Transparent Zebrafish Research Articles

Automated cell tracking using 3D nnUnet and Light Sheet Microscopy to quantify regional deformation in zebrafish.

Light Sheet Microscopy (LSM) in conjunction with embryonic zebrafish, is rapidly advancing three-dimensional, in vivo characterization of myocardial contractility. Preclinical cardiac deformation imaging is predominantly restricted to a low-order dimensionality image space (2i) or suffers from poor reproducibility. In this regard, LSM has enabled high throughput, non-invasive 4i (3d+time) characterization of dynamic organogenesis within the transparent zebrafish model. More importantly, LSM offers cellular resolution across large imaging Field-of-Views at millisecond camera frame rates, enabling single cell localization for global cardiac deformation analysis. However, manual labeling of cells within multilayered tissue is a time-consuming task and requires substantial expertise. In this study, we applied the 3i nnU-Net with Linear Assignment Problem (LAP) framework for automated segmentation and tracking of myocardial cells. Using binarized labels from the neural network, we quantified myocardial deformation of the zebrafish ventricle across 4-6 days post fertilization (dpf). Our study offers tremendous promise for developing highly scalable and disease-specific biomechanical quantification of myocardial microstructures.

Ellagic acid-enhanced biocompatibility and bioactivity in multilayer core-shell gold nanoparticles for ameliorating myocardial infarction injury

BackgroundMyocardial infarction (MI) is the main contributor to most cardiovascular diseases (CVDs), and the available post-treatment clinical therapeutic options are limited. The development of nanoscale drug delivery systems carrying natural small molecules provides biotherapies that could potentially offer new treatments for reactive oxygen species (ROS)-induced damage in MI. Considering the stability and reduced toxicity of gold-phenolic core-shell nanoparticles, this study aims to develop ellagic acid-functionalized gold nanoparticles (EA-AuNPs) to overcome these limitations.ResultsWe have successfully synthesized EA-AuNPs with enhanced biocompatibility and bioactivity. These core-shell gold nanoparticles exhibit excellent ROS-scavenging activity and high dispersion. The results from a label-free imaging method on optically transparent zebrafish larvae models and micro-CT imaging in mice indicated that EA-AuNPs enable a favorable excretion-based metabolism without overburdening other organs. EA-AuNPs were subsequently applied in cellular oxidative stress models and MI mouse models. We found that they effectively inhibit the expression of apoptosis-related proteins and the elevation of cardiac enzyme activities, thereby ameliorating oxidative stress injuries in MI mice. Further investigations of oxylipin profiles indicated that EA-AuNPs might alleviate myocardial injury by inhibiting ROS-induced oxylipin level alterations, restoring the perturbed anti-inflammatory oxylipins.ConclusionsThese findings collectively emphasized the protective role of EA-AuNPs in myocardial injury, which contributes to the development of innovative gold-phenolic nanoparticles and further advances their potential medical applications.Graphical

Highly sensitive response to the toxicity of environmental chemicals in transparent casper zebrafish

The response sensitivity to toxic substances is the most concerned performance of animal model in chemical risk assessment. Casper (mitfaw2/w2;mpv17a9/a9), a transparent zebrafish mutant, is a useful in vivo model for toxicological assessment. However, the ability of casper to respond to the toxicity of exogenous chemicals is unknown. In this study, zebrafish embryos were exposed to five environmental chemicals, chlorpyrifos, lindane, α-endosulfan, bisphenol A, tetrabromobisphenol A (TBBPA), and an antiepileptic drug valproic acid. The half-lethal concentration (LC50) values of these chemicals in casper embryos were 62–87 % of that in the wild-type. After TBBPA exposure, the occurrence of developmental defects in the posterior blood island of casper embryos was increased by 67–77 % in relative to the wild-type, and the half-maximal effective concentration (EC50) in casper was 73 % of that in the wild-type. Moreover, the casper genetic background significantly increased the hyperlocomotion caused by chlorpyrifos and lindane exposure compared with the wild-type. These results demonstrated that casper had greater susceptibility to toxicity than wild-type zebrafish in acute toxicity, developmental toxicity and neurobehavioral toxicity assessments. Our data will inform future toxicological studies in casper and accelerate the development of efficient approaches and strategies for toxicity assessment via the use of casper.

Phollow: Visualizing Gut Bacteriophage Transmission within Microbial Communities and Living Animals.

Bacterial viruses (known as "phages") shape the ecology and evolution of microbial communities, making them promising targets for microbiome engineering. However, knowledge of phage biology is constrained because it remains difficult to study phage transmission dynamics within multi-member communities and living animal hosts. We therefore created "Phollow": a live imaging-based approach for tracking phage replication and spread in situ with single-virion resolution. Combining Phollow with optically transparent zebrafish enabled us to directly visualize phage outbreaks within the vertebrate gut. We observed that virions can be rapidly taken up by intestinal tissues, including by enteroendocrine cells, and quickly disseminate to extraintestinal sites, including the liver and brain. Moreover, antibiotics trigger waves of interbacterial transmission leading to sudden shifts in spatial organization and composition of defined gut communities. Phollow ultimately empowers multiscale investigations connecting phage transmission to transkingdom interactions that have the potential to open new avenues for viral-based microbiome therapies.

Mapping the Metabolic Characteristics and Perturbation of Adult Casper Zebrafish by Ambient Mass Spectrometry Imaging.

Casper, a type of transparent mutant-line zebrafish, was generated to overcome the opaque trunk of an adult zebrafish for tumor modeling to realize real-time visualization of transplanted cells in vivo. However, the molecular information at the metabolic level has not received much attention. Herein, a spatially resolved metabolomics method based on an airflow-assisted desorption electrospray ionization-mass spectrometry imaging (AFADESI-MSI) system for whole-body zebrafish was used to investigate small molecules and the distribution of adult casper (Mitfaw2/w2, roya9/a9) and the differences from wild-type zebrafish. Finally, the spatial distribution information of more than 1500 endogenous ions was obtained in positive and negative detection modes, and 186 metabolites belonging to a variety of structural categories were identified or annotated. Compared with wild-type samples, 85 variables, including 37 known metabolites, were screened out. In addition, the disordered metabolic pathways caused by the genetic mutation were excavated, involving downregulation of purine metabolism and arachidonic acid metabolism, upregulation of glycerophospholipid metabolism, and biosynthesis of unsaturated fatty acids. All these results were observed in the most intuitive way through MSI. This study revealed important metabolic characteristics of and perturbation in adult casper zebrafish, and provides indispensable fundamental knowledge for tumor research based on it.

Abstract 1432: Development of transparent transgenic zebrafish model to study CNS microglia and NF-kB activity in glioblastoma

Abstract Microglia are resident macrophages of the central nervous system (CNS) and have crucial roles in normal brain homeostasis, function, and immune response. Microglia play key roles in the healthy brain, ranging from phagocytosis of dying neurons and CNS debris to modulating adaptability. The role of microglia has been well documented but their specific contributions to the development and progression of cancers, such as glioblastoma, have recently garnered wide attention. The zebrafish have proven novel tool for modeling innate immune responses. A macrophage-specific marker mpeg1 and its promoter are used in mpeg1-driven transgenes. mpeg1-driven transgenes mpeg1:mCherryFP are expressed in macrophage-lineage cells that do not express neutrophil-marking transgenes. These zebrafish transgenes contribute to the fields of inflammation, infection, and leukocyte biology. We have recently developed a novel transparent transgenic homozygous strain Tg(6xNF-kB:EGFP); Casper(roy−/−, nacre−/−) of zebrafish to the in-vivo study of NF-kB activity under normal and inflammation stage at the cellular, organ-specific and whole organism level. To perform an in-vivo study of microglial function, we are developing Casper;mpeg1:mCherryFP microglia specific strain and integrating with newly developed Tg(6xNF-kB:EGFP); Casper(roy−/−, nacre−/−) strain through crossbreeding of Casper and transgenic zebrafish. We have generated F1 generation of newly developed strains of Casper;mpeg1:mCherryFP and Tg(6xNF-kB:EGFP);Casper. The presence of fluorescence mCherry and GFP, in crossbred progeny was identified through the screening of larvae at 72 HPF stage. The expression of these fluorescent proteins determines the presence of the reporter genes. F1 generation confirmed heterozygous and allowed them to grow into adults to breed transparent transgenic strain to get F2 generation. This process will be continued to F2, F3, F4, and F5 generation until achieving homozygous progeny. This model allows for in-vivo examination of microglia functional morphology by using fluorescent and confocal microscopy. This novel transgenic model will be applied to identify novel anti-inflammatory drug inhibitors in NF-kB pathophysiology for glioblastoma and cardio-oncology experimental therapeutics. Citation Format: Surendra K. Rajpurohit, Sai Nasanally, Simran Hotwani, Taitum Gossman, Keshu Bhatt, Aiden Van Derhei, Rohan Pasi, Devan-Anmol Manhiani, Kirk Kimmerling, Balakrishna L. Lokeshwar. Development of transparent transgenic zebrafish model to study CNS microglia and NF-kB activity in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1432.

Abstract 1430: Development of transparent transgenic zebrafish model to study anti-cancer drug induced in vivo cardiotoxicity

Abstract Cardiotoxicity is the second leading cause of death among cancer survivors and can manifest as arrhythmia, acute coronary syndrome, acute heart failure, or pericarditis/myocarditis. Chronic myocyte damage and apoptosis can transition from an asymptomatic phase to adaptive myocardial hypertrophy, followed by decreased left ventricular ejection fraction, and eventual heart failure. Cardio-oncology is an emerging field that studies cancer therapy-associated cardiotoxicities. The development of reliable and effective preclinical models for cardiotoxicity prediction and prevention is essential to improving the long-term cardiac health of cancer patients. Zebrafish are a promising animal model for the field of cardio-oncology and have emerged as an invaluable tool for investigating genetic and molecular aspects of cardiovascular research. Zebrafish heart cells are like human heart cells at the molecular level, making them an ideal system for studying the genetic underpinning of cardiac function. Zebrafish myl7 gene, a myosin light chain-7 gene, is a regulatory gene of heart orthologs to human MYL7. In-vivo imaging is limited due to the opacification of skin and subdermal structures. To conduct in-vivo whole organism imaging studies, skin transparency is a primary requirement, necessitating the blocking of pigmentation. The Zebrafish Casper mutant maintains skin transparency throughout its life, offering the ideal combination of sensitivity and resolution for in-vivo analyses and imaging. In this study, we have developed a novel transparent transgenic zebrafish model and established time-lapse in-vivo confocal microscopy to study cellular phenotype/pathologies of cardiomyocytes. The newly developed homozygous transparent transgenic Casper/myl7:RFP; annexin-5:YFP/NF-kB: GFP/mpeg1:mcherryFP, allows us to study cell death process (Annexin5) and inflammatory activity (NF-kB) in microglia and cardiomyocytes. We have successfully established in vivo fluorescence and confocal microscopy in this newly developed strain, enabling the study of cellular morphology and function changes under various conditions. This will allow us to elucidate cell death patterns, and cardio-inflammatory and cardiotoxicity pathways. Citation Format: Surendra K. Rajpurohit, Taitum Gossman, Sai Nasanally, Keshu Bhatt, Aiden Van Derhei, Simran Hotwani, Devan-Anmol Manhiani, Rohan Pasi, Jiaqi Mi, Vishal Arora, Balakrishna Lokeshwar. Development of transparent transgenic zebrafish model to study anti-cancer drug induced in vivo cardiotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1430.

Abstract 1431: Generation of transparent transgenic zebrafish strain to study cardiovascular functionin translational cardio-oncology research

Abstract Transgenic zebrafish lines have been extensive use in biomedical research and their primary applications. The zebrafish has proven to be an ideal tool for direct In-vivo observations of developmental processes. In vertebrates, including zebrafish and humans, the ability to obtain high-resolution, in-vivo imaging is limited due to the natural opacification of skin and subdermal structures. To conduct in-vivo whole organism imaging studies, skin transparency is a primary requirement, necessitating blocking of pigmentation. Zebrafish casper mutant maintains skin transparency throughout its life, offering the ideal combination of sensitivity and resolution for in-vivo analyses and imaging. Our Laboratory is developing a novel transparent transgenic zebrafish model to address basic and translational cardio-oncology and experimental therapeutics. The fli1 promoter in zebrafish having an enhanced expression reporter gene of green fluorescent protein, GFP. This model allows for the in-vivo examination of the vascular system by using fluorescent and confocal microscopy. In this study, we developed transparent transgenic strain of Caspe/fli1:EGFP and integrating with recently developed casper/myl7/annexin-5:YFP and casper/fli1:EGFP strain through crossbreeding of casper and transgenic zebrafish. We have generated F1 generation of newly developed strains of Caspe/fli1:EGFP and casper/myl7/annexin-5:YFP and casper/fli1:EGFP. The presence of fluorescence expressions RFP, YFP and GFP, in crossbred progeny identified through the screening of larvae at 72 HPF stage. Expression of these fluorescent proteins determines the presence of the reporter genes. Positive transgenic larvae validated by confocal microscopy. The progenies of the F1 generation are heterozygous in nature and allow them to grow into adults to breed transparent transgenic strain to get F2 generation. This process will be continued to F2, F3, F4 and F5 generation until obtain homozygous progeny of newly developed strain. The development of casper/myl7/annexin-5:YFP x casper/fli1:EGFP will aid new dimensions in Cardio-oncology research for cardiac injury and Vasotoxicity induced by anticancer drug treatment in cancer patients. Citation Format: Surendra K. Rajpurohit, Taitum Gossman, Rohan Pasi, Sai Nasanally, Keshu Bhatt, Aiden Van Derhei, Simaran Hotwani, Devan-Anmol Manhiani, Kirk Kimmerling, Vishal Arora, Balakrishna L. Lokeshwar. Generation of transparent transgenic zebrafish strain to study cardiovascular functionin translational cardio-oncology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1431.

Magnetically powered microwheel thrombolysis of occlusive thrombi in zebrafish

Tissue plasminogen activator (tPA) is the only FDA-approved treatment for ischemic stroke but carries significant risks, including major hemorrhage. Additional options are needed, especially in small vessel thrombi which account for ~25% of ischemic strokes. We have previously shown that tPA-functionalized colloidal microparticles can be assembled into microwheels (µwheels) and manipulated under the control of applied magnetic fields to enable rapid thrombolysis of fibrin gels in microfluidic models of thrombosis. Transparent zebrafish larvae have a highly conserved coagulation cascade that enables studies of hemostasis and thrombosis in the context of intact vasculature, clotting factors, and blood cells. Here, we show that tPA-functionalized µwheels can perform rapid and targeted recanalization in vivo. This effect requires both tPA and µwheels, as minimal to no recanalization is achieved with tPA alone, µwheels alone, or tPA-functionalized microparticles in the absence of a magnetic field. We evaluated tPA-functionalized µwheels in CRISPR-generated plasminogen (plg) heterozygous and homozygous mutants and confirmed that tPA-functionalized µwheels are dose-dependent on plasminogen for lysis. We have found that magnetically powered µwheels as a targeted tPA delivery system are dramatically more efficient at plasmin-mediated thrombolysis than systemic delivery in vivo. Further development of this system in fish and mammalian models could enable a less invasive strategy for alleviating ischemia that is safer than directed thrombectomy or systemic infusion of tPA.

Zebrafish as a model for podocyte research.

Podocytes, specialized postmitotic cells, are central players in various kidney-related diseases. Zebrafish have become a valuable model system for studying podocyte biology because they are genetically easy to manipulate, transparent, and their glomerular structure is similar to that of mammals. This review provides an overview of the knowledge of podocyte biology in zebrafish larvae, with particular focus on their essential contribution to understanding the mechanisms that underlie kidney diseases as well as supporting drug development. In addition, special attention is given to advances in live-imaging techniques allowing the observation of dynamic processes, including podocyte motility, podocyte process behavior, and glomerulus maturation. The review further addresses the functional aspects of podocytes in zebrafish larvae. This includes topics such as glomerular filtration, ultrastructural analyses, and evaluation of podocyte response to nephrotoxic insults. Studies presented in this context have provided important insights into the maintenance and resistance of the glomerular filtration barrier in zebrafish larvae and explored the potential transferability of these findings to mammals such as mice, rats, and most importantly, humans. The recent ability to identify potential therapeutic targets represents a promising new way to identify drugs that could effectively treat podocyte-associated glomerulopathies in humans. In summary, this review gives an overview about the importance of zebrafish as a model for podocyte-related disease and targeted drug development. It also highlights the key role of advanced imaging techniques in transparent zebrafish larvae, improving our understanding of glomerular diseases and the significant potential for translation of these findings to humans.

Structural and Functional Organization of Visual Responses in the Inferior Olive of Larval Zebrafish.

The olivo-cerebellar system plays an important role in vertebrate sensorimotor control. Here, we investigate sensory representations in the inferior olive (IO) of larval zebrafish and their spatial organization. Using single-cell labeling of genetically identified IO neurons, we find that they can be divided into at least two distinct groups based on their spatial location, dendritic morphology, and axonal projection patterns. In the same genetically targeted population, we recorded calcium activity in response to a set of visual stimuli using two-photon imaging. We found that most IO neurons showed direction-selective and binocular responses to visual stimuli and that the functional properties were spatially organized within the IO. Light-sheet functional imaging that allowed for simultaneous activity recordings at the soma and axonal level revealed tight coupling between functional properties, soma location, and axonal projection patterns of IO neurons. Taken together, our results suggest that anatomically defined classes of IO neurons correspond to distinct functional types, and that topographic connections between IO and cerebellum contribute to organization of the cerebellum into distinct functional zones.

Full-field exposure of larval zebrafish to narrow waveband LED light sources at defined power and energy for optogenetic applications

BackgroundOptogenetic approaches in transparent zebrafish models have provided numerous insights into vertebrate neurobiology. The purpose of this study was to develop methods to activate light-sensitive transgene products simultaneously throughout an entire larval zebrafish. New methodWe developed a LED illumination stand and microcontroller unit to expose zebrafish larvae reproducibly to full field illumination at defined wavelength, power, and energy. ResultsThe LED stand generated a sufficiently flat illumination field to expose multiple larval zebrafish to high power light stimuli uniformly, while avoiding sample bath warming. The controller unit allowed precise automated delivery of predetermined amounts of light energy at calibrated power. We demonstrated the utility of the approach by driving photoconversion of Kaede (398 nm), photodimerization of GAVPO (450 nm), and photoactivation of dL5**/MG2I (661 nm) in neurons throughout the CNS of larval zebrafish. Observed outcomes were influenced by both total light energy and its rate of delivery, highlighting the importance of controlling these variables to obtain reproducible results. Comparison with existing methodsOur approach employs inexpensive LED chip arrays to deliver narrow-waveband light with a sufficiently flat illumination field to span multiple larval zebrafish simultaneously. Calibration of light power and energy are built into the workflow. ConclusionsThe LED illuminator and controller can be constructed from widely available materials using the drawings, instructions, and software provided. This approach will be useful for multiple optogenetic applications in zebrafish and other models.

The mesencephalic locomotor region recruits V2a reticulospinal neurons to drive forward locomotion in larval zebrafish

The mesencephalic locomotor region (MLR) is a brain stem area whose stimulation triggers graded forward locomotion. How MLR neurons recruit downstream vsx2+ (V2a) reticulospinal neurons (RSNs) is poorly understood. Here, to overcome this challenge, we uncovered the locus of MLR in transparent larval zebrafish and show that the MLR locus is distinct from the nucleus of the medial longitudinal fasciculus. MLR stimulations reliably elicit forward locomotion of controlled duration and frequency. MLR neurons recruit V2a RSNs via projections onto somata in pontine and retropontine areas, and onto dendrites in the medulla. High-speed volumetric imaging of neuronal activity reveals that strongly MLR-coupled RSNs are active for steering or forward swimming, whereas weakly MLR-coupled medullary RSNs encode the duration and frequency of the forward component. Our study demonstrates how MLR neurons recruit specific V2a RSNs to control the kinematics of forward locomotion and suggests conservation of the motor functions of V2a RSNs across vertebrates.

Exposure to the aryl hydrocarbon receptor agonist dioxin disrupts formation of the muscle, nerves, and vasculature in the developing jaw

Human exposure to environmental pollutants can disrupt embryonic development and impact juvenile and adult health outcomes by adversely affecting cell and organ function. Notwithstanding, environmental contamination continues to increase due to industrial development, insufficient regulations, and the mobilization of pollutants as a result of extreme weather events. Dioxins are a class of structurally related persistent organic pollutants that are highly toxic, carcinogenic, and teratogenic. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent dioxin compound and has been shown to induce toxic effects in developing organisms by activating the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor targeted by multiple persistent organic pollutants. Contaminant-induced AHR activation results in malformations of the craniofacial cartilages and neurocranium; however, the mechanisms mediating these phenotypes are not well understood. In this study, we utilized the optically transparent zebrafish model to elucidate novel cellular targets and potential transcriptional targets underlying TCDD-induced craniofacial malformations. To this end, we exposed zebrafish embryos at 4 h post fertilization to TCDD and employed a mixed-methods approach utilizing immunohistochemistry staining, transgenic reporter lines, fixed and in vivo confocal imaging, and timelapse microscopy to determine the targets mediating TCDD-induced craniofacial phenotypes. Our data indicate that embryonic TCDD exposure reduced jaw and pharyngeal arch Sox10+ chondrocytes and Tcf21+ pharyngeal mesoderm progenitors. Exposure to TCDD correspondingly led to a reduction in collagen type II deposition in Sox10+ domains. Embryonic TCDD exposure impaired development of tissues derived from or guided by Tcf21+ progenitors, namely: nerves, muscle, and vasculature. Specifically, TCDD exposure disrupted development of the hyoid and mandibular arch muscles, decreased neural innervation of the jaw, resulted in compression of cranial nerves V and VII, and led to jaw vasculature malformations. Collectively, these findings reveal novel structural targets and potential transcriptional targets of TCDD-induced toxicity, showcasing how contaminant exposures lead to congenital craniofacial malformations.

Abstract P2176: Development Of Integrated Transparent Transgenic Zebrafish Modeling System For Experimental Cardio-oncology Therapeutics

The zebrafish model has provided an excellent platform to study the genetic and molecular approach of basic and translational cardiac research. Zebrafish heart cells are similar to human heart cells at the molecular level and determine the function of genes that control cardiac function and dysfunction. In zebrafish, myl7 is myosin light chain-7 gene and identified as a regulatory gene of heart orthologs to human MYL7. In vertebrates, including zebrafish, murine and human systems, the in-vivo spatial resolution is limited due to the normal opacification of skin and subdermal structures. For in-vivo imaging the skin transparency is primary requirement and to maintain the transparency, blocking the pigmentation needs to maintain. Chemical inhibition treatment is temporary and possible till the organism treated with the chemical inhibitor agent. Zebrafish casper mutant maintain transparency throughout the life and serve as ideal combination of sensitivity and resolution for in-vivo stem cell analyses and in-vivo imaging. To maintain the skin transparency, in this study, we have developed transparent transgenic zebrafish model and established of time lapse in-vivo confocal microscopy to study of cellular phenotype/pathologies of the cardiomyocytes to quantify changes in cardiomyocyte morphology and function overtime by comparing control and cardiac injury by generating casper/ myl7:RFP; annexin-5:YFP transgenic zebrafish. In the field of cardio-oncology, we are developing in-vivo transparent transgenic cellular phenotype model of zebrafish to study of the cell death process (Annexin5) and Inflammatory activity (NFkB) in the microglia, and the resident macrophage of the brain and cardiomyocytes) homozygous casper/ myl7:RFP; annexin-5:YFP/NF-kB:GFP/mpeg1:mcherryFP transgenic strain zebrafish. By using this novel model our lab establishing the role of NFkB and microglia function under normal healthy status and in glioblastomas Our strategist approach to yield crucial new insights into in-vivo cardiomyocyte imaging by confocal microscopy to observe and track the cell death pattern and cardio inflammatory pathways in cardiomyocyte and develop novel therapeutic approaches to treat cardio inflammatory pathology and glioblastoma

Development of a Transparent Transgenic Zebrafish Cellular Phenotype Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-) to Study NF-kB Activity.

NF-κB signaling has broad effects on cell survival, tissue growth, and proliferation activities. It controls many genes that are involved in inflammation and thus is a key player in many inflammatory diseases. The elevation of NF-κB activators is associated with elevated mortality, especially in cancer and cardiovascular diseases. The zebrafish has emerged as an important model for whole-organism in vivo modeling in translational research. In vertebrates, in-vivo spatial resolution is limited due to normal opacification of skin and subdermal structure. For in vivo imaging, skin transparency by blocking the pigmentation via chemical inhibition is required and the maintenance of this transparency is vital. The Casper(roy-/-, nacre-/-) mutant of zebrafish maintains this transparency throughout its life and serves as an ideal combination of sensitivity and resolution for in vivo stem cell analyses and imaging. We developed an NF-kB:GFP/Casper transparent transgenic zebrafish cellular phenotype to study inflammatory processes in vivo. We outline the experimental setup to generate a transparent transgenic NF-kB/Casper strain of zebrafish through the cross-breeding of Casper and NF-kB transgenic adult fish and have generated F01 in the form of heterozygous progeny. The transgenic F01 progeny was further inbred to generate heterozygous progenies from F1 to F4 generations. Furthermore, it continued to successfully develop the homozygous strain Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-) in the F05 generation. This novel strain of F05 generation showed 100% homozygosity in the transgenic transparent progeny of Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-). The strain has been confirmed by generating the F06 generation of homozygous progeny and again verified and validated for its homogeneity in the F07 generation. The newly developed novel transparent transgenic strain of the NF-kB reporter line has been coined as "Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-)gmc1". We have established a newly generated phenotype of transparent transgenic zebrafish for time-lapse in vivo confocal microscopy to study the cellular phenotype and pathologies at the cellular level over time. This will allow for quantifying the changes in the NF-kB functional activities over time and allow the comparison of control and cardiac-oncology experimental therapeutics. We validated the newly developed Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-)gmc1 homozygous strain of zebrafish by studying the inflammatory response to bacterial lipopolysaccharide (LPS) exposure, tolerance, and the inhibitory role of a potential novel drug candidate against LPS-induced inflammation. The results establish the unique application of newly developed strains by identifying hit and lead drug candidates for experimental therapeutics.

Non-POU Domain-Containing Octomer-Binding (NONO) protein expression and stability promotes the tumorigenicity and activation of Akt/MAPK/β-catenin pathways in human breast cancer cells

Breast cancer is one of the most common cancers with a high mortality rate, underscoring the need to identify new therapeutic targets. Here we report that non-POU domain-containing octamer-binding (NONO) protein is overexpressed in breast cancer and validated the interaction of the WW domain of PIN1 with c-terminal threonine-proline (thr-pro) motifs of NONO. The interaction of NONO with PIN1 increases the stability of NONO by inhibiting its proteasomal degradation, and this identifies PIN1 as a positive regulator of NONO in promoting breast tumor development. Functionally, silencing of NONO inhibits the growth, survival, migration, invasion, epithelial to mesenchymal transition (EMT), and stemness of breast cancer cells in vitro. A human metastatic breast cancer cell xenograft was established in transparent zebrafish (Danio rerio) embryos to study the metastatic inability of NONO-silenced breast cancer cells in vivo. Mechanistically, NONO depletion promotes the expression of the PDL1 cell-surface protein in breast cancer cells. The identification of novel interactions of NONO with c-Jun and β-catenin proteins and activation of the Akt/MAPK/β-catenin signaling suggests that NONO is a novel regulator of Akt/MAPK/β-catenin signaling pathways. Taken together, our results indicated an essential role of NONO in the tumorigenicity of breast cancer and could be a potential target for anti-cancerous drugs.C-mep14XPctyTeLC54ZqwrVideo

A tessellated lymphoid network provides whole-body T cell surveillance in zebrafish

Homeostatic trafficking to lymph nodes allows T cells to efficiently survey the host for cognate antigen. Nonmammalian jawed vertebrates lack lymph nodes but maintain diverse T cell pools. Here, we exploit invivo imaging of transparent zebrafish to investigate how T cells organize and survey for antigen in an animal devoid of lymph nodes. We find that naïve-like T cells in zebrafish organize into a previously undescribed whole-body lymphoid network that supports streaming migration and coordinated trafficking through the host. This network has the cellular hallmarks of a mammalian lymph node, including naïve T cells and CCR7-ligand expressing nonhematopoietic cells, and facilitates rapid collective migration. During infection, T cells transition to a random walk that supports antigen-presenting cell interactions and subsequent activation. Our results reveal that T cells can toggle between collective migration and individual random walks to prioritize either large-scale trafficking or antigen search insitu. This lymphoid network thus facilitates whole-body T cell trafficking and antigen surveillance in the absence of a lymph node system.

Let's talk about sex… obesity and melanoma

The World Health Organization (WHO) has recently reported that more than 39% of adults are currently overweight or obese, globally, raising concerns for the prevalence of obesity-related conditions, such as certain cancers, including melanoma. Moreover, the WHO estimated that the global number of cases of melanoma – the most lethal skin cancer – are due to increase by more than 50% between 2020 and 2040. Despite this, the link between obesity and melanoma remains unclear, although some human studies have reported sex-dependent effects that impact this association.To examine this further, Emily Montal and colleagues in Richard White's lab investigated the effect of genetic- and diet-induced obesity, as well as sex, on melanoma initiation and progression in zebrafish. The authors overexpressed AgRP – a neuropeptide that regulates food intake – in embryonic zebrafish. AgRP overexpression increased several clinical signs of obesity when the fish reached adulthood and augmented these obesogenic effects in response to a high fat diet (HFD). Interestingly, males and females showed differences in these clinical signs of obesity. These zebrafish also harboured a mutated BRAF oncogene in the embryonic melanocytes to allow initiation of melanoma later in life. Montal and colleagues examined melanoma onset and progression by imaging the optically transparent casper zebrafish to assess hyperpigmentation and lesion growth, and found that obese male zebrafish overexpressing AgRP developed melanoma tumours significantly faster than their lean counterparts; however, this was not observed in females. The team then used a more-precise approach to induce melanoma by specifically activating BRAF in adult zebrafish skin cells. They found that, in male zebrafish, AgRP overexpression and HFD, alone or combined, enhanced tumour initiation and, to a lesser extent, tumour progression. However, in female zebrafish, enhanced tumour initiation was only detected when AgRP overexpression and HFD were combined, and no effect was observed on tumour progression at later stages.In their article, White and colleagues present the first account of melanoma initiation dependent on sex as well as obesity in an animal model, which crucially mirrors findings on sex differences in human studies. With rising prevalence of obesity and melanoma worldwide, this study sets the stage to delineate the mechanism underlying these important interactions. Furthermore, with historic sex-bias in scientific research causing disparity in patient treatment, studies that unravel sex-dependent mechanisms of disease are of utmost importance.

Proper modulation of AHR signaling is necessary for establishing neural connectivity and oligodendrocyte precursor cell development in the embryonic zebrafish brain.

2,3,7,8-tetrachlorodibenzo-[p]-dioxin (TCDD) is a persistent global pollutant that exhibits a high affinity for the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. Epidemiological studies have associated AHR agonist exposure with multiple human neuropathologies. Consistent with the human data, research studies using laboratory models have linked pollutant-induced AHR activation to disruptions in learning and memory as well as motor impairments. Our understanding of endogenous AHR functions in brain development is limited and, correspondingly, scientists are still determining which cell types and brain regions are sensitive to AHR modulation. To identify novel phenotypes resulting from pollutant-induced AHR activation and ahr2 loss of function, we utilized the optically transparent zebrafish model. Early embryonic TCDD exposure impaired embryonic brain morphogenesis, resulted in ventriculomegaly, and disrupted neural connectivity in the optic tectum, habenula, cerebellum, and olfactory bulb. Altered neural network formation was accompanied by reduced expression of synaptic vesicle 2. Loss of ahr2 function also impaired nascent network development, but did not affect gross brain or ventricular morphology. To determine whether neural AHR activation was sufficient to disrupt connectivity, we used the Gal4/UAS system to express a constitutively active AHR specifically in differentiated neurons and observed disruptions only in the cerebellum; thus, suggesting that the phenotypes resulting from global AHR activation likely involve multiple cell types. Consistent with this hypothesis, we found that TCDD exposure reduced the number of oligodendrocyte precursor cells and their derivatives. Together, our findings indicate that proper modulation of AHR signaling is necessary for the growth and maturation of the embryonic zebrafish brain.