Establishment of a standardized method for evaluating zebrafish innate color preference research
Although the zebrafish is a lower vertebrate, it has evolved four-color vision and can distinguish between ultraviolet and visible wavelengths. In view of the large discrepancies in the results of papers on the innate color preference of zebrafish reported, the light source, color intensity and other parameters (such as age, gender, and container size ratio) and the operation of different laboratories have produced inconsistent results of the color preference tests. Therefore, the development of standardization of equipment and methods for zebrafish's color preference and behavior-related stimulus response has considerable importance. Associate Professor Kelvin H.-C. Chen of Pingtung University, Associate Professor Jung-Ren Chen of I-Shou University, and Professor Chung-Der Hsiao of Chung Yuan Christian University have standardized the zebrafish color preference test for measuring light source position, light intensity, gender, age, animal size and spatial ratio. The results of the study showed that under standardized experimental conditions, zebrafish's color preference order (red>blue>green>yellow) was not affected by light position, spatial ratio, gender, strain, and age. In addition, this standardized method can also be used to test the order and index difference of color preference of other fish species (such as the tiger barb and the glass catfish). This study has been published in the Q1 index “Biomedicines” journal in the field of biomedicine in 2020, Associate Professor Kelvin H.-C. Chen, the moderator of the inter-university and cross-field integration team, said that this study has successfully established a standardized method for zebrafish color preference screening and has become an important tool for high-throughput neurotoxicology and pharmacology applications.
Which zebrafish strain is more suitable for behavioral research? Comprehensive comparison by phenotypic methods
In recent years, there has been a significant increase in research publications using zebrafish (Danio rerio) as experimental animals in the field of behavioral neuroscience research. Zebrafish have the advantages of low feeding costs, easy maintenance, small size, and transparency during embryonic periods. Wild-type (WT) zebrafish are often used in behavioral-related experiments, however, such as AB, Tübingen long fin (TL) and Wild Indian Karyotype (WIK) strains are also summarized in the definition of wild type. These different zebrafish strains are not only different in morphology, but also in physiological and genetic characteristics at the juvenile and adult stages, may contribute to the behavioral variations. Compared to rodents with extensive understanding of the characteristics of different outbred and inbred strains, only a few studies have focused on the behavioral difference between different zebrafish strains. In order to clearly establish the behavioral characteristics of different zebrafish strains, the Department head Jong-Chin Huang of the Department of Applied Chemistry, National Pingtung University and Professor Chung-Der Hsiao of Department of Bioscience Technology, Chung Yuan Christian University lead a research team to conduct a series of behavioral experimental tests on six different strains of zebrafish (thanks to the Academia Sinica Zebrafish Center for providing experimental fish species), and observed behavioral performance for each tested strain. At the same time, mathematical principal component analysis and cluster analysis were also conducted to reduce dimensional complexity. Results show the tested strains can be divided into two major clusters. Among them, AB and TL strains are classified as one group, while other strains (such as PET, absolute, golden and WIK) are grouped as another cluster. In addition, after calculating the behavioral coefficient of variation of each strain, it was interesting to find AB and TL strains have a relative lower coefficient of variation, highlighting that these two strains have relatively high repeatability and robustness in the behavioral test. These experimental results can help researchers determine which strain is more suitable for future behavioral tests, and help establish a basic information database between several common zebrafish strains and will help scientists on choosing appropriate strains and appropriate detection methods to conduct behavioral experiments. The results of this research have recently been successfully published in the Q1 Biology field research journal "Biology".
Interspecies Behavioral Variability of Medaka Fish Assessed by Comparative Phenomics
In recent years, based on the experimental animal 3R principle of "replacement (replacement), reduction (reduction) and optimization (optimization)" and many experimental advantages, model models have become widely used in toxicology and pharmacology. Compared with traditional animals such as rodents, the behavioral characteristics of fish, which include the rapid development process, and the low cost, short time and low manpower consumption, gradually promoting it becoming a good animal model for experimental conducting. The medaka fish is a small freshwater bony fish distributed in East Asia. It is often found in rice fields, so it is commonly known as rice field fish. One of the most popular laboratory animal options, especially the Japanese medaka (Osryzias latipes), has been studied for basic animal biology and behavioral research biological models. In addition, the use of medaka as a model organism has made considerable contributions to the knowledge of various research fields such as genetics and toxicology. Compared with another fish model organism, medaka has received relatively little attention in behavioral research, especially in terms of interspecies behavioral diversity. In order to provide more information about the behavior of medaka fish and verify the behavioral differences between different medaka fish, the research team compared the behaviors of four medaka fish, Oryzias latipes, Oryzias dancena, Oryzias woworae, and Oryzias sinensis. Behavioral and biochemical analysis of the biomarkers expressed in the brain of each medaka were also conducted. The research team found that medaka species clearly show different behaviors, which may be related to the different basic levels of biomarkers in the brain of medaka. The integration of various behavioral research results for phenotypic cluster analysis, and the use of sequence alignment of the mitochondrial nd2 gene for cluster analysis of genomic relationships also showed the significant differences between these medaka fish. This research was recently published in the Q1 ranking interdisciplinary research journal of "International Journal of Molecular Sciences". These results help to improve the medaka phenotype database and provide basic research background knowledge for future medaka phenotypes and toxicology-related experiments.
Residues of emerging environmental pollutants (psychotherapy drugs) in the environment can induce abnormal behavior in fish
Donepezil (DPZ) is an acetylcholine esterase inhibitor, which is clinically used in the treatment of mild cognitive impairment. DPZ has been found to have side effects such as abnormal heart rhythms, insomnia, vomiting and muscle cramps. Associate Professor Kelvin Chen of National Pingtung University, Professor Chung-Der Hsiao of Chung Yuan Christian University and the team of University of Santo Tomas in the Philippines, chronically exposed normal zebrafish to 1 ppm and 2.5 ppm DPZ. Experimental results show that DPZ can significantly elevate fish aggressiveness and anxiolytic behaviors, and found that these changes are related to increased oxytocin and decreased brain cortisol levels. In addition, the research team observed a significant increase in the content of reactive oxygen species (ROS) and malondialdehyde (MDA) in the muscle tissue, which in turn caused the zebrafish's exercise activity to be greatly reduced and abnormal exercise patterns. This research has recently been published in "Biomolecules", an index journal in the field of chemical biology. Associate Professor Kelvin Chen of the Department of Applied Chemistry, National Pingtung University, the leader of the inter-university and cross-field integration team, said that this study proved for the first time that chronic exposure to environmental level of DPZ, can seriously affect the normal behavior of fish. In the future, zebrafish and other more aquatic animal models should be studied for chronic exposure to psychotropic drugs to understand the ecological impact of emerging environmental pollutants.
The New Discovery of DNA Methyltransferase on Controlling Zebrafish Behaviors
Epigenesis is a mechanism that able to regulate gene expression without changing the DNA sequence. The DNA methylation is closely related to methyltransferase (DNMT) activity. Recent studies have shown that the degree of gene methylation is closely related to mental illness caused by stress. In particular, the amount of gene methylation and the differential methylation of non-coding regulatory regions of genes, is greatly related to brain pressure and related behavioral regulation. An inter-university and cross-disciplinary research team composed of Associate Professor Yu-Heng Lai from the Department of Chemistry of Chinese Cultural University, Professor Chung-Der Hsiao from the Department of Biotechnology Technology of Chung Yuan Christian University, and Director Ken-Hong Lim from the Department of Hematology and Oncology of Mackay Memorial Hospital utilized Behavioral, biochemistry and genome-wide methylation gene profiling approach to discover the new function of DNA methyltransferase DNMT3A gene function in zebrafish. It was found that when the zebrafish methyltransferase DNMT3A is missing, it will cause the zebrafish’s motor activity to be unstable, anxiety increased, social behavior abnormal and short-term memory decline. The most importance of this study is to use gene knock-out technology to confirm that the two duplicated genes of dnmt3aa and dnmt3ab play important but non-overlapping regulatory functions in behavior-related regulation. At the same time, whole-genome methylation sequencing is used to analyze the methylation gene profile, which provides a possible molecular mechanism for the abnormal behavior regulation after the inactivation of the methyltransferase DNMT3A. The results have been successfully published in the "Genes" journal recently.
Development of a modified T-maze to evaluate short-term memory loss after chronic exposure to heavy metals in zebrafish
A T-maze test is an experimental approach used to conduct congenital research. However, the food reward-based protocol for the T-maze test in fish has low efficiency and long training period drawbacks. An inter-university research team composed of Professor Jong-Chin Huang, the head of the Department of Applied Chemistry of National Pingtung University and Professor Chung-Der Hsiao from the Department of Biotechnology of Chung Yuan Christian University has developed an improved electric shock punishment T-maze that able to evaluate the learning and memory capacity of adult zebrafish within three days. In contrast, traditional food reward-based T-maze test often takes more than a week to complete the experiment. This innovative methodology has been published in the " International Journal of Molecular Sciences ". The research team further discovered that chronic exposure to PbCl2 at a low concentration can significantly induce a sharp reduction of aggressiveness and short-term memory and elevate the level of stress and anxiety. The biochemical assays showed elevated cortisol and reduced serotonin and melatonin levels in the brain, thus, altering the behavior of the PbCl2-exposed zebrafish. The highlight of this study is to determine the potential ecotoxicity of lead exposure to aquatic organisms through a variety of behavioral and biochemical tests. More importantly, even at a low concentration, long-term exposure to low dose lead could impair the memory and cause a decrease in the aggressiveness and exploratory activities of zebrafish, which may reduce their survival fitness. This research has been published in the " International Journal of Molecular Sciences ". Professor Jong-Chin Huang said that the ceramic tableware commonly used in daily life contains lead in the colored glaze, and many inferior toys have too high lead content. The use of inferior colored ceramic tableware and inferior toys should be avoided as possible as we can. If exposed to lead carelessly for a long time, it may cause reduction of short-term memory. In addition, some traditional drugs of unknown origin often contain excessive lead and should be paid more attention.
Carbon coating of magnetite particles can effectively improve its biocompatibility
The unique physical properties of magnetic nanoparticles (such as magnetite NPs) have shown great potential in biomedical engineering applications, such as bio separation, contrast enhancement for MRI, magnetic hyperthermia, and intracellular drug delivery. However, the uncoated magnetite NPs are unstable and might lead to easy aggregation and oxidation. Modifying the surface to increase their stability and biocompatibility has been one of the research focuses for scientists. Carbon is an excellent material with good biocompatibility and stability throughout the pH range. If a carbon shell coating may be formed, we hypothesize that the biocompatibility, stability, and surface functionality of magnetite NPs should be elevated. An inter-university and inter-departmental research team leading by Professor Jiann-Shing Lee from the Department of Applied Physics and Professor Chung-Der Hsiao from the Department of Bioscience Technology, Chung Yuan Christian University, and Associate Professor Tzong-Rong Ger from the Department of Biomedical Engineering, Chung Yuan Christian University, has utilized zebrafish to investigate the behavioral and biochemical alterations after waterborne exposed to magnetic nanoparticles. The research team used starch as a green and environmental- friendly carbon source and utilized the hydrothermal method to perform the carbon coating for magnetite NPs. The zebrafish were then waterborne exposed to NPs for chronic toxicity evaluation. The study’s major achievement showing the biocompatibility of magnetite NPs, can be greatly elevated by carbon coating. This conclusion is supported by evidence showing no apparent behavioral and biochemical alterations after two-week carbon-coated magnetite NPs exposure. In contrast, the uncoated magnetite NPs have a relatively high toxicity. The research team believes that the high biocompatibility may be associated with carbon coating in preventing the corrosion and oxidation of the magnetite core. This in vivo experimental evidence collected from zebrafish fully supports the high biocompatibility and high biosafety of carbon-coated magnetite NPs and enhances future application feasibility to humans. These interesting research results have recently been published in "Molecules", an indexed journal in chemical biology. At the same time, the author was also invited by "Molecules" to write a comprehensive review article to introduce the application and potential toxicity of iron oxide magnetic nanoparticles.
Little zebrafish and daphnia make great achievements: research team establishes new method for high-throughput screening of pesticide toxicity
Pesticide toxicity screening is an extremely important issues in the field of food safety. After experiencing many food safety crisis cases, Taiwanese society has paid much attention to and discussed food safety issues. According to the screening and assessment of the in vivo acute toxicity of pesticides, there is no fast and high-throughput model available so far. An inter-school research team organized by Professor Chung-Der Hsiao from Chung Yuan Christian University, Professor Hong-Thih Lai from National Chiayi University and Professor Kelvin Huang-Chou Chen from National Pingtung University compares the potential toxicity of pesticides by monitoring the variation of aquatic biological activity. In this research, zebrafish (vertebrate model) and daphnia (invertebrate model) were exposed to 12 different pesticides (include three fungicides and 9 insecticides) and then Zebrabox high-throughput facility was employed to measure biological movement distance, burst movement and rotation activities. Depends on the mathematical principal component analysis and cluster analysis, the data complexity can be reduced and receive the biological meaning. The experimental results show that zebrafish and daphnia will change the activity of the two animal models when exposed to pesticides. Interestingly, low-concentration pesticide exposure can cause hypoactivity of daphnia neonates but hyperactivity in zebrafish larvae. Professor Hong-Thih Lai mention that the most significant part of this study is to use both zebrafish and daphnia models in parallel to establish a high-throughput platform for pesticide toxicity assessment. It’s possible to conduct acute locomotor toxicity up to 96 animals triggered by pesticides exposure just in 1.5 hours. This research has successfully developed a high-throughput detection technology and potentially been widely used to screen and evaluate of the potential toxicity of pesticides and other emerging pollutants, and will be responsible for the health and safety of consumers. This research recently has been published in the high impact journal "Biomolecules" in the field of chemical biology.
The latest research shows that recombinant GHK peptide has high-potency on chelating heavy metal and reducing cardiotoxicity
In recent years, due to the increasing pollution of heavy metals in the environment, even the slow accumulation of low-dose heavy metals will pose a major threat and impact on human health. GHK (Glycyl-Histidyl-Lysine) tripeptide is a short peptide that naturally exists in the human body and is often used in cosmetic products to protect the skin and slow down the aging process. Due to the high price of the blue copper peptide on the market, the research team leaded by Professor Kelvin Huang-Chou Chen from National Pingtung University and Professor Chung-Der Hsiao from Chung Yuan Christian University attempted to use gene recombination, expression and purification technology to produce GHK tripeptide. First, GHK gene fragments were cloned into expressional plasmid with His-Tag and GST-Tag double tags at the N-terminus and TEV restriction site. Later, by using optimal E. coli fermentation conditions, bulky GHK blue copper peptide with high economic value was successfully produced. Subsequently, high-pressure liquid chromatography was used to isolate GHK peptide with high purity. Finally, UV-visible absorption spectroscopy and near-edge X-ray absorption spectroscopy were used to study the physical and chemical properties of the recombinant GHK blue copper peptide. Experimental evidence shows that each unit of GHK peptide can form a high-affinity GHK-Cu complex with divalent copper ions in a ratio of 1:1. The research team subsequently used zebrafish as an in vivo experimental model to validate the recombinant GHK blue copper peptide indeed can attenuate cardiotoxicity and arrhythmia caused by copper ion exposure. This research has recently been published in "Biomolecules", a high-impact journal in the field of chemical biology. Professor Kelvin Huang-Chou Chen from Department of Applied Chemistry, National Pingtung University, who is the leader of the inter-university and cross-field integration team, said that the importance of this research is to provide a method that able to effectively reduce the production cost of GHK blue copper peptides and provide direct and strong supporting evidence for the biological activity of GHK blue copper peptides on neutralizing the cardiotoxicity of heavy metals. In the meantime, due to the technology of the present invention has high market applicability, a number of biotechnology manufacturers have proceeded to contact the team for technology licensing negotiations.
Establish of zebrafish disease model carrying aging and behavioral abnormalities
Progeroid syndrome is a rare inherited disease. Patients suffering from the disease are characterized by developmental retardation, cutis laxa due to short of connective tissues, and accelerated aging. From previous studies we know that mutation in PYCR1 gene encoding proline synthetase causes progeroid syndrome in human. However, there was no optimal animal model to serve as a platform for mechanism study and therapeutic drug screening. The research teams of Professor Hsiao, Chung-Der in the Department of Bioscience Technology in CYCU and Dr. Lin, Dar-Shong in Mackay Memorial Hospital have been working on the research project related to progeroid syndrome for several years sponsored by MOST and MMH. Now we have successfully established the first zebrafish disease model simulating the human progeroid syndrome with deficiency of pycr1 gene. This fish model harboring pycr1 gene alteration, leading to insufficient expression and function of PYCR1 protein appears aging symptoms from the 4th month onward, including decreased fertility, reduced locomotion ability, aggression, passive avoidance and social interaction, as well as aberrant color discernibility and circadian rhythm. In addition, the body weight and length as well as survival rate in the model fish significantly decrease when compared with those of wild type fish. After biochemical analyses, we found the proline component, total antioxidant capacity, body energy and telomerase activity in the model fish decrease significantly when compared to those of wild type fish, suggesting the aging phenomenon be related to abnormality of mitochondrial functions. Dr. Lin indicates that establishment of the zebrafish progeroid model be beneficial to applications in academic and clinical research. In the future this model can serve to study the progeroid syndrome related mechanism and assist us in screening the therapeutic drugs (Cells, 2019)
Use zebrafish as a simple model to perform drug screening and toxicity test
Take the advantages of body transparency, large embryo production, easy maintenance and able to conduct high-throughput screening properties, we have established several easy methodologies to screen drugs on enhancing osteogenesis (Molecules, 2017, cooperated with I-Shou University) and promoting wound healing by using small molecular weight collagen extracted from tilapia skin (BBRC, 2018, cooperated with Chinese Culture University). In addition, we have also set up some easy protocols to evaluate the potential nephrotoxicity (Hum Exp Toxicol, 2015), hepatotoxicity (J Pharmacol Toxicol Methods, 2016) and cardiotoxicity (Inventions, 2018) (Cells, 2019) by using zebrafish embryos as a platform. For example, in the experiment to test how soon the small collagen molecules can promote the wound healing on fish skin, we injected the glacial acetic acid to induce wound on fish embryo skin and found that the zebrafish treated with small molecule collagen extracted from Tilapia showed a fast healing on the wound, decreased pigment accumulation, more cell proliferation, more new blood vessels formation, high clearance rate of dead cells and increased anti-inflammation reaction. These data indicated that the small molecule collagen extracted from Tilapia can promote the wound healing in zebrafish and also provided with the evidence at molecular and cellular levels to support the efficacy of small molecule collagen (BBRC, 2018).
Design simple and cost-effective devices for physiology and behavior studies in Zebrafish
In our lab, we have setup several simple and cost-effective devices to measure cardiac rhythm, blood flow, swimming activity and behavior in zebrafish. (1) For cardiac rhythm, we use conventional dissecting microscope mounted with CCD to capture digital videos. The video is analyzed by ImageJ software to obtain atrium and ventricle rhythm in wild-type zebrafish without fluorescent tag labeling or running scripts in software. (2) For blood flow velocity, we use inverted microscope mounted with high speed CCD to capture digital videos. The red blood cell movement is tracked by ImageJ software. This method can precisely measure blood flow rate in artery, vein and capillary. (3) For 2D/3D swimming analysis, we use single CCD to capture images and later use Matlab-based software to track swimming path. This setup can track multiple fish shoaling behavior in 2D/3D. (4) For swimming activity assay, we setup a simple swimming tunnel device to measure the swimming activity of adult zebrafish. By using this device, we can manipulate the water flow speed, water temperature or dissolved oxygen level and evaluate how zebrafish deal with those challenges. (5) For behavior assay, we set up several easy devices to measure active/passive avoidance, fear/anxiety, circadian rhythm and spatial memory behaviors. In conclusion, we have developed several simple and cost-effective devices to measure heartbeat, blood flow, swimming activity and behavior in zebrafish. We believe those settings will benefit zebrafish community in the fields of cardiovascular physiology, excise physiology and behavior studies. (Inventions 2018, 3:11 ; Inventions 2018, 3:21; Inventions 2018, 3:75; Biology Open 2019, In press)
Genome editing tools develop in Zebrafish
Following ZFN (zinc-finger nuclease) technology, TALEN (TALE nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats) are two newly developed technologies which provide with high efficiency of site specific modification of target genes. The most difficult point encountered by researchers when they are performing the knockout skills using the TALEN/CRISPR system is how to efficiently screen the wanted knockout genotypes. With regard to this point, we cooperated with Zgene Biotechnology companies through the collaboration projects between industry and university sponsored by MOST and developed the first set of CRISPR/Cas9 construction kit in Taiwan. We also set up a series of methods to fast screen the fish with wanted mutations, using the High Resolution Melting (HRM) and Next Generation Sequencing (NGS) to efficiently detect the genetic alterations such as insertions, deletions and point mutations, and saved a lot of money for Sanger Sequencing. Using the optimized genomic manipulation skills on zebrafish, we have successfully generated many transgenic and knockout zebrafish lines which appear the disease symptoms like premature ageing, immune-deficiency as well as obesity and might be used as tools to study human diseases.(IJMS, 2018, 19, 4038)(Cells 2019, 8, 453)
NGS analysis tools develop
In studies of animal/plant evolutions and population genetics, we often utilize many different molecular markers such as mitochondria DNA, chloroplast DNA, micro-satellite and nuclear DNA to perform the kinship/phylogeny analyses. We focused on developing methodology to fast, efficiently and economically grab the complete information. We performed low coverage sequencing for animals/plants using Illumina MiSeq/HiSeq NGS, followed by de novo assembling and genome mapping of these sequences. Thus far we have successfully completed the high throughput sequencing of mitochondria, chloroplasts, 18S-5.8S-28S and nuclear rDNA for dozens of high economical marine animals/plants and used these molecular markers in the evolutionary studies. We have successfully developed a simple analysis pipeline to grab large amount of molecular markers from the low coverage sequencing data of animals/plants to help identify the population genome and kinship/phylogeny. Taking the advantage of developing this technology, we have published dozens of scientific papers in recent two years with our cooperation teams including the Natural Science Museum, National Museum of Marine Biology and National Taiwan Ocean University. (IJMS, 2017, 18:2341)
Novel Zebrafish Skin Cancer Model
Aberration in DNA replication is a major cause to genome instability that is a hallmark of cancer cells. Cell division cycle 6 (Cdc6) and c-Myc have a critical role in the initiation of DNA replication. However, whether their interaction induces epithelial-mesenchymal transition (EMT) and promotes tumorigenesis in in vivo animal model remains unclear. Since using zebrafish as a cancer model has been restricted by the late onset of tumorigenesis and extreme difficulty in transformation on skin, we tried to establish a novel non-melanoma skin model in zebrafish to study their role in tumorigenesis. A stable transgenic zebrafish was created by using tol2 transposon, in which cdc6 and c-myc were co-overexpressed in epidermis driven by a skin-specific krt4 promoter. Intriguingly, co-overexpression of cdc6 and c-myc in transgenic zebrafish skin triggered tumor-like transformation, apoptosis attenuation, genomic instability, and EMT, hallmarks of malignant tumorigenesis. Our findings and other characteristics of zebrafish, including optical clarity and small molecule treatment, provide the future utility of this model for easy and non-invasive detection and for identification of new anti-cancer drug. (Oncotarget, 2014, 5:6300-6311)
Obesity is a complex, multifactorial disorder influenced by the interaction of genetic, epigenetic, and environmental factors. Obesity increases the risk of contracting many chronic diseases or metabolic syndrome. Researchers have established several mammalian models of obesity to study its underlying mechanism. However, a lower vertebrate model for conveniently performing drug screening against obesity remains elusive. The specific aim of this study was to create a zebrafish obesity model by over expressing the insulin signaling hub of the Akt1 gene. Skin oncogenic transformation screening shows that a stable zebrafish transgenic of Tg(krt4:Hsa.myrAkt1)cy18 displays severely obese phenotypes at the adult stage. In Tg(krt4:Hsa.myrAkt1)cy18, the expression of exogenous human constitutively active Akt1 (myrAkt1) can activate endogenous downstream targets of mTOR, GSK-3a/b, and 70S6K. During the embryonic to larval transitory phase, the specific over expression of myrAkt1 in skin can promote hypertrophic and hyperplastic growth. From 21 hour post-fertilization (hpf) onwards, myrAkt1 transgene was ectopically expressed in several mesenchymal derived tissues. This may be the result of the integration position effect. Tg(krt4:Hsa.myrAkt1)cy18 caused a rapid increase of body weight, hyperplastic growth of adipocytes, abnormal accumulation of fat tissues, and blood glucose intolerance at the adult stage. Real-time RT-PCR analysis showed the majority of key genes on regulating adipogenesis, adipocytokine, and inflammation are highly upregulated in Tg(krt4:Hsa.myrAkt1)cy18. In contrast, the myogenesis- and skeletogenesis-related gene transcripts are significantly downregulated in Tg(krt4:Hsa.myrAkt1)cy18 , suggesting that excess adipocyte differentiation occurs at the expense of other mesenchymal derived tissues. Collectively, the findings of this study provide direct evidence that Akt1 signaling plays an important role in balancing normal levels of fat tissue in vivo. The obese zebrafish examined in this study could be a new powerful model to screen novel drugs for the treatment of human obesity. (PLoS One. 2012. 7:e36474)
Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)cy17 (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR+ fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR+signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR+ fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR+ fluorescent signaling. The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo. (PLoS One. 2011. 6:e20654)