BACKGROUNDRetinoic acid (RA) is a potent signaling molecule that plays pleiotropic roles in patterning, morphogenesis, and organogenesis during embryonic development. The synthesis from retinol (vitamin A) to retinoic acid requires two sequential oxidative steps. The first step involves the oxidation of retinol to retinal through the action of retinol dehydrogenases. Retinol dehydrogenases1l (RDH1l) is a novel zebrafish retinol dehydrogenase. Herein we investigated the role of zebrafish RDH1l in heart development and cardiac performance in detail.

METHODSRDH1l specific morpholino was used to reduce the function of RDH1l in zebrafish. The gene expressions were observed by using whole mount in situ hybridization. Heart rates were observed and recorded under the microscope from 24 to 72 hours post fertilization (hpf). The cardiac performance was analyzed by measuring ventricular shortening fraction (VSF).

RESULTSThe knock-down of RDH1l led to abnormal neural crest cells migration and reduced numbers of neural crest cells in RDH1l morphant embryos. The reduced numbers of cardiac neural crest cells also can be seen in RDH1l morphant embryos. Furthermore, the morpholino-mediated knock-down of RDH1l resulted in the abnormal heart loop. The left-right determining genes expression pattern was altered in RDH1l morphant embryos. The impaired cardiac performance was observed in RDH1l morphant embryos. Taken together, these data demonstrate that RDH1l is essential for the heart development and cardiac performance in zebrafish.

CONCLUSIONSRDH1l plays a important role in the neural crest cells development, and then ultimately affects the heart loop and cardiac performance. These results show for the first time that an enzyme involved in the retinol to retinaldehyde conversion participate in the heart development and cardiac performance in zebrafish.

Alcohol Oxidoreductases ; genetics ; metabolism ; Animals ; Animals, Genetically Modified ; Heart ; embryology ; Zebrafish ; Zebrafish Proteins ; genetics ; metabolism

Animals ; Embryo, Nonmammalian ; drug effects ; metabolism ; Ethanol ; toxicity ; RNA, Messenger ; metabolism ; Resveratrol ; pharmacology ; Zebrafish ; Zebrafish Proteins ; metabolism

Animals ; Keratolytic Agents ; pharmacology ; Neural Crest ; embryology ; metabolism ; Signal Transduction ; drug effects ; Tretinoin ; pharmacology ; Zebrafish ; Zebrafish Proteins ; genetics ; metabolism

In order to develop a transgenic zebrafish line with green fluorescent protein (enhanced green fluorescent protein, EGFP) expressed specifically in muscle and heart, the recombinant expression vector constructed using the zebrafish ttn.2 gene promoter fragment and EGFP gene coding sequence and the capped mRNA of Tol2 transposase were co-injected into the zebrafish 1-cell stage embryos. The stable genetic Tg (ttn.2: EGFP) transgenic zebrafish line was successfully developed by fluorescence detection, followed by genetic hybridization screening and molecular identification. Fluorescence signals and whole-mount in situ hybridization showed that EGFP expression was located in muscle and heart, the specificity of which was consistent with the expression of ttn.2 mRNA. Inverse PCR showed that EGFP was integrated into chromosomes 4 and 11 of zebrafish in No. 33 transgenic line, while integrated into chromosome 1 in No. 34 transgenic line. The successful construction of this fluorescent transgenic zebrafish line, Tg (ttn.2: EGFP), laid a foundation for the research of muscle and heart development and related diseases. In addition, the transgenic zebrafish lines with strong green fluorescence can also be used as a new ornamental fish.
Animals ; Zebrafish/genetics* ; Animals, Genetically Modified/genetics* ; Green Fluorescent Proteins/metabolism* ; Zebrafish Proteins/genetics* ; Promoter Regions, Genetic

Motor neurons are an important type of neurons that control movement. The transgenic fluorescent protein (FP)-labeled motor neurons of zebrafish line is disadvantageous for studying the morphogenesis of motor neurons. For example, the individual motor neuron is indistinguishable in this transgenic line due to the high density of the motor neurons and the interlaced synapses. In order to optimize the in vivo imaging methods for the analysis of motor neurons, the present study was aimed to establish a microtubule-fluorescent fusion protein mosaic system that can label motor neurons in zebrafish. Firstly, the promotor of mnx1, which was highly expressed in the spinal cord motor neurons, was subcloned into pDestTol2pA2 construct combined with the GFP-α-Tubulin fusion protein sequence by Gateway cloning technique. Then the recombinant constructs were co-injected with transposase mRNA into the 4-8 cell zebrafish embryos. Confocal imaging analysis was performed at 72 hours post fertilization (hpf). The results showed that the GFP fusion protein was expressed in three different types of motor neurons, and individual motor neurons were mosaically labeled. Further, the present study analyzed the correlation between the injection dose and the number and distribution of the mosaically labeled neurons. Fifteen nanograms of the recombinant constructs were suggested as an appropriate injection dose. Also, the defects of the motor neuron caused by the down-regulation of insm1a and kif15 were verified with this system. These results indicate that our novel microtubule-fluorescent fusion protein mosaic system can efficiently label motor neurons in zebrafish, which provides a more effective model for exploring the development and morphogenesis of motor neurons. It may also help to decipher the mechanisms underlying motor neuron disease and can be potentially utilized in drug screening.
Animals ; Animals, Genetically Modified ; Green Fluorescent Proteins/pharmacology* ; Microtubules/metabolism* ; Motor Neurons ; Zebrafish/genetics* ; Zebrafish Proteins/genetics*

Objective: To investigate the toxicity of tris (2-chloropropyl) phosphate (TCIPP) and tributyl phosphate (TnBP) on the growth and development of zebrafish embryos, as well as to explore the underlying mechanisms at the transcriptional level. Methods: With zebrafish as a model, two hpf zebrafish embryos were exposed to TCIPP and TnBP (0.1, 1, 10, 100, 500, and 1 000 μmol/L) using the semi-static method, and their rates of lethality and hatchability were determined. The transcriptome changes of 120 hpf juvenile zebrafish exposed to environmentally relevant concentrations of 0.1 and 1 μmol/L were measured. Results: The 50% lethal concentrations (LC₅₀) of TCIPP and TnBP for zebrafish embryos were 155.30 and 27.62 μmol/L (96 hpf), 156.5 and 26.05 μmol/L (120 hpf), respectively. The 72 hpf hatching rates of TCIPP (100 μmol/L) and TnBP (10 μmol/L) were (23.33±7.72)% and (91.67±2.97)%, which were significantly decreased compared with the control group (P<0.05). Transcriptome analysis showed that TnBP had more differential genes (DEGs) than TCIPP, with a dose-response relationship. These DEGs were enriched in 32 pathways in total, including those involved in oxidative stress, energy metabolism, lipid metabolism, and nuclear receptor-related pathways, using the IPA pathway analysis. Among them, three enriched pathways overlapped between TCIPP and TnBP, including TR/RXR activation and CAR/RXR activation. Additionally, DEGs were also mapped onto pathways of LXR/RXR activation and oxidative stress for TnBP exposure only. Conclusion: Both TCIPP and TnBP have growth and developmental toxicities in zebrafish embryos, with distinct biomolecular mechanisms, and TnBP has a stronger effect than TCIPP.
Animals ; Zebrafish/metabolism* ; Embryo, Nonmammalian/metabolism* ; Transcriptome ; Oxidative Stress ; Water Pollutants, Chemical/metabolism*

OBJECTIVETo investigate the effect of lead exposure on the gene expression of fibroblast growth factor 3 (Fgf3) in zebrafish embryonic development and the mechanism of lead-induced embryonic developmental toxicity.

METHODSThe embryos of zebrafish (wild types A and B) were exposed to lead acetate (PbAc) at the doses of 0, 0.1, 0.5, 2.5, and 12.5 µmol/L separately. Total RNA was extracted from each treatment group of zebrafish embryos at 8, 12, 16, 24, 36, 48, and 72 hours post fertilization (hpf). The total mRNA expression of Fgf3 was measured by real-time quantitative PCR. The spatial expression of Fgf3 in zebrafish embryos was determined by whole-mount in situ hybridization using synthesized Fgf3 RNA probe.

RESULTSThe mRNA expression of Fgf3 in each group peaked at 12 hpf (P < 0.01). With the increase in PbAc concentration, the mRNA expression of Fgf3 rose. Compared with the mRNA expression level of Fgf3 in the control group, the relative mRNA expression levels of Fgf3 in the 0.1, 0.5, 2.5, and 12.5 µmol/L PbAc exposure groups were 1.02 ± 0.24, 1.05 ± 0.26, 1.22 ± 0.46, and 1.25 ± 0.38, respectively, and the 2.5 and 12.5 µmol/L PbAc exposure groups showed significantly higher Fgf3 expression than the control group (P < 0.05). The whole-mount in situ hybridization results showed that Fgf3 expression occurred mainly in the head and tail in the early stage of embryonic development and in the midbrain, fin bud, and pharyngeal arch in the middle/late stage of embryonic development; there were the most significant regions and intensities of positive hybridization signals at 12 hpf; but no significant differences were found between the control group and exposure groups in the location and intensity of Fgf3 expression

CONCLUSIONLead exposure can result in the upregulation of Fgf3 expression in zebrafish embryonic development, which might contribute to lead-induced embryonic developmental toxicity.

Animals ; Embryonic Development ; drug effects ; Fibroblast Growth Factor 3 ; genetics ; metabolism ; Gene Expression ; Organometallic Compounds ; adverse effects ; Signal Transduction ; Zebrafish ; embryology ; genetics ; metabolism ; Zebrafish Proteins ; genetics ; metabolism

Mechanistic target of rapamycin (mTOR) signaling governs important physiological and pathological processes key to cellular life. Loss of mTOR negative regulators and subsequent over-activation of mTOR signaling are major causes underlying epileptic encephalopathy. Our previous studies showed that UBTOR/KIAA1024/MINAR1 acts as a negative regulator of mTOR signaling, but whether UBTOR plays a role in neurological diseases remains largely unknown. We therefore examined a zebrafish model and found that ubtor disruption caused increased spontaneous embryonic movement and neuronal activity in spinal interneurons, as well as the expected hyperactivation of mTOR signaling in early zebrafish embryos. In addition, mutant ubtor larvae showed increased sensitivity to the convulsant pentylenetetrazol, and both the motor activity and the neuronal activity were up-regulated. These phenotypic abnormalities in zebrafish embryos and larvae were rescued by treatment with the mTORC1 inhibitor rapamycin. Taken together, our findings show that ubtor regulates motor hyperactivity and epilepsy-like behaviors by elevating neuronal activity and activating mTOR signaling.
Animals ; Hyperkinesis/genetics* ; Mutation/genetics* ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism* ; Zebrafish/metabolism*

OBJECTIVETo investigate the expression pattern of hoxd3 gene during early embryogenesis and angiogenesis of wild-type zebrafish.

METHODSTotal RNA was extracted from embryos of zebrafish in different development stages by trizol. The cDNA of hoxd3 gene was amplified by RT-PCR. The RT-PCR product was ligated to pCS(2+) vector by T4 DNA ligatase polymerase and sequenced. T3 RNA polymerase in vitro transcription system was used to obtain the probe of digoxin-labeled anti-sense mRNA of hoxd3 gene. The expression pattern of hoxd3 was detected by whole embryo in situ hybridization (WISH) with anti-sense mRNA probe.

RESULTSpCS(2+)-hoxd3 plasmid was successfully constructed, which was used to prepare anti-sense mRNA probe of hoxd3 in vitro. Expression pattern of hoxd3 gene was detected by WISH during zebrafish early embryogenesis and angiogenesis. It was observed that hoxd3 mRNA was expressed at the junction region of midbrain and hindbrain in wild-type zebrafish in embryos at 24 ≊72h postfertilization(hpf).

CONCLUSIONhoxd3 gene is mainly expressed in nervous system of wide-type zebrafish embryos.

Animals ; Cloning, Molecular ; Gene Expression Regulation, Developmental ; Genetic Vectors ; Homeodomain Proteins ; genetics ; metabolism ; In Situ Hybridization ; Plasmids ; genetics ; RNA, Messenger ; genetics ; Transfection ; Zebrafish ; embryology ; genetics ; Zebrafish Proteins ; genetics ; metabolism

Objective: To detect the therapeutic efficacy of FGF21 analogues on the zebrafish model of non-alcoholic fatty liver disease. Methods: A zebrafish model of non-alcoholic fatty liver disease was established by providing the normal diet fed to wild-type zebrafish three times daily. PF-05231023 was administered exogenously at a final concentration of 0.5 μmol/L. Body length, body weight, triglycerides, and other indexes were measured after 20 days. Pathological changes were evaluated in liver tissue sections by HE staining. Quantitative PCR was used to identify expressional changes in genes related to lipid metabolism, endoplasmic reticulum stress, and inflammation. Results: QPCR and immunofluorescence staining results showed that FGF21 was highly expressed in the zebrafish model group. The addition of the FGF21 analogue PF-05231023 significantly reduced the body length and body weight (P < 0.01), and the triglyceride content (P < 0.05) in the zebrafish model group. The liver HE staining results showed that PF-05231023 had alleviated the large and tiny bullae fat, lesions, and others in the zebrafish model group. The quantitative PCR results demonstrated that PF-05231023 reduced the expression of lipogenic factors (P < 0.01), inflammatory-related factors (P < 0.001), and genes related to endoplasmic reticulum stress (P < 0.05), but raised lipid-oxidation-related factors (P < 0.05) in the zebrafish model group. The addition of PF-05231023 reduced oleic acid-induced lipid and triglyceride levels in HepG2 cells. Conclusion: FGF21 analogue addition can improve indexes in the zebrafish disease model of non-alcoholic fatty liver disease.
Animals ; Body Weight ; Diet, High-Fat ; Lipids ; Liver/pathology* ; Non-alcoholic Fatty Liver Disease/pathology* ; Triglycerides/metabolism* ; Zebrafish/metabolism* ; Zebrafish Proteins