Zebrafish Strains Research Articles

Anosmin-1-Like Effect of UMODL1/Olfactorin on the Chemomigration of Mouse GnRH Neurons and Zebrafish Olfactory Axons Development.

The impairment of development/migration of hypothalamic gonadotropin-releasing hormone (GnRH) neurons is the main cause of Kallmann's syndrome (KS), an inherited disorder characterized by hypogonadism, anosmia, and other developmental defects. Olfactorin is an extracellular matrix protein encoded by the UMODL1 (uromodulin-like 1) gene expressed in the mouse olfactory region along the migratory route of GnRH neurons. It shares a combination of WAP and FNIII repeats, expressed in complementary domains, with anosmin-1, the product of the ANOS1 gene, identified as the causative of KS. In the present study, we have investigated the effects of olfactorin in vitro and in vivo models. The results show that olfactorin exerts an anosmin-1-like strong chemoattractant effect on mouse-immortalized GnRH neurons (GN11 cells) through the activation of the FGFR and MAPK pathways. In silico analysis of olfactorin and anosmin-1 reveals a satisfactory similarity at the N-terminal region for the overall arrangement of corresponding WAP and FNIII domains and marked similarities between WAP domains’ binding modes of interaction with the resolved FGFR1–FGF2 complex. Finally, in vivo experiments show that the down-modulation of the zebrafish z-umodl1 gene (orthologous of UMODL1) in both GnRH3:GFP and omp 2k :gap-CFP rw034 transgenic zebrafish strains leads to a clear disorganization and altered fasciculation of the neurites of GnRH3:GFP neurons crossing at the anterior commissure and a significant increase in olfactory CFP + fibers with altered trajectory. Thus, our study shows olfactorin as an additional factor involved in the development of olfactory and GnRH systems and proposes UMODL1 as a gene worthy of diagnostic investigation in KS.

Genetic and Functional Differences between Duplicated Zebrafish Genes for Human SCN1A.

There are currently seven different zebrafish strains that model Dravet Syndrome, a severe childhood form of epilepsy. These models are based on a set of duplicated genes, scn1laa and scn1lab, which are the homologs for human SCN1A. Disrupting one of the genes would mimic a heterozygous disease state in humans, as the paralog gene is still present. While this ‘disease-state model’ is widely accepted, there is also evidence that the function of these genes might not be completely the same. By analyzing the functional domains, we discovered several hotspots in the protein that are not conserved, indicating a functional difference. Based on this, we generated scn1Laa knockout zebrafish and compared their phenotype to scn1lab knockouts. The genetic and functional differences we discovered can have implications for the use of zebrafish as a model for Dravet Syndrome.

Triclosan induced zebrafish immunotoxicity by targeting miR-19a and its gene socs3b to activate IL-6/STAT3 signaling pathway

As a broad-spectrum antibacterial agent, triclosan (TCS) has been confirmed to possess potential immunotoxicity to organisms, but the underlying mechanisms remains unclear. Herein, with the aid of transgenic zebrafish strains Tg (coro1A: EGFP) and Tg (rag2: DsRed), we intuitively observed acute TCS exposure caused the drastic differentiation, abnormal development and distribution of innate immune cells, as well as barriers to formation of adaptive immune T cells. These abnormalities implied occurrence of the cytokine storm, which was further evidenced by expression changes of immune-related genes, and functional biomarkers. Based on transcriptome deep sequencing, target gene prediction and dual luciferase validation, the highly conservative and up-regulated miR-19a was chosen as the research target. Under TCS exposure, miR-19a up-regulation triggered down-regulation of its target gene socs3b, and simultaneously activated the downstream IL-6/STAT3 signaling pathway. Artificial over-expression and knock-down of miR-19a was realized by microinjecting agomir and antagomir, respectively, in 1–2-cell embryos. The miR-19a up-regulation inhibited socs3b expression to activate IL-6/STAT3 pathway, and yielded abnormal changes in the functional cytokine biomarkers, along with the sharp activation of immune responses. These findings disclose the molecular mechanisms regarding TCS-induced immunotoxicity, and offer important theoretical guidance for healthy safety evaluation and disease early warning from TCS pollution.

Different wild type strains of zebrafish show divergent susceptibility to TNBS-induced intestinal inflammation displaying distinct immune cell profiles

Little is known about the diversity in immune profile of the different wild type strains of zebrafish (Danio rerio), despite its growing popularity as an animal model to study human diseases and drug testing. In the case of data resulting from modeling human diseases, differences in the background Danio fishes have rarely been taken into consideration when interpreting results and this is potentially problematic, as many studies not even mention the source and strain of the animals. In this study, we hypothesized that different wild type zebrafish strains could present distinct immune traits. To address the differences in immune responses between two commonly used wild type strains of zebrafish, AB and Tübingen (TU), we used an intestinal inflammation model induced by 2,4,6-Trinitrobenzenesulfonic acid (TNBS) and characterized the susceptibility and immune profile in these two strains. Our data demonstrates significant differences in survival between AB and TU strains when exposed to TNBS, suggesting important physiological differences in how these strains respond to inflammatory challenges. We observed that the AB strain presented increased mortality, higher neutrophilic intestinal infiltration, decreased goblet cell numbers and decreased IL-10 expression when exposed to TNBS, compared to the TU strain. In summary, our study demonstrates strain-specific immunological responses in AB and TU animals. Finally, the significant variations in strain-related susceptibility to inflammation and the differences in the immune profile shown here, highlight that the background of each strain need to be considered when utilizing zebrafish to model diseases and for drug screening purposes, thus better immune characterization of the diverse wild type strains of zebrafish is imperative.

Establishment of a Dihydrofolate Reductase Gene Knock-In Zebrafish Strain to Aid Preliminary Analysis of Congenital Heart Disease Mechanisms.

Background: The dihydrofolate reductase (DHFR) gene is imperative in development, therefore it is essential to explore its effects on heart development. Thus, here a dhfr zebrafish knock-in (KI) strain was constructed.Methods: CRISPR/Cas9 technology was used to establish the dhfr KI zebrafish strain. This strain was hybridized with TgG fluorescent strain zebrafish to observe the phenotypes of heart shape, size, and circularization direction. Wild-type (WT) and KI zebrafish were then dissected and histologically stained to observe pathological changes. Western blot analysis was used to verify the increased expressions of zebrafish genes after KI. Hybridization experiments were used to confirm the presence of abnormal gonadal dysplasia.Results: The zebrafish dhfr KI strain was successfully constructed through CRISPR/Cas9 technology. At 6 days post fertilization (dpf), microscopic examinations of KI (homozygous) specimens revealed pericardial effusions, heart compressions, and curled tails. Compared with WT, the Hematoxylin and Eosin (H&E) tissue sections of KI-homozygous zebrafish showed defects such as reduced atria and ventricles. Western blot analysis indicated that the expression of the DHFR protein increased in both heterozygotes and homozygotes of dhfr KI zebrafish. Hybridization experiments revealed that dhfr KI may affect gonadal function.Conclusion: The DHFR gene plays an important regulatory role in the process of heart development, and copy number variations (CNVs) of this gene may constitute a new pathogenic mechanism of congenital heart disease (CHD).

Evaluation of endogenous miRNA reference genes across different zebrafish strains, developmental stages and kidney disease models

The majority of kidney diseases arise from the loss of podocytes and from morphological changes of their highly complex foot process architecture, which inevitably leads to a reduced kidney filtration and total loss of kidney function. It could have been shown that microRNAs (miRs) play a pivotal role in the pathogenesis of podocyte-associated kidney diseases. Due to their fully functioning pronephric kidney, larval zebrafish have become a popular vertebrate model, to study kidney diseases in vivo. Unfortunately, there is no consensus about a proper normalization strategy of RT-qPCR-based miRNA expression data in zebrafish. In this study we analyzed 9 preselected candidates dre-miR-92a-3p, dre-miR-206-3p, dre-miR-99-1, dre-miR-92b-3p, dre-miR-363-3p, dre-let-7e, dre-miR-454a, dre-miR-30c-5p, dre-miR-126a-5p for their capability as endogenous reference genes in zebrafish experiments. Expression levels of potential candidates were measured in 3 different zebrafish strains, different developmental stages, and in different kidney disease models by RT-qPCR. Expression values were analyzed with NormFinder, BestKeeper, GeNorm, and DeltaCt and were tested for inter-group differences. All candidates show an abundant expression throughout all samples and relatively high stability. The most stable candidate without significant inter-group differences was dre-miR-92b-3p making it a suitable endogenous reference gene for RT-qPCR-based miR expression zebrafish studies.

Vesicular monoamine transporter 2 (SLC18A2) regulates monoamine turnover and brain development in zebrafish.

We aimed at identifying potential roles of vesicular monoamine transporter 2, also known as Solute Carrier protein 18 A2 (SLC18A2) (hereafter, Vmat2), in brain monoamine regulation, their turnover, behaviour and brain development using a novel zebrafish model. A zebrafish strain lacking functional Vmat2 was generated with the CRISPR/Cas9 system. Larval behaviour and heart rate were monitored. Monoamines and their metabolites were analysed with high-pressure liquid chromatography. Amine synthesising and degrading enzymes, and genes essential for brain development, were analysed with quantitative PCR, in situ hybridisation and immunocytochemistry. The 5-bp deletion in exon 3 caused an early frameshift and was lethal within 2weeks post-fertilisation. Homozygous mutants (hereafter, mutants) displayed normal low locomotor activity during night-time but aberrant response to illumination changes. In mutants dopamine, noradrenaline, 5-hydroxytryptamine and histamine levels were reduced, whereas levels of dopamine and 5-hydroxytryptamine metabolites were increased, implying elevated monoamine turnover. Consistently, there were fewer histamine, 5-hydroxytryptamine and dopamine immunoreactive cells. Cellular dopamine immunostaining, in wild-type larvae more prominent in tyrosine hydroxylase 1 (Th1)-expressing than in Th2-expressing neurons, was absent in mutants. Despite reduced dopamine levels, mutants presented upregulated dopamine-synthesising enzymes. Further, in mutants the number of histidine decarboxylase-expressing neurons was increased, notch1a and pax2a were downregulated in brain proliferative zones. Lack of Vmat2 increases monoamine turnover and upregulates genes encoding amine-synthesising enzymes, including histidine decarboxylase. Notch1a and pax2a, genes implicated in stem cell development, are downregulated in mutants. The zebrafish vmat2 mutant strain may be a useful model to study how monoamine transport affects brain development and function, and for use in drug screening.

Behavioral effects of acute ethanol in larval zebrafish (D. rerio) depend on genotype and volume of experimental well

Ethanol consumption is a worldwide problem. Sensitivity to acute effects of ethanol influences the development of chronic ethanol abuse and ethanol dependence. Environmental and genetic factors have been found to contribute to differential effects of acute ethanol. Animal models have been employed to investigate these factors. An increasingly frequently utilized animal model in ethanol research is the zebrafish. A large proportion of ethanol studies with zebrafish have been conducted with adult zebrafish. However, high throughput drug and mutation screens are particularly well adapted to larval zebrafish. These studies are often carried out using the 96-well-plate that allows monitoring large numbers of fish efficiently. Here, we investigate the effects of acute (30 min long) ethanol exposure in 8-day post-fertilization (dpf) old zebrafish. We compare four genetically distinct populations (strains) of zebrafish, measuring numerous parameters of their swim path in two well sizes, i.e., in the 96-well-plate (small volume wells) and in the 6-well-plate (large volume wells). In general, we found that the highest dose of ethanol (1% vol/vol) reduced swim speed, increased duration of immobility, increased turn angle, and increased intra-individual variance of turn angle, while the intermediate dose (0.5%) had a less strong effect, compared to control. However, we also found that these ethanol effects were strain dependent and, in general, were better detected in the larger volume well. We conclude that larval zebrafish are appropriate for quantification of acute ethanol effects and also for the analysis of environmental and genetic factors that influence these effects. We also speculate that using larger wells will likely increase sensitivity of detection and precision in screening applications.

Development of Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP); Casper(roy-/-,nacre-/-) Transparent Transgenic In Vivo Zebrafish Model to Study the Cardiomyocyte Function.

The zebrafish provided an excellent platform to study the genetic and molecular approach of cellular phenotype-based cardiac research. We designed a novel protocol to develop the transparent transgenic zebrafish model to study annexin-5 activity in the cardiovascular function by generating homozygous transparent skin Casper(roy−/−,nacre−/−); myl7:RFP; annexin-5:YFP transgenic zebrafish. The skin pigmentation background of any vertebrate model organism is a major obstruction for in vivo confocal imaging to study the transgenic cellular phenotype-based study. By developing Casper(roy−/−,nacre−/−); myl7; annexin-5 transparent transgenic zebrafish strain, we established time-lapse in vivo confocal microscopy to study cellular phenotype/pathologies of cardiomyocytes over time to quantify changes in cardiomyocyte morphology and function over time, comparing control and cardiac injury and cardio-oncology. Casper contributes to the study by integrating a transparent characteristic in adult zebrafish that allows for simpler transparent visualization and observation. The Casper(roy−/−,nacre−/−) transgenic progenies developed through cross-breeding with the transgenic strain of Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP). Confocal and fluorescent microscopy were being used to obtain accurate, precise imaging and to determine fluorescent protein being activated. This study protocol was conducted under two sections; 1.1: Generation of homozygous Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP); Casper(roy−/−,nacre−/−) zebrafish (generation F01-F06) and 1.2: Screening and sorting the transparent transgenic progeny and in vivo imaging to validate cardiac morphology through in vivo confocal imaging. We coined the newly developed strain as Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP); Casper(roy−/−,nacre−/−)gmc1. Thus, the newly developed strain maintains transparency of the skin throughout the entire life of zebrafish and is capable of application of a non-invasive in vivo imaging process. These novel results provide an in vivo whole organism-based platform to design high-throughput screening and establish a new horizon for drug discovery in cardiac cell death and cardio-oncology therapeutics and treatment.

Using Zebrafish to Elucidate Glial-Vascular Interactions During CNS Development.

An emerging area of interest in Neuroscience is the cellular relationship between glia and blood vessels, as many of the presumptive support roles of glia require an association with the vasculature. These interactions are best studied in vivo and great strides have been made using mice to longitudinally image glial-vascular interactions. However, these methods are cumbersome for developmental studies, which could benefit from a more accessible system. Zebrafish (Danio rerio) are genetically tractable vertebrates, and given their translucency, are readily amenable for daily live imaging studies. We set out to examine whether zebrafish glia have conserved traits with mammalian glia regarding their ability to interact with and maintain the developing brain vasculature. We utilized transgenic zebrafish strains in which oligodendrocyte transcription factor 2 (olig2) and glial fibrillary acidic protein (gfap) identify different glial populations in the zebrafish brain and document their corresponding relationship with brain blood vessels. Our results demonstrate that olig2+ and gfap+ zebrafish glia have distinct lineages and each interact with brain vessels as previously observed in mouse brain. Additionally, we manipulated these relationships through pharmacological and genetic approaches to distinguish the roles of these cell types during blood vessel development. olig2+ glia use blood vessels as a pathway during their migration and Wnt signaling inhibition decreases their single-cell vessel co-option. By contrast, the ablation of gfap+ glia at the beginning of CNS angiogenesis impairs vessel development through a reduction in Vascular endothelial growth factor (Vegf), supporting a role for gfap+ glia during new brain vessel formation in zebrafish. This data suggests that zebrafish glia, akin to mammalian glia, have different lineages that show diverse interactions with blood vessels, and are a suitable model for elucidating glial-vascular relationships during vertebrate brain development.

Lipolysis and lipophagy play individual and interactive roles in regulating triacylglycerol and cholesterol homeostasis and mitochondrial form in zebrafish

Neutral lipases-mediated lipolysis and acid lipases-moderated lipophagy are two main processes for degradation of lipid droplets (LDs). However, the individual and interactive roles of these metabolic pathways are not well known across vertebrates. This study explored the roles of lipolysis and lipophagy from the aspect of neutral and acid lipases in zebrafish. We established zebrafish strains deficient in either adipose triglyceride lipase (atgl−/−; AKO fish) or lysosomal acid lipase (lal−/−; LKO fish) respectively, and then inhibited lipolysis in the LKO fish and lipophagy in the AKO fish by feeding diets supplemented with the corresponding inhibitors Atglistatin and 3-Methyladenine, respectively. Both the AKO and LKO fish showed reduced growth, swimming activity, and oxygen consumption. The AKO fish did not show phenotypes in adipose tissue, but mainly accumulated triacylglycerol (TAG) in liver, also, they had large LDs in the hepatocytes, and did not stimulate lipophagy as a compensation response but maintained basal lipophagy. The LKO fish reduced total lipid accumulation in the body but had high cholesterol content in liver; also, they accumulated small LDs in the hepatocytes, and showed increased lipolysis, especially Atgl expression, as a compensatory mechanism. Simultaneous inhibition of lipolysis and lipophagy in zebrafish resulted in severe liver damage, with the potential to trigger mitophagy. Overall, our study illustrates that lipolysis and lipophagy perform individual and interactive roles in maintaining homeostasis of TAG and cholesterol metabolism. Furthermore, the interactive roles of lipolysis and lipophagy may be essential in regulating the functions and form of mitochondria.

MO030IDENTIFICATION OF REGULATED MRNAS AND MIRNAS IN GLOMERULI ISOLATED FROM AN FSGS-LIKE ZEBRAFISH MODEL

Abstract Background and Aims The zebrafish (Danio rerio) is a powerful animal model to study glomerular morphology and the function of the permselectivity of the glomerular filtration barrier. Since zebrafish larvae develop quickly and can be bred to become transparent, in vivo observation of these animals is possible. At 48 hours post fertilization, zebrafish larvae develop a single glomerulus which is attached to a pair of tubules. Like in mammals, the glomerular filtration barrier consists of a fenestrated endothelium, the glomerular basement membrane and interdigitating podocyte foot processes bridged by a slit diaphragm. By using genetically modified zebrafish strains with fluorescently labeled podocytes, it is possible to study alterations of the glomerulus during the development of renal disease like focal segmental glomerulosclerosis (FSGS) directly in vivo. FSGS is characterized by podocyte loss, the effacement of their foot processes as well as scarring of the glomerulus. To study FSGS in zebrafish larvae, we induced podocyte detachment by the use of a zebrafish strain expressing the enzyme nitroreductase converting metronidazole into a toxic substance specifically in podocytes. The aim of our study was to collect glomeruli for the identification of mRNAs as well as miRNAs by RNA_Seq that are up- and down-regulated in the glomeruli of this FSGS-like disease model. Method The transgenic zebrafish strain Cherry (Tg(nphs2:GAL4); Tg(UAS:Eco.nfsB-mCherry); mitfaw2/w2; mpv17a9/a9) which expresses the prokaryotic enzyme nitroreductase (NTR) fused to mCherry, a red fluorescent protein, under the control of the podocyte-specific podocin (nphs2) promoter in a transparent zebrafish strain, was utilized. After addition of metronidazole (MTZ) into the tank water, MTZ is converted into a cytotoxin by NTR leading to dose-dependent apoptosis exclusively in podocytes. Cherry larvae were treated at 4 days post fertilization (dpf) for 48 h with 80 µM MTZ. MTZ-treated and control larvae were homogenized at 6 dpf. The cell suspension was diluted, and red-fluorescent glomeruli were collected using a micropipette and a microscope. Total RNA was isolated, and integrity was checked by a Bioanalyzer. Libraries were generated with a MACE kit and True Quant small RNA seq kit by GenXPro. Constructs were amplified by PCR and sequenced on an Illumina Hiseq 2000. Normalization and statistical analysis for differential gene expression were done using DESeq2. Results Zebrafish larvae showed severe whole-body edema, proteinuria, loss of podocytes and an increased mortality rate after MTZ-treatment. The glomerular histology resembled mammalian FSGS. We found that only the RNA of manually collected glomeruli had an excellent quality. Using RNA_Seq, we identified a total of 16941 genes. DESeq2 analysis showed 494 up-regulated and 473 down-regulated genes. Gene ontology (GO) enrichment analysis of up-regulated genes revealed a total of 167 that are significantly enriched in GO terms (e.g. metabolic processes, immune response and ion transport). Down-regulated genes were enriched in 14 GO terms and most of them are linked to normal glomerular function and the slit diaphragm. DESeq2 analysis identified 200 miRNAs of 777 small RNAs. Some of these miRNA are already described to be regulated in different glomerular diseases like FSGS, lupus nephritis, IgA nephropathy and diabetic nephropathy. Conclusion We analyzed isolated glomeruli from transgenic zebrafish larvae that developed a FSGS-like disease. By sequencing, we have found mRNAs and miRNAs that were significantly regulated after the onset of disease. Detailed knowledge of these mRNAs and miRNA-based gene regulation will help to uncover the pathomechanism as well as to develop therapeutics for the treatment of FSGS.

Label-free photoacoustic microscopy: a potential tool for the live imaging of blood disorders in zebrafish

The zebrafish has emerged as a useful model for human hematological disorders. Transgenic zebrafish that express green fluorescence protein (GFP) in red blood cells (RBCs) visualized by fluorescence microscopy (FLM) is a fundamental approach in such studies to understand the cellular processes and biological functions. However, additional and cumbersome efforts are required to breed a transgenic zebrafish line with reliable GFP expression. Further, the yolk autofluorescence and finite GFP fluorescence lifetimes also have an adverse impact on the observation of target signals. Here, we investigate the identification of intracerebral hemorrhage (ICH) and hemolytic anemia (HA) in zebrafish embryos using label-free photoacoustic microscopy (PAM) for imaging. First, ICH and HA in transgenic LCR-EGFP zebrafish are mainly studied by PAM and FLM. The results show that PAM is comparable to FLM in good identification of ICH and HA. Besides, PAM is more advantageous in circumventing the issue of autofluorescence. Secondly, ICH and HA in the transparent casper zebrafish without fluorescent labeling are imaged by PAM and bright-field microscopy (BFM). Because of the high contrast to reveal RBCs, PAM obviously outperforms BFM in the identification of both ICH and HA. Note that FLM cannot observe casper zebrafish due to its lack of fluorescent labeling. Our work proves that PAM can be a useful tool to study blood disorders in zebrafish, which has advantages: (i) Reliable results enabled by intrinsic absorption of RBCs; (ii) wide applicability to zebrafish strains (no requirement of a transgene); (iii) high sensitivity in identification of ICH and HA compared with BFM.

Development of Transparent Transgenic Zebrafish Strain to Study NF-KB, Annexin 5, and the Microglia

Development of Transparent Transgenic Zebrafish Strain to Study NF-KB, Annexin 5, and the Microglia

Midkine-a Regulates the Formation of a Fibrotic Scar During Zebrafish Heart Regeneration.

Unlike the hearts of mammals, the adult zebrafish heart regenerates after injury. Heart cryoinjury in zebrafish triggers the formation of a fibrotic scar that gradually degrades, leading to regeneration. Midkine-a (Mdka) is a multifunctional cytokine that is activated after cardiac injury. Here, we investigated the role of mdka in zebrafish heart regeneration. We show that mdka expression was induced at 1-day post-cryoinjury (dpci) throughout the epicardial layer, whereas by 7 dpci expression had become restricted to the epicardial cells covering the injured area. To study the role of mdka in heart regeneration, we generated mdka-knock out (KO) zebrafish strains. Analysis of injured hearts showed that loss of mdka decreased endothelial cell proliferation and resulted in an arrest in heart regeneration characterized by retention of a collagenous scar. Transcriptional analysis revealed increases in collagen transcription and intense TGFβ signaling activity. These results reveal a critical role for mdka in fibrosis regulation during heart regeneration.

Biochemical characterization of zebrafish Prss59.1

Eleven genes, including prss59.1, were selected as candidate ovulation-inducing genes on the basis of microarray analysis and RNA sequencing in our previous study. To address the role of prss59.1, the prss59.1 gene knock-out zebrafish strain is currently being established by genome editing. In this study, for further phenotypic analysis of prss59.1, biochemical characterization of Prss59.1 was conducted using recombinant protein.A C-terminal histidine-tagged version of zebrafish Prss 59.1 was constructed. Although E. coli-produced recombinant Prss59.1 showed almost no activity, peptidase activities appeared after denaturation and renaturation. Zebrafish Prss59.1 showed the highest activity against Lys-MCA. The optimal temperature and pH of the activity toward Lys-MCA were 37 °C and pH 8.0, respectively. The Km value was 0.17 mM. Thus, zebrafish Prss59.1 possesses the closed character of trypsin, as expected from the DNA sequence.

Investigating Opioid‐induced Respiratory Depression and Analgesia Using Larval Zebrafish

Introduction Opioid drugs produce both analgesia and severe respiratory depression which can be lethal. In 2019, over 50,000 North Americans died of an opioid overdose. Our understanding on how to differentiate between these two effects is limited due to a lack of simple animal models. Therefore, we aim to understand respiratory depression and analgesia by opioids using novel zebrafish models, allowing for drug screening. Using these models, we tested a variety of drugs including serotoninergic agonists, glutamatergic modulators, and calcium channel activators, for their ability to reverse opioid-induced respiratory depression. Methods We have established phenotype-based approaches using in-vivo zebrafish models of respiratory depression and analgesia by fentanyl. To quantify respiratory depression, we measured respiratory activity by quantifying mandible movements in 12-14 day post-fertilization larvae. To quantify analgesia, we measured the escape swimming response to nociceptive stimuli such as formalin or allyl isothiocyanate (AITC) combined with fentanyl to induce analgesia. We then introduced various candidate drugs to measure their effects on respiratory depression and analgesia. Results Respiratory rate was normalized to each fish's baseline (baseline=100%). Zebrafish strains were found to be differentially sensitive to opioids, where Tübingen (TU) fish did not respond to fentanyl (1µM, p=0.4240, n=9) and AB fish showed significant respiratory rate depression (p<0.001, n=11). AB and TU crosses did not show respiratory depression (n=7). In AB zebrafish, a dose-dependent decrease in respiratory rate was observed with 1μM and 3µM fentanyl (p=0.01, n=11 and p<0.001, n=27, respectively). The opioid antagonist naloxone (20µM) and selective antagonist CTAP (4μM) reversed respiratory depression by fentanyl (p<0.001, n=9 and p=0.049, n=11, respectively). Respiratory depression by 1μM fentanyl was reversed by the AMPA receptor positive allosteric modulator CX614 (5μM, p<0.001, n=9) but not the serotoninergic 5-HT4 agonist BIMU8 (10μM, n=21). Calcium channel activators nefiracetam (1µM) and FLP-64176 (1µM) reversed respiratory depression by fentanyl (1µM, p=0.005, n=11 and3µM, p<0.001, n=15, respectively). Formalin (0.075%) and AITC (100µM) significantly increased swimming velocity (p=0.005, n=21 and p<0.001, n=23, respectively), which was reduced by fentanyl (4μM, p<0.001, n=12and 6µM, p=0.004, n=14, respectively). FLP-64176 reversed fentanyl analgesia when administered with formalin (p<0.001, n=10). Discussion: Our models show that respiratory depression and analgesia by opioids can be measured in zebrafish larvae. The strain differences point to a potential genetic protection which we are investigating using quantitative real-time polymerase chain reaction (qPCR). Our novel zebrafish models can be used to investigate the effects of opioids, as well as for drug screening to find ways to treat respiratory depression while preserving analgesia.

70274 TL1 team approach to investigating the adhesin gene fimH in adherent invasive E. coli induced inflammation and colorectal cancer development

ABSTRACT IMPACT: We are developing the 3D perfusion system for use with patient-derived bacteria to further characterize the mechanism behind bacterial-induced inflammation and cancer. OBJECTIVES/GOALS: We previously reported the adherent invasive E. coli NC101 promote colorectal cancer (CRC) in mice. FimH, a mannose-specific adhesin on type 1 fimbriae, is involved in bacterial surface adhesion. Herein, we investigated the role of FimH in E. coli NC101-induced adherence and carcinogenesis in a novel 3D perfusion culture imaging plate. METHODS/STUDY POPULATION: E. coli NC101 gene fimH was deleted byï ŲRed Recombinase System. Biofilm formation was assessed by crystal violet and congo red staining. 5 dpf (wild-type strain) zebrafish embryos were infected in 6x107 cfu/ml wild type (WT) or fimH-deleted (ï ,,fimH) E. coli NC101 for 24hr and gut dissected for bacterial culture. A 2D/3D infection culture system for IEC-6 and HT-29 cells was infected for 4 hr and imaged and then DNA damage examined by comet assay, cell cycle andÎ3H2AX accumulation. Germ-free (GF) Il10-/- (colitis) mice were orally gavaged with 108 cfu WT orï ,,fimH E. coli NC101 for 16 weeks. E. coli colonization were quantified by plate culture and qPCR. Lipocalin2 was quantified by ELISA. PCNA and β-catenin were evaluated by immunohistochemistry (IHC). RESULTS/ANTICIPATED RESULTS: Biofilm formation was reduced by more than 40% (p&lt;0.05) in E. coli NC101ï ,,fimH compared to WT strain. Zebrafish larvae showed a 41% decrease in intestinal colonization ofï ,,fimH compared to WT (p&lt;0.05). E. coli NC101-induced DNA damage was reduced by 67% (p&lt;0.0001) in HT-29 cells infected withï ,,fimH compared to WT strain. Using the 3D infection system, a 46% decrease in yH2AX (p&lt;0.05) and 42% decrease in G2 cell cycle arrest (p&lt;0.05) was observed inï ,,fimH infected IEC-6 cells compared to WT strain. Furthermore, ï ,,fimH infected Il10-/- mice showed decreased colonization (p&lt;0.01), decreased intestinal inflammation (p&lt;0.05), decreased stool lipocalin2 level (p&lt;0.01), and reduction of PCNA positive cells in the intestine (p&lt;0.05) compared to mice infected with WT strain. DISCUSSION/SIGNIFICANCE OF FINDINGS: Adhesin protein FimH is required by E. coli NC101 to colonize and promote colitis and carcinogenesis both in a 3D perfusion culture and in mice and may serve as potential therapeutic target.

Influence of Prednisolone and Alendronate on the de novo Mineralization of Zebrafish Caudal Fin.

ABSTRACTDysregulated balance between bone resorption and formation mediates the onset and progression of osteoporosis. The administration of prednisolone is known to induce osteoporosis, whereas alendronate is commonly used to reverse the process. However, the assessment of the effects of such medicines on the nanostructure of bone remodeling and mechanical properties remains a major technical challenge. The aim of this study was to apply various analytical approaches to evaluate the compositional, morphological, and mechanical properties of regenerative zebrafish caudal fin bony rays affected by prednisolone and alendronate. Adult wild‐type AB strain zebrafish were first exposed to 125μM of prednisolone for 14 days to develop glucocorticoid‐induced osteoporosis. Fish fins were then amputated and let to regenerate for 21 days in tank water containing 30μM of alendronate or no alendronate. The lepidotrichia in the proximal and distal regions were evaluated separately using confocal microscope, scanning electron microscope, electron‐dispersive spectroscopy, Raman spectroscopy, atomic force microscopy, and a triboindenter. As expected, prednisolone led to significant osteoporotic phenotypes. A decrease of Ca/P ratio was observed in the osteoporotic subjects (1.46 ± 0.04) as compared to the controls (1.74 ± 0.10). Subsequent treatment of alendronate overmineralized the bony rays during regeneration. Enhanced phosphate deposition was detected in the proximal part with alendronate treatment. Moreover, prednisolone attenuated the reduced elastic modulus and hardness levels (5.60 ± 5.04 GPa and 0.12 ± 0.17 GPa, respectively), whereas alendronate recovered them to the pre‐amputation condition (8.68 ± 8.74 GPa and 0.34 ± 0.47 GPa, respectively). As an emerging model of osteoporosis, regrowth of zebrafish caudal fin was shown to be a reliable assay system to investigate the various effects of medicines in the de novo mineralization process. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

A genetic modifier of venous thrombosis in zebrafish reveals a functional role for fibrinogen AαE in early hemostasis

AbstractPlasma fibrinogen molecules comprise 2 copies of Aα, Bβ, and γ chains folded into a hexameric protein. A minor fibrinogen isoform with an extended Aα chain (AαE) is more abundant in newborn human blood than in adults. Larval zebrafish produce predominantly AαE-containing fibrinogen, but its functional significance is unclear. In 3-day-old zebrafish, when hemostasis is reliant on fibrinogen and erythrocyte-rich clotting but is largely thrombocyte-independent, we measured the time to occlusion (TTO) in a laser-induced venous thrombosis assay in 3 zebrafish strains (AB, TU, and AB × TL hybrids). AB larvae showed delayed TTO compared with the TU and AB × TL strains. Mating AB with TU or TL produced larvae with a TU-like TTO. In contrast to TU, AB larvae failed to produce fibrinogen AαE, due to a mutation in the AαE-specific coding region of fibrinogen α-chain gene (fga). We investigated whether the lack of AαE explained the delayed AB TTO. Transgenic expression of AαE, but not Aα, shortened the AB TTO to that of TU. AαE rescued venous occlusion in fibrinogen mutants or larvae with morpholino-targeted fibrinogen α-chain messenger RNA, but Aα was less effective. In 5-day-old larvae, circulating thrombocytes contribute to hemostasis, as visualized in Tg(itga2b:EGFP) transgenics. Laser-induced venous thrombocyte adhesion and aggregation is reduced in fibrinogen mutants, but transgenic expression of Aα or AαE restored similar thrombocyte accumulation at the injury site. Our data demonstrate a genetic modifier of venous thrombosis and a role for fibrinogen AαE in early developmental blood coagulation, and suggest a link between differentially expressed fibrinogen isoforms and the cell types available for clotting.