together, the present study demonstrates that MC-LR can promote tumor in kras V12 transgenic zebrafish and that the upregulation of prmt5 expression might contribute to MC-LR-mediated promotion of liver tumorigenesis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

A previous study reported that exposure to tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) could promote the progression of hepatocellular carcinoma (HCC) in female HCC model zebrafish. Due to the existence of gender disparity in the development of HCC between females and males, whether the promotion effect of TDCIPP still exists in male HCC model zebrafish remains unclear. In this study, Tg(fabp10:rtTA2s-M2; TRE2:EGFP-kras G12V ), referred as kras transgenic zebrafish which was shown to be an inducible liver tumor model, was applied as experimental model to assess the promotion potential of TDCIPP for HCC in males. In brief, kras males were exposed to 20 mg/L doxycycline (DOX), 0.3 mg/L TDCIPP and a binary mixture of 20 mg/L DOX with 0.3 mg/L TDCIPP, and after exposure liver size, histopathology and transcriptional profiles of liver from these treatments were examined. With the involvement of TDCIPP, the liver size was significantly increased and the lesion of hepatocyte became more aggressive. Furthermore, expressions of genes involved in DNA replication and inflammatory response were simultaneously up-regulated in the treatment of TDCIPP compared with the solvent control and in the treatment of the binary mixture of the two chemicals compared to the single DOX treatment. Overall, our results suggested that TDCIPP had promotion effect on the progression of liver tumor in kras males., Competing Interests: Declaration of competing interest All of the authors declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Polyhalogenated carbazoles (PHCZs), which have the similar structure of dioxin, have been reported ubiquitous in the environments and drawn wide concerns. However, their potential ecological and health risks are still poorly understood. Here, wildtype zebrafish embryos were used to evaluate the environmental risks of 2,7-dibromocarbazole (2,7-DBCZ), 3,6-dibromocarbazole (3,6-DBCZ), and 3,6-dichlorocarbazole (3,6-DCCZ). 2,7-DBCZ was the most toxic compound with the 96-h LC 50 value of 581.8 ± 29.3 μg·L -1 and the EC 50 value of 201.5 ± 6.5 μg·L -1 for pericardial edema. The teratogenic effects of 2,7-DBCZ were tested using transgenic zebrafish larvae. The transcriptomic analysis revealed that 90 genes in zebrafish expressed differently after exposure to 2,7-DBCZ, and many pathways were related to aryl hydrocarbon receptor (AhR) activation. The qRT-PCR also showed that expression levels of AhR1 and CYP1 A in zebrafish were significantly up-regulated after exposure to 2,7-DBCZ. In conclusion, 2,7-DBCZ exhibited more potent toxicity and cardiac teratogenic effects, and presented developmental toxicity partially consistent with AhR activation. Our results will be of great help to the risk assessment and regulation-making of PHCZs. Meanwhile, further studies should be promoted to illustrate the potential mechanism between PHCZs and AhR in the near future., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Five picrotoxane sesquiterpenoids belonging to the unusual dendrobine-type (1 and 4) and the picrotoxinin-type (2, 3, and 5) were isolated from the stems of Dendrobium nobile Lindl. Their structures were established by spectroscopic analyses and physical properties. Compound 1 was a new dendrobine analogue. Although the planar structure of 2 and 3 had been reported, their absolute configurations were first determined by single-crystal X-ray diffraction and circular dichroism. Compound 2 exhibited angiogenesis effect against sunitinib-induced damage on intersegmental blood vessels in Tg (flk1: EGFP) and Tg (fli1: nEGFP) transgenic zebrafish at concentrations of 3.13, 6.25, 12.50, and 25.00μM., (Copyright © 2017. Published by Elsevier B.V.)

Temporal and spatial control of gene expression is important for studying the molecular and cellular mechanisms of development, physiology, and disease. We used the doxycycline (Dox)-inducible, Tet-On system to develop transgenic zebrafish for inducible, cell specific control of gene expression in the ultraviolet (UV) cone photoreceptors. Two constructs containing the reverse tetracycline-controlled transcriptional transactivator (rtTA) gene driven by the UV opsin-specific promoter (opn1sw1) were used to generate stable transgenic zebrafish lines using the Tol2-based transgenesis method. One construct included a self-reporting GFP (opn1sw1:rtTA, TRE:GFP) and the other incorporated an epitope tag on the rtTA protein (opn1sw1:rtTA(flag)). UV cone-specific expression of TRE-controlled transgenes was induced by Dox treatment in larvae and adults. Induction of gene expression was observed in 96% of all larval UV cones within 16 h of Dox treatment. UV cone-specific expression of two genes from a bidirectional TRE construct injected into one-cell Tg(opn1sw1:rtTA(flag)) embryos were also induced by Dox treatment. In addition, UV cone-specific expression of Crb2a(IntraWT) was induced by Dox treatment in progeny from crosses of the TRE-response transgenic line, Tg(TRE:HA-Crb2a(IntraWT)), to the Tg(opn1sw1:rtTA, TRE:GFP) line and the Tg(opn1sw1:rtTA(flag)) line. These lines can be used in addition to the inducible, rod-specific gene expression system from the Tet-On Toolkit to elucidate the photoreceptor-specific effects of genes of interest in photoreceptor cell biology and retinal disease., (Copyright © 2014 Elsevier B.V. All rights reserved.)