Zebrafish 동물모델에서 human HtrA2의 expression system 정립에 관한 연구

조성원,박효진,김구영,남민경,김호영,고인호,김철희,임향숙,Cho, Sung-Won,Park, Hyo-Jin,Kim, Goo-Young,Nam, Min-Kyung,Kim, Ho-Young,Ko, In-Ho,Kim, Cheol-Hee,Rhim, Hyang-Shuk 한국생명과학회 2006 생명과학회지 Vol.16 No.4

Mitochondrial serine protease로 알려진 human HtrA2 (hHtrA2)는 apoptosis 유도 과정에서 중요한 역할을 담당하고 있을 뿐만 아니라 hHtrA2가 motor neuron degeneration과 관련이 있다는 최근 연구 결과가 있으나, hHtrA2의 생리적 기능은 아직 명확하게 밝혀져 있지 않다. 이와 같이 생체내에서 필수적인 업무를 담당하는 hHtrA2의 기능을 심도 있게 연구하기 위해서는 적절한 동물모델 시스템이 필요하나 이에 대한 연구도 미흡한 실정이다. 따라서 본 연구에서는 hHtrA2의 기능 분석을 위한 기본적인 실험으로 zebrafish라는 동물모델을 선택하여 hHtrA2의 발현 시스템을 정립하였다. 먼저 zebrafish에 hHtrA2를 발현시키기 위하여 zebrafish에서 일반적으로 사용되는 발현 시스템인 pCS2+ vector에 hHtrA2와 GFP를 cloning하고 plasmid를 HEK293 cell에 transfection한 후, hHtrA2-GFP fusion 단백질의 발현을 immunoblot과 immunofluorescence staining assay로 확인한 바 약 64 kDa의 hHtrA2 단백질의 발현을 확인할 수 있었다. Zebrafish에서 hHtrA2-GFP fusion 단백질의 발현양상은 immunofluorescence microscope으로 확인하였다. hHtrA2-GFP DNA와 mRNA를 zebrafish embyro에 microinjection하여 두 가지 component의 발현을 비교 분석한 결과, DNA는 dot 형태로 mRNA는 몸 전체에 퍼져보이는 형태로 발현 양상의 차이는 있었으나 둘 다 zebrafish embryo에서 잘 발현되는 것을 알 수 있다. 다음 DNA를 주 component로 microinjection하여 zebrafish embryo에서 발현을 확인한 결과 hHtrA2는 72 hpf 까지 발현이 지속되는 것을 확인하였다. 본 연구에서 정립한 hHtrA2의 zebrafish 발현 조건은 앞으로 zebrafish에서 hHtrA2의 생리적 기능을 심도있고 정확하게 연구하는 데 있어 기본적인 자료로 활용 할 수 있을 것이다. HtrA2/Omi, a mitochondrial trypsin-like serine protease, is pivotal in regulating apoptotic cell death. Several lines of recent evidence suggest that HtrA2 is associated with the pathogenesis of neurodegenerative disorders; however, the physiological function of HtrA2 still remains elusive. For studying physiological function of HtrA2 in depth, it is necessary to develop a suitable expression system in the model animal. We therefore utilized the zebrafish as a model animal to establish expression of human HtrA2 (hHtrA2) in vivo. For expression of mature HtrA2 as GFP fusion in zebrafish embryos, the HtrA2 (WT) or (S306A) cDNAs with the C-terminal GFP tag were inserted into the pCS2+ plasmid. Expression patterns of HtrA2 in HEK293 cells were first monitored by immunofluorescence staining and immunoblot assays, showing approximately 64 kDa of the HtrA2-GFP fusion proteins. Subsequently, the hHtrA2 plasmid DNA or in vitro transcribed mRNA was microinjected into zebrafish embryos. The expression patterns of HtrA2 in Zebrafish embryos were monitored by GFP fluorescence in 24 hours-post-fertilization (hpf). Although expression patterns of HtrA2-GFP in developing embryos were different between the injected DNA and mRNA, both nucleic acids revealed good expression levels to further study the physiological role of HtrA2 in vivo. This study provides a suitable condition for expressing hHtrA2 in the zebrafish embryos as well as a method for generating useful system to investigate physiological properties of the specific human genes.

mRNA expression and metabolic regulation of <i>npy</i> and <i>agrp1/2</i> in the zebrafish brain

Jeong, Inyoung,Kim, Eunmi,Kim, Suhyun,Kim, Hwan-Ki,Lee, Dong-Won,Seong, Jae Young,Park, Hae-Chul Elsevier 2018 Neuroscience Letters Vol.668 No.-

<P><B>Abstract</B></P> <P>Neuropeptide Y (NPY) is an evolutionarily conserved neuropeptide implicated in feeding regulation in vertebrates. In mammals, NPY neurons coexpress Agouti-related protein (AgRP) in the arcuate nucleus of the hypothalamus, and NPY/AgRP neurons activate orexigenic signaling to increase food intake. Zebrafish express <I>npy</I> and two <I>agrp</I> genes, <I>agrp1</I> and <I>agrp2</I>, in the brain. Similar to mammals, NPY and AgRP1 act as orexigenic factors in zebrafish, but the exact distribution of NPY and AgRP neurons in the zebrafish brain and the regulation of these genes by metabolic states remain unclear. In this study, we analyzed the tissue distribution of <I>npy, agrp1</I>, and <I>agrp2</I> mRNA in the brain of larval and adult zebrafish. We detected the expression of <I>agrp1</I>, but not <I>npy</I>, in the hypothalamus of larval zebrafish. In the adult zebrafish brain, <I>npy</I> mRNA expression was detected in the dorsal area of the periventricular and lateral hypothalamus, but fasting induced upregulation of <I>npy</I> only in the lateral hypothalamus, indicating that NPY neurons in this area are implicated in feeding regulation. However, consistent with the findings in larval zebrafish, NPY neurons in the hypothalamus did not coexpress AgRP1. In contrast, fasting resulted in a dramatic increase in AgRP1 neurons in the ventral periventricular hypothalamus, which do not coexpress NPY. In addition, we found for the first time that <I>npy</I>- and <I>agrp1</I>-expressing neurons function as GABAergic inhibitory neurons in zebrafish, as they do in mammals. Taken together, our results show that the zebrafish NPY/AgRP system is involved in appetite regulation. In addition, our data suggest that although <I>npy</I> and <I>agrp1</I> were initially expressed in distinct neurons, evolution has resulted in their coexpression in mammalian hypothalamic neurons.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We analyzed <I>npy, agrp1,</I> and <I>agrp2</I> mRNA distribution in larval/adult zebrafish brains. </LI> <LI> NPY and AGRP1 neurons function as GABAergic inhibitory neurons in larval zebrafish. </LI> <LI> <I>Npy</I> and <I>agrp1/2</I> are expressed in distinct neurons in the hypothalamus. </LI> <LI> Metabolic state regulated NPY neurons in the lateral hypothalamus of adult zebrafish. </LI> <LI> Metabolic state regulated AgRP1 neurons in the adult zebrafish ventral hypothalamus. </LI> </UL> </P>

Generation of cd63-deficient zebrafish to analyze the role of cd63 in viral infection

Sumi Jung,Myoung-Jin Kim,Sarithaa Sellaththurai,Suna Kim,Seongdo Lee,Jehee Lee 제주대학교 해양과학연구소 2021 해양과환경연구소 연구논문집 Vol.45 No.-

The tetraspanin superfamily proteins are transmembrane proteins identified in a diverse range of eukaryotic organisms. Tetraspanins are involved in a variety of essential biological functions, including cell differentiation, adhesion, migration, signal transduction, intracellular trafficking, and immune responses. For an infection to occur, viruses must interact with various cell surface components, including receptors and signaling molecules. Tetraspanin CD63 is involved in the organization of the cell membrane and trafficking of cellular transmembrane proteins that interact with many viruses. In this study, the cd63 gene was characterized by studying its expression and function in a zebrafish model. The functional domains and structural features of Cd63, such as the Cys-Cys- Gly (CCG) motif in the large extracellular loop and cysteine residues, are conserved in zebrafish. We confirmed that cd63 was expressed in immune system organs, such as the axial vein and pronephric duct, during the embryonic development of zebrafish. To better understand the role of cd63 in the zebrafish immune system, we established cd63-deficient zebrafish lines using the clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 (CRISPR/Cas9) system. A 19 bp insertion mutation was generated in single guide RNA (sgRNA) target sequence of exon 3 of the cd63 gene, to create a pre-mature stop codon. We then analyzed the expression of cd63-related genes cxcr4a and cxcr4b in wild type (WT) and cd63-deficient zebrafish. We believe our study provides an important model that could be used to investigate the roles of cd63 in viral infection in vivo.

Palmitate induces nitric oxide production and inflammatory cytokine expression in zebrafish

Cha, Seon-Heui,Hwang, Yongha,Kim, Kil-Nam,Jun, Hee-Sook Elsevier 2018 FISH AND SHELLFISH IMMUNOLOGY Vol.79 No.-

<P><B>Abstract</B></P> <P>Inflammation markers in zebrafish embryos reflect a toxic response that is common to other animal models and humans. Free fatty acids (FFAs) are known to cause damage in various tissues by inducing inflammation. In this study, we investigated whether a FFA (palmitate) induces inflammation in zebrafish embryos. Nitrous oxide (NO) production and cyclooxygenase-2 (COX-2) mRNA expression were increased in palmitate-treated zebrafish embryos in a dose-dependent manner. mRNA expression of pro-inflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF- α), were also increased. Additionally, the mRNA expression of p65 nuclear factor-kB and I-kB-α were significantly increased after palmitate-treatment. Increased reactive oxygen species (ROS) expression was observed in palmitate-treated zebrafish embryos as well as pericardial edema. Additionally, mRNA expression of pro-inflammatory cytokines were increased in zebrafish liver and pancreas fed with palmitate-contained diet. Taken together, these results indicated that palmitate increases pro-inflammatory mediators in zebrafish embryos, suggesting that zebrafish could be an alternative animal model for inflammatory disease including diabetes.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Palmitate is a fatty acid, one of the major causing factor of diabetes. </LI> <LI> Palmitate induces inflammation, but it is not known in zebrafish. </LI> <LI> Zebrafish is useful as an alternative <I>in vivo</I> model for disease research. </LI> <LI> Palmitate induced inflammation in zebrafish. </LI> <LI> Zebrafish would be possible diabetic model to study palmitate-induced. </LI> </UL> </P>

Oxytetracycline의 단기 노출이 제브라피쉬의 행동 및 내분비 반응에 미치는 영향

고은성(Eun Seong Ko),이승헌(Seungheon Lee) 한국생명과학회 2015 생명과학회지 Vol.25 No.2

Zebrafish는 약리학 연구에 널리 사용되는 실험 동물 중 하나이다. Oxytetracycline은 넓은 항균 범위를 가지는 항생제로써 단백질 합성을 억제하여 정균 작용을 가진 항생제이다. 본 연구의 목적은 OTC에 노출이 되었을 때 zebrafish에게서 나타나는 행동변화와 내분비계의 변화를 관찰하였다. OTC (50, 100 or 200 mg/l)에 노출된 zebrafish의 행동변화를 확인하기 위해 novel tank test와 open field test를 진행하였다. 또한 내분비계의 변화를 확인하기 위하여 whole-body cortisol의 수치를 cortisol kit를 이용하여 측정하였다. Novel tank test 결과 상층부에서 머문 시간과 부동 시간에서 OTC를 약욕하였을 때 통계적으로 유의성이 있는 증가현상이 확인되었다. 반면에, 총 이동거리, 과행동 시간, 유영 속도 그리고 상하 변위에서 OTC를 약욕하였을 때 통계적으로 유의성 있는 감소현상이 확인되었다. Open field test 결과 부동시간에서 OTC를 약욕 하였을 때 통계적으로 유의성 있는 증가 현상이 확인 되었다. 그러나 OTC를 약욕 하였을 때 총 이동거리, 움직인 시간, 유영 속도에서 통계적으로 유의성이 있는 감소 현상이 확인 되었다. Whole-body cortisol에서는 zebrafish에게 약욕을 하였을 때 증가하는 경향이 관찰되었다. 이러한 결과를 통하여 OTC가 zebrafish에게 화학적 스트레스로 작용한 것으로 사료된다. Zebrafish (Danio rerio) has been more widely used to study pharmacology. Oxytectracycline (OTC) is a broad-spectrum antibiotic and works by interfering with the ability to produce essential proteins of bacteria. The aim of this study was to identify the effects of exposure to OTC on behavioral changes or endocrine response in zebrafish. The behavioral effects of exposure to OTC (50, 100 or 200 mg/l) were characterized in several novelty-based paradigms such as the novel tank or open field test in zebrafish. Moreover, to investigate effects of exposure to OTC on endocrine response, we measured whole-body cortisol level using cortisol ELISA kit. As results of novel tank test, duration in top and immobile duration were significantly increased by the exposure to OTC in a concentration-dependent manner (p<0.05). In addition, moving distance, highly mobile, velocity and zone transition were significantly decreased by the exposure to OTC in a concentration-dependent manner (p<0.05). As results of open field test, the exposure to OTC increased immobile duration significantly (p<0.05). However, moving distance, mobile duration and velocity were significantly decreased by the exposure to OTC in a concentration-dependent manner (p<0.05). Besides, the exposure to OTC elevated whole-body cortisol levels in zebrafish. These results suggest that the exposure to OTC may induce chemical stress in zebrafish.

Development of a Zebrafish Larvae Model for Diabetic Heart Failure With Reduced Ejection Fraction

Inho Kim,Seung Hyeok Seok,Hae-Young Lee 대한심장학회 2023 Korean Circulation Journal Vol.53 No.1

Background and Objectives: Diabetes mellitus (DM)-associated heart failure (HF) causes high morbidity and mortality. In this study, we established a zebrafish larvae model for in vivo research on diabetic HF. Methods: DM-like phenotypes were induced by treating zebrafish larvae with a combination of D-glucose (GLU) and streptozotocin (STZ). HF was induced by treatment with terfenadine (TER), a potassium channel blocker. Additionally, myocardial contractility, motility, and viability were evaluated. Results: The zebrafish larvae treated with a combination of GLU and STZ showed significantly higher whole-body glucose concentrations, lower insulin levels, and higher phosphoenolpyruvate carboxykinase levels, which are markers of abnormal glucose homeostasis, than the group treated with only GLU, with no effect on viability. When treated with TER, DM zebrafish showed significantly less myocardial fractional shortening and more irregular contractions than the non-DM zebrafish. Furthermore, in DM-HF with reduced ejection fraction (rEF) zebrafish, a significant increase in the levels of natriuretic peptide B, a HF biomarker, markedly reduced motility, and reduced survival rates were observed. Conclusions: We established a DM-HFrEF zebrafish model by sequentially treating zebrafish larvae with GLU, STZ, and TER. Our findings indicate the potential utility of the developed zebrafish larvae model not only in screening studies of new drug candidates for DM-HFrEF but also in mechanistic studies to understand the pathophysiology of DM-HFrEF.

Bisphenol A 가 zebrafish의 발달단계에 미치는 영향

여민경 한국환경과학회 2003 한국환경과학회지 Vol.12 No.6

The effects of bisphenol A on the catalase activities in the development stage of zebrafish were investigated. In this study, the catalase activities for zebrafish fries exposed to bisphenol A of 1 x 10^(-10) g/ℓ during 1 week, 2 week, and 4 week post-hatching were examined. Also, the changes of organs weight and the catalase activities for adult zebrafishes exposed to bisphenol A during 3 weeks were investigated. Catalase activities for zebrafish fries exposed to bisphenol A of 1 x 10^(-10) g/ℓ during 1 week post-hatching were significantly lower, compared to the control. Somewhat, for zebrafish fries exposed to bisphenol A during 4 week post-hatching, catalase activities were significantly increased. For adult zebrafishes, the effects of bisphenol A were higher for female than male. Specially, catalase activities were significantly increased in the ovary of zebrafishes exposed to bisphenol A during 3 weeks. The ovary weight were increased for zebrafishes exposed to bisphenol A during 3 weeks. Catalase activities were increased in the intestine of female exposed to bisphenol A during 3 weeks. Catalase activities were increased in testis exposed to bisphenol A during 3 weeks but there was no significance. In conclusion, the damages of an endocrine disrupter were higher in the earlier development stage compared with adult. The damages were higher for female exposed to an endocrine disrupter compared with male.

Pharmacological (ethanol) and mutation (<i>sam2</i> KO) induced impairment of novelty preference in zebrafish quantified using a new three-chamber social choice task

Ariyasiri, Krishan,Choi, Tae-Ik,Kim, Oc-Hee,Hong, Ted Inpyo,Gerlai, Robert,Kim, Cheol-Hee Elsevier 2019 Progress in neuro-psychopharmacology & biological Vol.88 No.-

<P><B>Abstract</B></P> <P>Social behavior is a fundamental aspect of our own species, a feature without which our society would not function. There are numerous human brain disorders associated with abnormal social behavior, among them are the autism spectrum disorders whose causal factors include a genetic component. Environmental factors, including drugs of abuse such as alcohol, also contribute to numerous abnormalities related to social behavior. Several such disorders have been modeled using laboratory animals. Perhaps one of the newest among them is the zebrafish. However, the paucity of standardized behavioral assays specifically developed for the zebrafish have hindered progress. Here, we present a newly developed zebrafish behavioral paradigm, the three-chamber social choice task. This task, which was adapted from a murine model, assesses sociality and social novelty preference in zebrafish in three phases: habituation, phase-I to evaluate sociality, and phase-II to quantify social novelty preference. Test fish are placed in the middle chamber, while conspecifics are introduced to the flanking chambers during phase-I and II. Both male and female zebrafish displayed sociality (preference for conspecifics) during phase-I and social novelty preference (preference for unfamiliar conspecifics) during phase-II. We found the paradigm to be able to detect both environmentally (alcohol) as well as genetically (targeted knock out of <I>sam2</I>) induced alterations of behavioral phenotypes. Although ethanol-treated fish displayed similar levels of sociality to those of control (not alcohol exposed) male and female zebrafish, they were found to exhibit significantly impaired social novelty preference, a finding compatible with altered motivational or perhaps mnemonic processes. Moreover, we found that knock out of <I>sam2</I>, previously shown to lead to emotional dysregulation, also disrupted social novelty preference, while leaving sociality relatively intact. We conclude that our novel behavioral paradigm is appropriate for the modeling and quantification of social behavior deficits in zebrafish.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A novel 3-chamber social choice task adopted from the mouse literature is developed for zebrafish. </LI> <LI> Preference for a conspecific, and choice between familiar and unfamiliar conspecifics are quantified. </LI> <LI> Male and female zebrafish displayed strong social behavior and social novelty preference. </LI> <LI> Ethanol treatment impaired social novelty preference but did not affect social behavior. </LI> <LI> Knock out of <I>sam2</I> gene, disrupted both social behavior and social novelty preference. </LI> </UL> </P>

Zebrafish needle EMG: a new tool for high-throughput drug screens

Cho, Sung-Joon,Nam, Tai-Seung,Byun, Donghak,Choi, Seok-Yong,Kim, Myeong-Kyu,Kim, Sohee American Physiological Society 2015 journal of neurophysiology Vol.114 No.3

<P>Zebrafish models have recently been highlighted as a valuable tool in studying the molecular basis of neuromuscular diseases and developing new pharmacological treatments. Needle electromyography (EMG) is needed not only for validating transgenic zebrafish models with muscular dystrophies (MD), but also for assessing the efficacy of therapeutics. However, performing needle EMG on larval zebrafish has not been feasible due to the lack of proper EMG sensors and systems for such small animals. We introduce a new type of EMG needle electrode to measure intramuscular activities of larval zebrafish, together with a method to hold the animal in position during EMG, without anesthetization. The silicon-based needle electrode was found to be sufficiently strong and sharp to penetrate the skin and muscles of zebrafish larvae, and its shape and performance did not change after multiple insertions. With the use of the proposed needle electrode and measurement system, EMG was successfully performed on zebrafish at 30 days postfertilization (dpf) and at 5 dpf. Burst patterns and spike morphology of the recorded EMG signals were analyzed. The measured single spikes were triphasic with an initial positive deflection, which is typical for motor unit action potentials, with durations of ∼10 ms, whereas the muscle activity was silent during the anesthetized condition. These findings confirmed the capability of this system of detecting EMG signals from very small animals such as 5 dpf zebrafish. The developed EMG sensor and system are expected to become a helpful tool in validating zebrafish MD models and further developing therapeutics.</P>

Increased cell proliferation and neural activity by physostigmine in the telencephalon of adult zebrafish

Lee, Yunkyoung,Lee, Bongkyu,Jeong, Sumin,Park, Ji-Won,Han, Inn-Oc,Lee, Chang-Joong Elsevier 2016 Neuroscience Letters Vol.629 No.-

<P><B>Abstract</B></P> <P>Physostigmine, an acetylcholinesterase inhibitor, is known to affect the brain function in various aspects. This study was conducted to test whether physostigmine affects cell proliferation in the telencephalon of zebrafish. BrdU-labeled cells was prominently observed in the ventral zone of the ventral telencephalon of zebrafish. The increased number of BrdU- and proliferating cell nuclear antigen-labeled cells were shown in zebrafish treated with 200μM physostigmine, which was inhibited by pretreatment with 200μM scopolamine. iNOS mRNA expression was increased in the brain of zebrafish treated with 200μM physostigmine. Consistently, aminoguanidine, an iNOS inhibitor, attenuated the increase in the number of BrdU-labeled cells by physostigmine treatment. Zebrafish also showed seizure-like locomotor activity characterized by a rapid and abrupt movement during a 30min treatment with 200μM physostigmine. Neural activity in response to an electrical stimulus was increased in the isolated telencephalon of zebrafish continuously perfused with 200μM physostigmine. None of the number of BrdU-labeled cells, neural activity, or locomotor activity was affected by treatment with 20μM physostigmine. These results suggest that 200μM physostigmine increased neural activity and induced cell proliferation via nitric oxide production in zebrafish.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Physostigmine increased the cell proliferation and neural activity in the zebrafish brain. </LI> <LI> Scopolamine suppressed the increased cell proliferation by physostigmine. </LI> <LI> The increased cell proliferation by physostigmine was accompanied with the increased expression level of iNOS mRNA. </LI> <LI> The increased cell proliferation by physostigmine was suppressed by an iNOS inhibitor. </LI> </UL> </P>