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Abstract CHD2‐related epilepsy is characterized by early‐onset photosensitive myoclonic epilepsy with developmental delay and a high rate of pharmacoresistance. We sought to evaluate the efficacy of acetazolamide (ACZ) in CHD2‐related epilepsy, due to ACZ's unexpected efficacy in our first patient harboring a pathogenic CHD2 variant. We collected patients from different Eastern European countries with drug‐resistant CHD2‐related epilepsy who were then treated with ACZ. Patients underwent video EEG before and during ACZ treatment. In a zebrafish model of CHD2‐related epilepsy, ictal‐like events were recorded 5 days post‐fertilization after overnight ACZ exposure. Developmental delay preceded the onset of seizures in 10 of the 12 patients. Four had ataxia, and 6 exhibited autistic features. Seizures, primarily myoclonic, began at an average age of 3.4 years and were photosensitive in all 12 patients. Add‐on ACZ treatment controlled photosensitive seizures in all patients: 6 became seizure‐free, and in the remaining 6, seizure frequency decreased by over 75%. Four patients transitioned to ACZ monotherapy. The median follow‐up was 13 months. In the zebrafish model, ACZ exposure reduced ictal‐like events by 72%. ACZ, a well‐tolerated and cost‐effective medication, could be a good option for CHD2‐related epilepsy, predominantly manifesting with myoclonic seizures and photosensitivity. Plain Language Summary Epilepsy associated with CHD2 mutations is often pharmacoresistant and associated with developmental delay and eventually ataxia. There are several generalized seizure types, including generalized tonic–clonic seizures, but the most characteristic are jerks triggered by light stimulation. We collected 12 patients who received acetazolamide, a drug usually given as a diuretic and registered as a mild antiseizure medication. All jerks triggered by light disappeared while the frequency of spontaneous seizures decreased by over 75%. Further studies are needed to confirm this promising finding and identify the mechanism by which an old compound seems to have such a specific antiseizure effect.

Although the concept of endocrine disruptors first appeared almost 30 years ago, the relatively recent involvement of these substances in the etiology of metabolic pathologies (obesity, diabetes, hepatic steatosis, etc.) has given rise to the concept of Metabolic Disrupting Chemicals (MDCs). Organs such as the liver and adipose tissue have been well studied in the context of metabolic disruption by these substances. The intestine, however, has been relatively unexplored despite its close link with these organs. In vivo models are useful for the study of the effects of MDCs in the intestine and, in addition, allow investigations into interactions with the rest of the organism. In the latter respect, the zebrafish is an animal model which is used increasingly for the characterization of endocrine disruptors and its use as a model for assessing effects on the intestine will, no doubt, expand. This review aims to highlight the importance of the intestine in metabolism and present the zebrafish as a relevant alternative model for investigating the effect of pollutants in the intestine by focusing, in particular, on cytochrome P450 3A (CYP3A), one of the major molecular players in endogenous and MDCs metabolism in the gut.

ABSTRACT Tin (Sn) is one of the heavy metals to which various functions in biological development are attributed in small quantities, for example, a role in cell structure, enzyme activities, and protein and carbohydrate metabolism in fish. Tributyltin (TBT) and triphenyltin (TPT) are endocrine disrupting chemicals that are widely distributed in the aquatic environment. The aim of this study was to investigate the effects of TBT, TPT, and SnCl2 on the embryonic development of zebrafish. In this study, zebrafish embryos were used to observe the acute toxicity of TBT, TPT, and SnCl2. Fish embryo toxicity analysis was performed for different TBT, TPT, and SnCl2 concentrations (1, 5, 10, 15, and 20 ng/L). Fertilized eggs in 24‐well plates (20 eggs in each concentration) were incubated at 26°C for 4 days, and embryo clotting, embryo heartbeat, and morphological abnormalities were recorded after 96 h. Coagulation increased significantly in a dose‐dependent manner, and TBT was able to induce coagulation in zebrafish embryos. Heartbeat changes significantly decreased (p < 0.05) in a dose‐dependent manner at different TBT doses (p < 0.05). The percentage of mortality was higher in embryos at trace levels of TBT, indicating that the embryos are more sensitive to the toxicity of TBT. TBT is therefore extremely toxic and can have fatal consequences for zebrafish embryos, ultimately leading to the extinction of species and a decline in biodiversity in the aquatic environment. After analyzing the images 96 h after fertilization and converting them to a teratogenic index score, it was found that the index for the two compounds TPT and TBT was 4.6 and 9.6 ng/L, respectively.

Abstract Although bold and shy behavioral phenotypes in zebrafish (Danio rerio) have been selectively bred and maintained over multiple generations, it is unclear if they are underscored by different gut microbiota. Using the microbiota–gut–brain concept, we examined the relationship between gut microbiota and the behavioral phenotypes within this model animal system to assess possible gut microbe‐mediated effects on host behavior. To this end, we amplified and sequenced 16S rRNA gene amplicons from the guts of bold and shy zebrafish individuals using the Illumina Miseq platform. We did not record any significant differences in within‐group microbial diversity nor between‐group community composition of the two behavioral phenotypes. Interestingly, though not statistically different, we determined that the gut microbial community of the bold phenotype was dominated by Burkholderiaceae, Micropepsaceae, and Propionibacteriaceae. In contrast, the shy phenotype was dominated by Beijerinckaceae, Pirelullacaeae, Rhizobiales_Incertis_Sedis, and Rubinishaeraceae. The absence of any significant difference in gut microbiome profiles between the two phenotypes would suggest that in this species, there might exist a stable core gut microbiome, regardless of behavioral phenotypes, and possibly, a limited role for the gut microbiota in modulating this selected‐for host behavior. This study characterized the gut microbiomes of distinct innate behavioral phenotypes of the zebrafish (that are not considered dysbiotic states) and did not rely on antibiotic or probiotic treatments to induce changes in behavior. Such studies are crucial to our understanding of the modulating impacts of the gut microbiome on normative animal behavior.

Abstract Human exposure to Vibrio vulnificus, a gram‐negative, halophilic environmental pathogen, is increasing. Despite this, the mechanisms of its pathogenicity and virulence remain largely unknown. Each year, hundreds of infections related to V. vulnificus occur, leading to hospitalization in 92% of cases and a mortality rate of 35%. The infection is severe, typically contracted through the consumption of contaminated food or exposure of an open wound to contaminated water. This can result in necrotizing fasciitis and the need for amputation of the infected tissue. Although several genes (rtxA1, vvpE, and vvhA) have been implicated in the pathogenicity of this organism, a defined mechanism has not been discovered. In this study, we examine environmentally isolated V. vulnificus strains using a zebrafish model (Danio rerio) to investigate their virulence capabilities. We found significant variation in virulence between individual strains. The commonly used marker gene of disease‐causing strains, vcgC, did not accurately predict the more virulent strains. Notably, the least virulent strain in the study, V. vulnificus Sept WR1‐BW6, which tested positive for vcgC, vvhA, and rtxA1, did not cause severe disease in the fish and was the only strain that did not result in any mortality. Our study demonstrates that virulence varies greatly among different environmental strains and cannot be accurately predicted based solely on genotype.

Abstract Animal‐based research and drug safety studies are essential to understanding the mysteries of nature and the long‐term survival of humans. Due to the rapid increase in the global human population, conflict‐ and economically driven human migration, tourism‐related activities, densely populated metropolitan areas, and local policies, humans will be affected by a multitude of novel disease‐causing microorganisms and civilizational diseases. Despite disparities among countries, recent and planned changes in regulations concerning animal research and drug safety studies could have detrimental effects on both the animal research community and nations lacking sufficient social support systems. Based on existing scientific literature, I argue that we need animal research encompassing aspects such as animal development, behavior, drug safety studies, and for the understanding of future civilizational diseases. Depending on the nature of the research questions and local challenges, a suitable animal model organism should be made mandatory.

Abstract Lactobacillus rhamnosus GG (LGG), the well‐characterized human‐derived probiotic strain, possesses excellent properties in the maintenance of intestinal homeostasis, immunoregulation and defense against gastrointestinal pathogens in mammals. Here, we demonstrate that the SpaC pilin of LGG causes intestinal epithelium injury by inducing cell pyroptosis and gut microbial dysbiosis in zebrafish. Dietary SpaC activates Caspase‐3−GSDMEa pathways in the intestinal epithelium, promotes intestinal pyroptosis and increases lipopolysaccharide (LPS)‐producing gut microbes in zebrafish. The increased LPS subsequently activates Gaspy2−GSDMEb pyroptosis pathway. Further analysis reveals the Caspase‐3−GSDMEa pyroptosis is initiated by the species‐specific recognition of SpaC by TLR4ba, which accounts for the species‐specificity of the SpaC‐inducing intestinal pyroptosis in zebrafish. The observed pyroptosis‐driven gut injury and microbial dysbiosis by LGG in zebrafish suggest that host‐specific beneficial/harmful mechanisms are critical safety issues when applying probiotics derived from other host species and need more attention.

Abstract This trial was performed in order to investigate the response of zebrafish (Danio rerio) to dietary exposure to polystyrene nanoplastics (PS‐NPs) under laboratory conditions on fish growth and health. Healthy zebrafish (n=240) were divided into 12 tanks and fed with diets including 0 (T0), 100 (T1), 500 (T2), and 1000 (T3) mg kg−1 synthesized polystyrenes nanoplastics (PS‐NPs) for 30 days. At the end of trial, fish fed the PS‐NPs supplementation showed weight gain percentages of 79.45%, 70.35%, and 61.88% for T1, T2, and T3 groups, respectively, compared with 87.39% in the control fish. The SOD, GPX and MDA activities and cortisol levels increased by 47%, 32% and 35% and 46%, respectively, especially at high‐dose administration (p

In vertebrates, nutritional tastants, such as amino acids and sugars, are recognized by G‐protein‐coupled receptors of the taste receptor type 1 (T1R) family. Previous studies have shown that fish T1Rs are functionally distinct from mammalian T1Rs in certain regards. Here, we report the existence of oral receptors with high sensitivity to amino acids in zebrafish and medaka fish. We describe the construction of multiple cell lines stably expressing functional T1Rs (from medaka fish or zebrafish) with a chimeric G‐protein (G16gust44) using the Flp‐In system. Through functional assays with these cell lines, medaka fish and zebrafish were confirmed to possess particular T1Rs highly sensitive to l‐proline, possibly reflecting the physiological importance of l‐proline in teleosts, in line with previous studies.

Abstract Recently, zebrafish has received more and more attention due to its wide range of applications such as regeneration promoting therapeutics and drug discovery. Therefore, vision‐based trackers are utilized to record the swimming trajectory of zebrafish. In this paper, a re‐association method is introduced in the 3D reconstruction process to generate missed targets caused by occlusion. Since the variation of the overall tracking targets has the property of continuity and stability, a topology association model (TAM) is proposed by point group similarity into the tracking framework. TAM describes the movement of zebrafish from the macroscopic level and utilizes the changes of the point group structure for tracking. Experimental results show that the tracking framework enhances the overall performance and promotes the trajectory integrity. On the latest 3D‐ZeF20 benchmark, state‐of‐the‐art results are achieved. In addition, TAM tracking framework is applied to 2D general tracking to prove that the method is useful and have great advantage in other scenarios with relatively stable amount of targets as well.

Abstract CRISPR‐Cas9–generated zebrafish carrying a 12 base‐pair deletion in stxbpb1b, a paralog sharing 79% amino acid sequence identity with human, exhibit spontaneous electrographic seizures during larval stages of development. Zebrafish stxbp1b mutants provide an efficient preclinical platform to test antiseizure therapeutics. The present study was designed to test antiseizure medications approved for clinical use and two recently identified repurposed drugs with antiseizure activity. Larval homozygous stxbp1b zebrafish (4 days postfertilization (dpf)) were agarose‐embedded and monitored for electrographic seizure activity using a local field recording electrode placed in midbrain. Frequency of ictal‐like events was evaluated at baseline and following 45 min of continuous drug exposure (1 mM, bath application). Analysis was performed on coded files by an experimenter blinded to drug treatment and genotype. Phenytoin (PHT), valproate (VPA), ethosuximide (ESX), levetiracetam (LEV), and diazepam (DZP) had no effect on the ictal‐like event frequency in stxbp1b mutant zebrafish. Clemizole and trazodone decreased ictal‐like event frequency in stxbp1b mutant zebrafish by 80% and 83%, respectively. These results suggest that repurposed drugs with serotonin receptor–binding affinities could be effective antiseizure treatments. Clemizole and trazodone were previously identified in a larval zebrafish model for Dravet syndrome. Based primarily on these preclinical zebrafish studies, compassionate‐use and double‐blind clinical trials with both drugs have progressed. The present study extends this approach to a preclinical zebrafish model representing STXBP1 (syntaxin‐binding protein 1)‐related disorders and suggests that future clinical studies may be warranted.

Abstract Background Aconitum carmichaelii, the mother root of Aconitum, has a long‐applied history for treating many diseases in China. Increased use of it has prompted significant concerns regarding its extensive cardiotoxicity and neurotoxicity. The molecular mechanisms underlying A. carmichaelii‐induced neurotoxicity are poorly understood. Methods We took advantage of the zebrafish model to investigate the neurotoxic mechanism of A. carmichaelii. In addition to the behavior and neuronal activity testing, gene expression of neuronal oxidative stress, and apoptosis levels were also analyzed. Results In contrast to the excitatory effect of low‐dose A. carmichaelii decoction, hypoactivity of locomotor behavior, and neural activity, especially telencephalon, were detected in the 20 mg/mL group. High doses of A. carmichaelii induced excessive ROS by downregulating DJ‐1 and activating Nox5, and further triggered cell apoptosis through the bax/bcl2a‐caspase‐9 pathway in zebrafish larva. Mitochondrial protection‐related genes PinK1 and Parkin were upregulated to protect against mitochondrial dysfunction during cellular stress. The ROS scavenger‐NAC significantly alleviated neurotoxicity in the 20 mg/mL group. Conclusion The study reveals the potential mechanism of A. carmichaelii‐induced neurotoxicity and provides new insights into a significant risk marker of A. carmichaelii poisoning.

Tollip is a multifunctional adaptor protein implicated in innate immunity, lysosomal trafficking/autophagy of protein aggregates and various signaling processes in mammalian models. To verify evolutionary conservation of these functions, we used CRISPR/Cas9 editing to construct a zebrafish line bearing a stable tollip knockout. In contrast to previously reported tollip morphants, Tollip‐deficient fish display normal development until adulthood, are fertile, and have no apparent physiological defects. When challenged with lipopolysaccharide (LPS), inflammatory gene expression is unaffected. Moreover, Tollip deficiency does not aggravate swimming deficiency resulting from lysosomal dysfunction and proteotoxicity in a fish model of Gaucher disease. Thus, individual functions of Tollip may be organism‐specific or manifest only upon certain conditions/challenges or disease backgrounds.

Abstract Zebrafish (Danio rerio) have attracted much attention over the past decade as a reliable model for gut microbiome research. Owing to their low cost, strong genetic and development coherence, efficient preparation of germ‐free (GF) larvae, availability in high‐throughput chemical screening, and fitness for intravital imaging in vivo, zebrafish have been extensively used to investigate microbiome‐host interactions and evaluate the toxicity of environmental pollutants. In this review, the advantages and disadvantages of zebrafish for studying the role of the gut microbiome compared with warm‐blooded animal models are first summarized. Then, the roles of zebrafish gut microbiome on host development, metabolic pathways, gut‐brain axis, and immune disorders and responses are addressed. Furthermore, their applications for the toxicological assessment of aquatic environmental pollutants and exploration of the molecular mechanism of pathogen infections are reviewed. We highlight the great potential of the zebrafish model for developing probiotics for xenobiotic detoxification, resistance against bacterial infection, and disease prevention and cure. Overall, the zebrafish model promises a brighter future for gut microbiome research.

Abstract Background TDCPP is one of the major chemical of organophosphate flame retardants (OPFRs) that has been detected ubiquitously in both the environment and biota. Previously we observed that it influenced the concentrations of sex and thyroid hormones in a sex‐dependent pattern, leading to reproductive impairments after short‐term exposure in zebrafish. Here we investigate the consequences of longer‐term exposure to TDCPP on the hypothalamic‐pituitary‐gonad (HPG), hypothalamic‐pituitary‐interrenal (HPI), and hypothalamic‐pituitary‐thyroid (HPT) axes of zebrafish (Danio rerio). Methods A 120‐day exposure test to 0.005, 0.05 and 0.5 mg/L TDCPP was initiated with fertilized eggs. Sex steroid hormones in the treated fishes were measured and transcriptional changes were analyzed. Results In female fish, exposure to TDCPP resulted in increases in plasma cortisol, follicle stimulating hormone (FSH), luteinizing hormone (LH), 17β‐estradiol (E2), cortisol, thyroxine (T4), and triiodothyronine (T3). Transcription of most target genes along HPG, HPI and HPT axes were increased by the exposure. While in male fish the exposure led to decreases in cortisol, FSH, LH, T4, T3, testosterone (T), and 11‐ketotestosterone (11‐KT). Transcription of genes along HPG, HPI and HPT axes, especially steroidogenic genes, were inhibited in male zebrafish. While, E2/T or E2/11‐KT ratio was increased in both female and females. The sex‐dependent changes in hormones might be due to differential responses to TDCPP induced stresses. An increase in cortisol level coincided with increases in E2 and THs in female fish, while in males decreases in cortisol as well as T, 11‐KT and THs were observed. Long‐term exposure to TDCPP at very low (μg/L) concentrations could disrupt hormone balances in a sex dependent way. Conclusion This study revealed that TDCPP could affect endocrine axes – HPG, HPI and HPT – in zebrafish, and impair zebrafish development.

Abstract The pharyngeal arch (PA) is a neural crest (NC)‐derived organ that is transiently developed during embryogenesis and is required for the subsequent development of various tissues. However, the role of zinc during PA differentiation from NC progenitor cells is unknown. Here, we found that the metal transporters Slc30a1a and Slc30a1b mediate zinc homeostasis during PA differentiation. Slc30a1‐deficient zebrafish develop zinc accumulation in NC cells, with increased expression of stemness markers and PA dorsal genes, and SMART‐seq analyses revealed that the genes snai2 and jag1b may serve as downstream targets. Furthermore, functional studies showed that knocking down either snai2 or jag1b rescues PA development in Slc30a1‐deficient zebrafish. Notably, we identified the double zinc‐finger domain in the transcription factor Snai2 as a zinc‐responsive element that regulates jag1b expression. Our findings indicate that the Slc30a1/zinc‐snai2‐jag1b axis is an essential regulatory network controlling PA differentiation, shedding new light on the function of zinc homeostasis in maintaining NC cell stemness and multipotency in vertebrates.

H 2 O 2 is a significant reactive oxygen species (ROS) that hinders redox-mediated processes and contributes to oxidative stress and neurodegenerative disorders. Oxidative stress causes impairment of cell macromolecules, which results in cell dysfunction and neurodegeneration. Alzheimer's disease and other neurodegenerative diseases are serious conditions linked to oxidative stress. Antioxidant treatment approaches are a novel and successful strategy for decreasing neurodegeneration and reducing oxidative stress. This study explored the antioxidant and neuroprotective characteristics of KK14 peptide synthesized from LEAP 2B (liver-expressed antimicrobial peptide-2B) derived from Cyprinus carpio L. Molecular docking studies were used to assess the antioxidant properties of KK14. The peptide at concentrations 5-45 μM was examined by using in vitro and in vivo assessment. Analysis was done on the developmental and neuroprotective potential of KK14 peptide treatment in H 2 O 2 -exposed zebrafish larvae which showed Nonlethal deformities. KK14 improves antioxidant enzyme activity like catalase and superoxide dismutase. Furthermore, it reduces neuronal damage by lowering lipid peroxidation and nitric oxide generation while increasing acetylcholinesterase activity. It improved the changes in swimming behavior and the cognitive damage produced by exposure to H 2 O 2 . To further substantiate the neuroprotective potential of KK14, intracellular ROS levels in zebrafish larvae were assessed. This led to a reduction in ROS levels and diminished lipid peroxidation. The KK14 has upregulated the antioxidant genes against oxidative stress. Overall, this study proved the strong antioxidant activity of KK14, suggesting its potential as a strong therapeutic option for neurological disorders caused by oxidative stress., (© 2024 Wiley Periodicals LLC.)

Background: FOXE1 mutations in humans are associated with cleft palate and hypothyroidism. We previously developed a foxe1 mutant zebrafish demonstrating mineralization defects in larvae. In the present study, we investigate the thyroid status and skeletal phenotype of adult foxe1 mutants., Results: Mutant fish have increased expression of tshβ in the pituitary, and of hepatic dio1 and dio2. In plasma, we found higher Mg levels. Together these findings are indicative of hypothyroidism. We further observed mineralization defects in scales due to enhanced osteoclast activity as measured by increased expression levels of tracp, ctsk, and rankl. Gene-environment interactions in the etiology of FOXE1-related craniofacial abnormalities remain elusive, which prompts the need for models to investigate genotype-phenotype associations. We here investigated whether ethanol exposure increases the risk of developing craniofacial malformations in foxe1 mutant larvae that we compared to wild types. We found in ethanol-exposed mutants an increased incidence of developmental malformations and marked changes in gene expression patterns of cartilage markers (sox9a), apoptotic markers (casp3b), retinoic acid metabolism (cyp26c1), and tissue hypoxia markers (hifaa, hifab)., Conclusion: Taken together, this study shows that the foxe1 mutant zebrafish recapitulates phenotypes associated with FOXE1 mutations in human patients and a clear foxe1-ethanol interaction., (© 2024 The Author(s). Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Renowned for their role in haemostasis and thrombosis, platelets are also increasingly recognized for their contribution in innate immunity, immunothrombosis and inflammatory diseases. Platelets express a wide range of receptors, which allows them to reach a variety of activation endpoints and grants them immunomodulatory functions. Activated platelets release extracellular vesicles (PEVs), whose formation and molecular cargo has been shown to depend on receptor-mediated activation and environmental cues. This study compared the immunomodulatory profiles of PEVs generated via activation of platelets by different receptors, glycoprotein VI, C-type lectin-like receptor 2 and combining all thrombin-collagen receptors. Functional assays in vivo in zebrafish and in vitro in human macrophages highlighted distinct homing and secretory responses triggered by the PEVs. In contrast, omics analyses of protein and miRNA cargo combined with physicochemical particle characterization found only subtle differences between the activated PEV types, which were insufficient to predict their different immunomodulatory functions. In contrast, constitutively released PEVs, formed in the absence of an exogenous activator, displayed a distinct immunomodulatory profile from the receptor-induced PEVs. Our findings underscore that PEVs are tunable through receptor-mediated activation. To truly comprehend their role(s) in mediating platelet functions among immune cells, conducting functional assays is imperative., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Background: Anemia is defined as a lack of erythrocytes, low hemoglobin levels, or abnormal erythrocyte morphology. Diamond-Blackfan anemia (DBA) is a rare and severe congenital hypoplastic anemia that occurs due to the dominant inheritance of a ribosomal protein gene mutation. Even rarer is a case described as Diamond-Blackfan anemia like (DBAL), which occurs due to a loss-of-function EPO mutation recessive inheritance. The effective cures for DBAL are bone marrow transfusion and treatment with erythropoiesis-stimulating agents (ESAs). To effectively manage the condition, construction of DBAL models to identify new medical methods or screen drugs are necessary., Results: Here, an epoa-deficient mutant zebrafish called epoa szy8 was generated to model DBAL. The epoa-deficiency in zebrafish caused developmental defects in erythroid cells, leading to severe congenital anemia. Using the DBAL model, we validated a loss-of-function EPO mutation using an in vivo functional analysis and explored the ability of ESAs to alleviate congenital anemia., Conclusions: Together, our study demonstrated that epoa deficiency in zebrafish leads to a phenotype resembling DBAL. The DBAL zebrafish model was found to be beneficial for the in vivo assessment of patient-derived EPO variants with unclear implications and for devising potential therapeutic approaches for DBAL., (© 2024 American Association for Anatomy.)

Toll-like receptor 2 (TLR2) belongs to the TLR protein family that plays an important role in the immune and inflammation response system. While TLR2 is predominantly expressed in immune cells, its expression has also been detected in the brain, specifically in microglia and astrocytes. Recent studies indicate that genomic deletion of TLR2 can result in impaired neurobehavioural function. It is currently not clear if the genomic deletion of TLR2 leads to any alterations in the microstructural features of the brain. In the current study, we noninvasively assess microstructural changes in the brain of TLR2-deficient (tlr2 -/- ) zebrafish using state-of-the art magnetic resonance imaging (MRI) methods at ultrahigh magnetic field strength (17.6 T). A significant increase in cortical thickness and an overall trend towards increased brain volumes were observed in young tlr2 -/- zebrafish. An elevated T 2 relaxation time and significantly reduced apparent diffusion coefficient (ADC) unveil brain-wide microstructural alterations, potentially indicative of cytotoxic oedema and astrogliosis in the tlr2 -/- zebrafish. Multicomponent analysis of the ADC diffusivity signal by the phasor approach shows an increase in the slow ADC component associated with restricted diffusion. Diffusion tensor imaging and diffusion kurtosis imaging analysis revealed diminished diffusivity and enhanced kurtosis in various white matter tracks in tlr2 -/- compared with control zebrafish, identifying the microstructural underpinnings associated with compromised white matter integrity and axonal degeneration. Taken together, our findings demonstrate that the genomic deletion of TLR2 results in severe alterations to the microstructural features of the zebrafish brain. This study also highlights the potential of ultrahigh field diffusion MRI techniques in discerning exceptionally fine microstructural details within the small zebrafish brain, offering potential for investigating microstructural changes in zebrafish models of various brain diseases., (© 2024 The Author(s). NMR in Biomedicine published by John Wiley & Sons Ltd.)

Backgound: Skeletal and cardiac muscles are contractile tissues whose development and function are dependent on genetic programs that must be precisely orchestrated in time and space. In addition to transcription factors, RNA-binding proteins tightly regulate gene expression by controlling the fate of RNA transcripts, thus specific proteins levels within the cell. Rbm24 has been identified as a key player of myogenesis and cardiomyogenesis in several vertebrates, by controlling various aspects of post-transcriptional regulation, including pre-mRNA alternative splicing and mRNA stabilization. In zebrafish, knockdown of rbm24a or rbm24b also causes skeletal and cardiac muscle phenotypes, but how their combined loss affects muscle integrity and function remains elusive., Results: By genome editing, we have generated rbm24a and rbm24b single mutants as well as double mutants. Structural analyses indicate that homozygous rbm24a and rbm24b double mutants exhibit severe somitic muscle and cardiac phenotypes, although rbm24b single mutants are obviously normal. We further show that the loss of rbm24a and rbm24b disrupts sarcomere organization, impairing functional contractility and motility of skeletal and cardiac muscles., Conclusion: The rbm24 mutant zebrafish represents a new genetic tool for in-depth studies of Rbm24-mediated post-transcriptional regulation of skeletal and cardiac muscle development, disease and regeneration., (© 2024 The Author(s). Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Background: The vertebrate enteric nervous system (ENS) consists of a series of interconnected ganglia within the gastrointestinal (GI) tract, formed during development following migration of enteric neural crest cells (ENCCs) into the primitive gut tube. Much work has been done to unravel the complex nature of extrinsic and intrinsic factors that regulate processes that direct migration, proliferation, and differentiation of ENCCs. However, ENS development is a complex process, and we still have much to learn regarding the signaling factors that regulate ENCC development., Results: Here in zebrafish, through transcriptomic, in situ transcript expression, immunohistochemical analysis, and chemical attenuation, we identified a time-dependent role for bone morphogenetic protein (BMP) in the maintenance of Phox2bb + enteric progenitor numbers and/or time of differentiation of the progenitor pool. In support of our in silico transcriptomic analysis, we identified expression of a novel ENS ligand-encoding transcript, bmp5, within developmental regions of ENCCs. Through generation of a novel mutant bmp5 wmr2 and bmp5 crispants, we identified a functional role for BMP5 in proper GI tract colonization, whereby phox2bb + enteric progenitor numbers were reduced., Conclusion: Altogether, this work identified time-dependent roles for BMP signaling and a novel extrinsic factor, BMP5, that is necessary for vertebrate ENS formation., (© 2024 American Association for Anatomy.)

The vertebrate retina is a tractable system for studying control of cell neurogenesis and cell fate specification. During embryonic development, retinal neurogenesis is under strict temporal regulation, with cell types generated in fixed but overlapping temporal intervals. The temporal sequence and relative numbers of retinal cell types generated during development are robust and show minimal experience-dependent variation. In many cold-blooded vertebrates, acute retinal injury induces a different form of neurogenesis, where Müller glia reprogram into retinal progenitor-like cells that selectively regenerate retinal neurons lost to injury. The extent to which the molecular mechanisms controlling developmental and injury-induced neurogenesis resemble one another has long been unclear. However, a recent study in zebrafish has shed new light on this question, using single-cell multiomic analysis to show that selective loss of different retinal cell types induces the formation of fate-restricted Müller glia-derived progenitors that differ both from one another and from progenitors in developing retina. Here, we discuss the broader implications of these findings, and their possible therapeutic relevance., (© 2024 Wiley Periodicals LLC.)

Abstract To harmoniously coordinate the activities of all its different cell types, a multicellular organism critically depends on intercellular communication. One recently discovered mode of intercellular cross‐talk is based on the exchange of "extracellular vesicles" (EVs). EVs are nano‐sized heterogeneous lipid bilayer vesicles enriched in a variety of biomolecules that mediate short‐ and long‐distance communication between different cells, and between cells and their environment. Numerous studies have demonstrated important aspects pertaining to the dynamics of their release, their uptake, and sub‐cellular fate and roles in vitro. However, to demonstrate these and other aspects of EV biology in a relevant, fully physiological context in vivo remains challenging. In this review we analyze the state of the art of EV imaging in vivo, focusing in particular on zebrafish as a promising model to visualize, study, and characterize endogenous EVs in real‐time and expand our understanding of EV biology at cellular and systems level.

Abstract Objective A significant number of epileptic patients fail to respond to available anticonvulsive medications. To find new anticonvulsive medications, we evaluated FDA‐approved drugs not known to be anticonvulsants. Using zebrafish larvae as an initial model system, we found that the opioid antagonist naltrexone exhibited an anticonvulsant effect. We validated this effect in three other epilepsy models and present naltrexone as a promising anticonvulsive candidate. Methods Candidate anticonvulsant drugs, determined by our prior transcriptomics analysis of hippocampal tissue, were evaluated in a larval zebrafish model of human Dravet syndrome (scn1Lab mutants), in wild‐type zebrafish larvae treated with the pro‐convulsant drug pentylenetetrazole (PTZ), in wild‐type C57bl/6J acute brain slices exposed to PTZ, and in wild‐type mice treated with PTZ in vivo. Abnormal locomotion was determined behaviorally in zebrafish and mice and by field potential in neocortex layer IV/V and CA1 stratum pyramidale in the hippocampus. Results The opioid antagonist naltrexone decreased abnormal locomotion in the larval zebrafish model of human Dravet syndrome (scn1Lab mutants) and wild‐type larvae treated with the pro‐convulsant drug PTZ. Naltrexone also decreased seizure‐like events in acute brain slices of wild‐type mice, and the duration and number of seizures in adult mice injected with PTZ. Significance Our data reveal that naltrexone has anticonvulsive properties and is a candidate drug for seizure treatment.

Abstract Implementing different tools and injury mechanisms in multiple animal models of retina regeneration, researchers have discovered the existence of retinal stem/progenitor cells. Although they appear to be distributed uniformly across the vertebrate lineage, the reparative potential of the retina is mainly restricted to lower vertebrates. Regenerative repair post‐injury requires the creation of a proliferative niche, vital for proper stem cell activation, propagation, and lineage differentiation. This seems to be lacking in mammals. Hence, in this review, we first discuss the many forms of retinal injuries that have been generated using animal models. Next, we discuss how they are utilized to stimulate regeneration and mimic eye disease pathologies. The key to driving stem cell activation in mammals relies on the information we can gather from these models. Lastly, we present a brief update about the genes, growth factors, and signaling pathways that have been brought to light using these models.

Abstract Background Myosin family genes, including those encoding myosin heavy chain 6, myosin heavy chain 7, myosin light chain 3, and myosin light chain 2 (MYL2), are important genetic factors in congenital heart disease (CHD). However, how these genes contribute to CHD in the Han Chinese population remains unclear. Methods We sequenced myosin family genes in a Han Chinese cohort comprising 412 CHD patients and 213 matched controls in the present study. A zebrafish model was used to evaluate the pathogenicity of rare mutations in MYL2. Results We identified 30 known mutations and 12 novel mutations. Furthermore, the contributions of two novel mutations, MYL2 p.Ile158Thr and p.Val146Met, to CHD were analyzed. The p.Ile158Thr mutation increased MYL2 expression. In zebrafish embryos, injection of myl2b‐targeting morpholinos led to aberrant cardiac structures, an effect that was reversed by expression of wild‐type MYL2 but not MYL2 p.Ile158Thr and pVal146Met. Conclusions Overall, our findings suggest that MYL2 p.Ile158Thr and p.Val146Met contribute to the etiology of CHD. The results also indicate the importance of MYL2 in heart formation.

Abstract The obesity epidemic is concerning as obesity appears to negatively impact cognition and behavior. Furthermore, some studies suggest that this negative effect could be carried across generations from both mothers and fathers although evidence is not consistent. Here, we attempt to address how obesogenic diets in the parental generation (F0) can impact offspring's cognition and anxiety intergenerationally (F1) in a zebrafish model. We compare both mean trait values and their variances. Using a multifactorial design, we created a total of four groups: F1T (treatment mothers × treatment fathers); F1M (treatment mothers × control fathers); F1P (treatment fathers × control mothers); and F1C (control mothers × control fathers, F1C); and subjected them to anxiety tank tests and aversive learning assays. When both parents were exposed, offspring (F1T) displayed the poorest aversive learning, while offspring that only had one parent exposed (F1P and F1M) learnt the aversive learning task the best. Zebrafish in all groups displayed no statistically significant differences in anxiety‐associated behaviors. Males and females also performed similarly in both anxiety and aversive learning assays. While all F1 groups had similar levels of fasting blood glucose, variance in glucose levels were reduced in F1P and F1T indicating the importance of investigating heteroskedasticity between groups. Furthermore, anxiety behaviors of these two groups appeared to be less repeatable. To our knowledge, this is the first study to test the intergenerational effects of an obesogenic diet on zebrafish cognition. Our multifactorial design as well as repeated tests also allowed us to disentangle maternal and paternal effects (as well as combined effects) and accurately detect subtle information such as between‐individual variation.

Abstract Aims/Introduction β‐Cell dysfunction is a hallmark of type 2 diabetes. In a previous pilot study, we identified an association between genetic variants within the human DACH1 gene and young‐onset type 2 diabetes. Here, we characterized the function of dachb, the only dach homologue to be expressed in the pancreas, in developing zebrafish embryos. Materials and Methods We injected one‐cell stage embryos with a dachb‐morpholino (MO) or with the dachb‐MO and dachb messenger ribonucleic acid, and determined the effect on the development of the pancreatic islet. We also carried out quantitative polymerase chain reaction and ribonucleic acid sequencing on the dachb‐MO group to determine the effect of dachb knockdown on gene expression. Results MO‐mediated dachb knockdown resulted in impaired islet cell development, with a significant decrease in both the β‐cell and islet cell numbers. This islet developmental defect was rescued when embryos were co‐injected with dachb‐MO and dachb messenger ribonucleic acid. Knockdown of dachb was associated with a significant downregulation of the β‐cell specific marker gene, insa, and the somatostatin cell marker, sst2, as well as regulators of pancreas development, ptf1a, neuroD, pax6a and nkx6.1, and the cell cycle gene, insm1a. Furthermore, ribonucleic sequencing analysis showed an upregulation of genes enriched in the forkhead box O and mitogen‐activated protein kinase signaling pathways in the dachb‐MO group, when compared with the control groups. Conclusions Together, our results suggest the possible role of dachb in islet development in zebrafish.

Thyroid dysgenesis (TD) is a major cause of primary congenital hypothyroidism; however, the molecular mechanism underlying this process is unclear. Current knowledge regarding the morphogenesis of the thyroid gland and vascular anomalies affecting thyroid development is limited. To monitor the early stages of thyroid gland development, we generated double transgenic zebrafish embryos Tg(tg:mCherry/flk1:EGFP). We described the volume of the thyroid from 2 days postfertilization (dpf) to 5 dpf using 3D reconstruction images. We treated zebrafish embryos with the fibroblast growth factor (FGF) inhibitor PD166866 to better understand the impact of vascular defects on thyroid development and the effects of drug administration at specific time periods on different stages of thyroid development. The 3D reconstruction data revealed that the thyroid glands underwent significant transformation at critical time points. PD166866 treatment from 48 to 72 hours postfertilization (hpf) and from 72 to 96 hpf did not cause obvious reductions in thyroid volume but did result in observable abnormalities in thyroid morphology. The treatment also affected thyroid volume from 36 to 48 hpf, thus indicating that there are time‐point‐specific effects of drug administration during thyroid development. Three‐dimensional image reconstruction provides a comprehensive picture of thyroid anatomy and can be used to complement anatomical fluorescence information. The effects of an FGF pathway inhibitor on thyroid development were determined to be time‐point‐dependent.

Abstract Aims/Introduction Diabetic cardiomyopathy is a type of myocardial disease. It causes left ventricular hypertrophy, followed by diastolic and systolic dysfunction, eventually leading to congestive heart failure. However, the underlying mechanism still requires further elucidation. Materials and Methods A high‐glucose zebrafish model was constructed by administering streptozocin intraperitoneally to enhance the development of cardiomyopathy and then treated with adenosine monophosphate‐activated protein kinase (AMPK) activator. Cardiac structure and function, and protein and gene expression were then analyzed. Cardiomyocytes (CMs) culture in vitro using lentivirus were used for detection of AMPK, p53 and Krüppel‐like factor 2a (klf2a) gene expression. Results In the hyperglycemia group, electrocardiogram findings showed arrhythmia, echocardiography results showed heart enlargement and dysfunction, and many differences, such as increased apoptosis and myocardial fiber loss, were observed. The phospho‐AMPK and klf2a expression were downregulated, and p53 expression was upregulated. Activation of phospho‐AMPK reduced p53 and increased klf2a expression, alleviated apoptosis in CMs and improved cardiac function in the hyperglycemic zebrafish. In vitro knockdown system of AMPK, p53 and klf2a using lentivirus illustrated an increased p53 expression and decreased klf2a expression in CMs by inhibiting AMPK. Repression of p53 and upregulation of klf2a expression were observed, but no changes in the expression of AMPK and its phosphorylated type. Conclusions In the model of streptozocin‐induced hyperglycemia zebrafish, the reduction of phosphorylated AMPK increased p53, which led to KLF2a decrease to facilitate apoptosis of CMs, inducing the cardiac remodeling and cardiac dysfunction. These results can be reversed by AMPK activator, which means the AMPK–p53–klf2a pathway might be a potential target for diabetic cardiomyopathy intervention.

Abstract Large‐scale screening of molecules heavily relies on phenotyping of small living organisms during preclinical development. However, deep profiling candidate therapeutics on whole animals typically requires laborious manipulations and anesthetic treatment using traditional techniques or automated tools. Here, a novel fish capsule system that combines automated zebrafish encapsulating technology and droplet microarray strategy for in vivo functional screening of mono/polytherapies is described. This platform enables automated, rapid zebrafish orientation and immobilization in agarose to generate large‐scale fish capsules by using a microfluidic device. Based on the effect of discontinuous dewetting, the prompt trapping of fish capsules in the aqueous arrays is successfully demonstrate. This system provides the capability to integrate pharmaceutical treatments with real‐time multispectral microscopic imaging in a simple, pipetting‐free and highly parallel manner. Coupling with machine learning algorithms, a small library of compounds is screened and analyzed, and clues about how to exploit compound combinations as therapeutic candidates are obtained. It is believed that this proposed strategy can be readily applied to multiple fields and is especially useful in the exploration of combinatorial drugs with limited amounts of samples and resources to accelerate the identification of novel therapeutics for precision medicines.

Alzheimer's disease (AD) affects millions of people and has limited treatment options, thus making it a global health concern. Amyloid β (Aβ), a disrupted cholinergic system with high acetylcholinesterase (AChE), oxidative stress (OS), reduced antioxidants, and neuroinflammation are key factors influencing AD progression. Prior research has shown that AChE can interact with Aβ and increase its accumulation and neurotoxicity, so targeting AChEs and Aβ could be a potential therapeutic approach for AD treatment. It has been known that nonsteroidal anti-inflammatory drugs (NSAIDs) can inhibit Aβ accumulation. Previously, TKM01, a derivative of 4-anilinoquinazoline, has demonstrated inhibitory effects against GSK-3β-a regulator in AD progression. The current research included molecular docking studies of NSAIDs and TKM01 with Aβ and AChEs as targets. TKM01 exhibited a higher binding affinity with Aβ among all tested compounds. Molecular dynamic (MD) simulations confirmed the stability of the protein-TKM01 complexes. TKM01 also exhibited favorable drug-likeness properties, and no hepatoxicity was visualized in comparison with other compounds. Further, in vitro assay showed an inhibitory action of TKM01 (50-1200 µg/mL) on AChEs. In the in vivo studies on zebrafish larvae brains, we found that TKM01 (120 and 240 µg/mL) reduced the levels of AChEs and lipid peroxidation (LPO) and increased antioxidant superoxide dismutase (SOD) and catalase (CAT) in AlCl 3 (80 µM)-induced AD-like model. Additionally, TKM01 treatment was found to decrease pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. The current study demonstrates that TKM01 can be used to treat AD. Nonetheless, experimental validation is needed to reveal the cellular, sub-cellular, and molecular mechanisms and possible implications at a clinical stage., (© 2024 Wiley Periodicals LLC.)

Background and Purpose: The oxidant sensor transient receptor potential ankyrin 1 (TRPA1) channel expressed by Schwann cells (SCs) has recently been implicated in several models of neuropathic pain in rodents. Here we investigate whether the pro-algesic function of Schwann cell TRPA1 is not limited to mammals by exploring the role of TRPA1 in a model of chemotherapy-induced peripheral neuropathy (CIPN) in zebrafish larvae., Experimental Approach: We used zebrafish larvae and a mouse model to test oxaliplatin-evoked nociceptive behaviours. We also performed a TRPA1 selective silencing in Schwann cells both in zebrafish larvae and mice to study their contribution in oxaliplatin-induced CIPN model., Key Results: We found that zebrafish larvae and zebrafish TRPA1 (zTRPA1)-transfected HEK293T cells respond to reactive oxygen species (ROS) with nociceptive behaviours and intracellular calcium increases, respectively. TRPA1 was found to be co-expressed with the Schwann cell marker, SOX10, in zebrafish larvae. Oxaliplatin caused nociceptive behaviours in zebrafish larvae that were attenuated by a TRPA1 antagonist and a ROS scavenger. Oxaliplatin failed to produce mechanical allodynia in mice with Schwann cell TRPA1 selective silencing (Plp1 + -Trpa1 mice). Comparable results were observed in zebrafish larvae where TRPA1 selective silencing in Schwann cells, using the specific Schwann cell promoter myelin basic protein (MBP), attenuated oxaliplatin-evoked nociceptive behaviours., Conclusion and Implications: These results indicate that the contribution of the oxidative stress/Schwann cell/TRPA1 pro-allodynic pathway to neuropathic pain models seems to be conserved across the animal kingdom., (© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Abstract Liver cancer is the third leading cause of cancer‐related deaths throughout the world, and more than 0.6 million people die from liver cancer annually. Therefore, novel therapeutic strategies to eliminate malignant cells from liver cancer patients are urgently needed. Recent advances in high‐throughput genomic technologies have identified de novo candidates for oncogenes and pharmacological targets. However, testing and understanding the mechanism of oncogenic transformation as well as probing the kinetics and therapeutic responses of spontaneous tumors in an intact microenvironment require in vivo examination using genetically modified animal models. The zebrafish (Danio rerio) has attracted increasing attention as a new model for studying cancer biology since the organs in the model are strikingly similar to human organs and the model can be genetically modified in a short time and at a low cost. This review summarizes the current knowledge of epidemiological data and genetic alterations in hepatocellular carcinoma (HCC), zebrafish models of HCC, and potential therapeutic strategies for targeting HCC based on knowledge from the models.

Abstract Triple‐negative breast cancer (TNBC) is a basal‐like cancer which is considered to be more intrusive, have a poorer prognosis and chemoresistance. TNBC is characterized by the presence of epithelial to mesenchymal transition (EMT) that plays a major role in the progression of the cancer. In the present study, we first use a classic prescription of Chinese medicine Fangjihuangqi Decoction to treat TGFβ1‐induced MDA‐MB‐231 cells in vitro. Our data showed that TGFβ1‐induced MDA‐MB‐231 cell morphology change, promoted MDA‐MB 231 invasion, increased Vimentin expression, and decreased E‐cadherin expression. Further, Fangjihuangqi Decoction‐medicated serum (FHS) treated both MDA‐MB 231 cells and TGFβ1‐induced MDA‐MB‐231 cells. Results showed that Fangjihuangqi Decoction could inhibit cell proliferation, reduce cell invasion, increase E‐cadherin expression, and decrease EMT markers. Secondly, we established a xenograft tumor zebrafish model to assess Fangjihuangqi Decoction inhibition of cancer cell proliferation and invasion. Our results indicated that Fangjihuangqi Decoction could inhibit tumor growth, restrain the sprouts number of tumor neovascularization, and reduce the length of tumor neoplastic lymphatics by increasing E‐cadherin expression and decreasing EMT markers in TNBC xenograft tumor zebrafish model. Overall, our studies provide evidences that Fangjihuangqi Decoction could inhibit TNBC, reverse EMT, and contribute to antimetastasis by increasing E‐cadherin expression and decreasing EMT markers, which provide an experimental basis for clinical application of Fangjihuangqi Decoction on TNBC treatment.

Abstract Sex determination in zebrafish by manual approaches according to current guidelines relies on human observation. These guidelines for sex recognition have proven to be subjective and highly labor‐intensive. To address this problem, we present a methodology to automatically classify the phenotypic sex using two machine learning methods: Deep Convolutional Neural Networks (DCNNs) based on the whole fish appearance and Support Vector Machine (SVM) based on caudal fin coloration. Machine learning techniques in sex classification provide potential efficiency with the advantage of automatization and robustness in the prediction process. Furthermore, since developmental plasticity can be influenced by environmental conditions, we have investigated the impact of elevated water temperature during embryogenesis on sex and sex‐related differences in color intensity of adult zebrafish. The estimated color intensity based on SVM was then applied to detect the association between coloration and body weight and length. Phenotypic sex classifications using machine learning methods resulted in a high degree of association with the real sex in nontreated animals. In temperature‐induced animals, DCNNs reached a performance of 100%, whereas 20% of males were misclassified using SVM due to a lower color intensity. Furthermore, a positive association between color intensity and body weight and length was observed in males. Our study demonstrates that high ambient temperature leads to a lower color intensity in male animals and a positive association of male caudal fin coloration with body weight and length, which appears to play a significant role in sexual attraction. The software developed for sex classification in this study is readily applicable to other species with sex‐linked visible phenotypic differences.

This study explores the 24-h rhythmic cycle of protein O-GlcNAcylation within the brain and highlights its crucial role in regulating the circadian cycle and neuronal function based on zebrafish as an animal model. In our experiments, disruption of the circadian rhythm, achieved through inversion of the light-dark cycle or daytime melatonin treatment, not only impaired the rhythmic changes of O-GlcNAcylation along with altering expression patterns of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in zebrafish brain but also significantly impeded learning and memory function. In particular, circadian disruption affected rhythmic expression of protein O-GlcNAcylation and OGT in the nuclear fraction. Notably, the circadian cycle induces rhythmic alterations in O-GlcNAcylation of H2B histone protein that correspond to changes in H3 trimethylation. Disruption of the cycle interfered with these periodic histone code alterations. Pharmacological inhibition of OGT with OSMI-1 disrupted the wake-sleep patterns of zebrafish without affecting expression of circadian rhythm-regulating genes. OSMI-1 inhibited the expression of c-fos, bdnf, and calm1, key genes associated with brain function and synaptic plasticity, and decreased the binding of O-GlcNAcylated H2B and OGT to promoter regions of these genes. The collective findings support the potential involvement of circadian cycling of the O-GlcNAc histone code in regulating synaptic plasticity and brain function. Overall, data from this study provide evidence that protein O-GlcNAcylation serves as a pivotal posttranslational mechanism integrating circadian signals and neuronal function to regulate rhythmic physiology., (© 2024 The Author(s). Journal of Pineal Research published by John Wiley & Sons Ltd.)

Background: Meis family of transcription factors operates in Pbx-Meis-Hox regulatory network controlling development of various tissues including eye, limbs, heart, hindbrain or craniofacial skeletal elements originating from the neural crest. Although studies in mouse provide abundant information about Meis factors function in embryogenesis, little is known about their role in zebrafish., Results: We generated zebrafish lines carrying null mutations in meis1a, meis1b, meis2a, and meis2b genes. Only meis1b mutants are lethal at larval stage around 13 dpf whereas the other mutant lines are viable and fertile. We focused on development of neural crest-derived craniofacial structures such as tendons, cranial nerves, cartilage and accompanying muscles. Meis1b mutants displayed morphogenetic abnormalities in the cartilage originating from the first and second pharyngeal arches. Meckel's cartilage was shorter and wider with fused anterior symphysis and abnormal chondrocyte organization. This resulted in impaired tendons and muscle fiber connections while tenocyte development was not largely affected., Conclusions: Loss-of-function mutation in meis1b affects cartilage morphology in the lower jaw that leads to disrupted organization of muscles and tendons., (© 2024 American Association for Anatomy.)

The compound Honokiol, derived from the bark of Magnolia officinalis, possesses the ability to induce apoptosis and inhibit cellular damage caused by reactive oxygen species. The objective of this study was to investigate the toxicological and histopathological effects of Honokiol on zebrafish (Danio rerio) through conducting a semistatic acute toxicity test involving immersion in an Honokiol-containing solution. The results showed that the toxic effects of Honokiol on zebrafish were primarily manifested in the liver and gills. When exposed to 0.6 mg/L of Honokiol, it could lead to liver hemorrhage as well as swelling and necrosis of gill tissues, and high concentrations of Honokiol could trigger inflammatory responses. Additionally, research found that Honokiol could induce apoptosis in liver and gill tissues through the P53 pathway and possessed the ability to enhance antioxidation. The present findings significantly contribute to a more profound understanding of the toxic impact of Honokiol and its underlying mechanism, thereby providing a valuable reference for the future safe utilization of Honokiol and related pharmaceutical advancements., (© 2024 Wiley Periodicals LLC.)

American ginseng (AG) has been reported to have anti-inflammatory effects in many diseases, but the key molecules and mechanisms are unclear. This study aims to evaluate the anti-inflammatory mechanism of AG and identify the key molecules by in vivo and in vitro models. Zebrafish was employed to assess the anti-inflammatory properties of AG and the compounds. Metabolomics was utilized to identify potential anti-inflammatory molecules in AG, while molecular dynamics simulations were conducted to forecast the interaction capabilities of these compounds with inflammatory targets. Additionally, macrophage cell was employed to investigate the anti-inflammatory mechanisms of the key molecules in AG by enzyme-linked immunosorbent assay and western blotting. Seven potential anti-inflammatory molecules were discovered in AG, with ginsenoside Rg1, ginsenoside Rs3 (G-Rs3), and oleanolic acid exhibiting the strongest affinity for signal transducer and activator of transcription 3. These compounds demonstrated inhibitory effects on macrophage migration in zebrafish models and the ability to regulate ROS levels in both zebrafish and macrophages. The cell experiments found that ginsenoside Rg1, ginsenoside Rs3, and oleanolic acid could promote macrophage M2/M1 polarization ratio and inhibit phosphorylation overexpression of signal transducer and activator of transcription 3. This study revealed the key anti-inflammatory molecules and mechanisms of AG, and provided new evidence of anti-inflammatory for the scientific use of AG., (© 2024 John Wiley & Sons Ltd.)

Background: During neurogenesis, growing axons must navigate through the complex extracellular environment and make correct synaptic connections for the proper functioning of neural circuits. The mechanisms underlying the formation of functional neural networks are still only partially understood., Results: Here we analyzed the role of a novel gene si:ch73-364h19.1/drish in the neural and vascular development of zebrafish embryos. We show that drish mRNA is expressed broadly and dynamically in multiple cell types including neural, glial, retinal progenitor and vascular endothelial cells throughout the early stages of embryonic development. To study Drish function during embryogenesis, we generated drish genetic mutant using CRISPR/Cas9 genome editing. drish loss-of-function mutant larvae displayed defects in early retinal ganglion cell, optic nerve and the retinal inner nuclear layer formation, as well as ectopic motor axon branching. In addition, drish mutant adults exhibited deficient retinal outer nuclear layer and showed defective light response and locomotory behavior. However, vascular patterning and blood circulation were not significantly affected., Conclusions: Together, these data demonstrate important roles of zebrafish drish in the retinal ganglion cell, optic nerve and interneuron development and in spinal motor axon branching., (© 2024 American Association for Anatomy.)

Background and Purpose: Parkinson's disease (PD) is a prevalent neurodegenerative movement disorder characterized by motor dysfunction. Environmental factors, especially manganese (Mn), contribute significantly to PD. Existing therapies are focused on motor coordination, whereas nonmotor features such as neuropsychiatric symptoms are often neglected. Daidzein (DZ), a phytoestrogen, has piqued interest due to its antioxidant, anti-inflammatory, and anxiolytic properties. Therefore, we anticipate that DZ might be an effective drug to alleviate the nonmotor symptoms of Mn-induced Parkinsonism., Experimental Approach: Naïve zebrafish were exposed to 2 mM of Mn for 21 days and intervened with DZ. Nonmotor symptoms such as anxiety, social behaviour, and olfactory function were assessed. Acetylcholinesterase (AChE) activity and antioxidant enzyme status were measured from brain tissue through biochemical assays. Dopamine levels and histology were performed to elucidate neuroprotective mechanism of DZ., Key Results: DZ exhibited anxiolytic effects in a novel environment and also improved intra and inter fish social behaviour. DZ improved the olfactory function and response to amino acid stimuli in Mn-induced Parkinsonism. DZ reduced brain oxidative stress and AChE activity and prevented neuronal damage. DZ increased DA level in the brain, collectively contributing to neuroprotection., Conclusion and Implications: DZ demonstrated a promising effect on alleviating nonmotor symptoms such as anxiety and olfactory dysfunction, through the mitigation of cellular damage. These findings underscore the therapeutic potential of DZ in addressing nonmotor neurotoxicity induced by heavy metals, particularly in the context of Mn-induced Parkinsonism., (© 2024 British Pharmacological Society.)

Background: The two-pore domain potassium (K2P) channels are a major type of potassium channels that maintain the cell membrane potential by conducting passive potassium leak currents independent of voltage change. They play prominent roles in multiple physiological processes, including neuromodulation, perception of pain, breathing and mood control, and response to volatile anesthetics. Mutations in K2P channels have been linked to many human diseases, such as neuronal and cardiovascular disorders and cancers. Significant progress has been made to understand their protein structures, physiological functions, and pharmacological modifiers. However, their expression and function during embryonic development remain largely unknown., Results: We employed the zebrafish model and identified 23 k2p genes using BLAST search and gene cloning. We first analyzed vertebrate K2P channel evolution by phylogenetic and syntenic analyses. Our data revealed that the six subtypes of the K2P genes have already evolved in invertebrates long before the emergence of vertebrates. Moreover, the vertebrate K2P gene number increased, most likely due to two whole-genome duplications. Furthermore, we examined zebrafish k2p gene expression during early embryogenesis by in situ hybridization. Each subgroup's genes showed similar but distinct gene expression domains with some exceptions. Most of them were expressed in neural tissues consistent with their known function of neural excitability regulation. However, a few k2p genes were expressed temporarily in specific tissues or organs, suggesting that these K2P channels may be needed for embryonic development., Conclusions: Our phylogenetic and developmental analyses of K2P channels shed light on their evolutionary history and potential roles during embryogenesis related to their physiological functions and human channelopathies., (© 2024 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)

Introduction: Polygonum amplexicaule D. Don var. sinense Forb (PAF), a medicinal plant, has the effect of promoting blood circulation and removing blood stasis. However, the active compounds and targets of its anticoagulant effect are still unclear., Objectives: This study aims to establish an effective reversely thrombin-targeted screening method for anticoagulant active components in PAF by affinity ultrafiltration (AUF) coupled with ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectroscopy (UPLC-Q-TOF-MS)., Methods: Different polar parts of PAF were screened for potential thrombin ligands by AUF-HPLC and identified by UPLC-Q-TOF-MS. After studying the affinity between ligands and thrombin by molecular docking, the antithrombotic activity of ligands was detected in vivo by zebrafish thrombus model, and in vitro by chromogenic substrate method. The mechanism of such ligands on thrombin was further studied by coagulation factor assay., Results: Eleven potential thrombin ligands from PAF were screened by the AUF-UPLC-Q-TOF-MS method, and two compounds (butyl gallate and β-sitosterol) with significant anticoagulant activity were discovered via in vitro and in vivo activity testing., Conclusion: A method system based on AUF-UPLC-Q-TOF-MS, molecular docking and in vivo and in vitro experiments also provided a powerful tool for further exploration of anticoagulant active components in PAF., (© 2024 John Wiley & Sons Ltd.)

Background: Previous studies have claimed that pharyngeal teeth in medaka (Oryzias latipes) are induced independent of retinoic acid (RA) signaling, unlike in zebrafish (Danio rerio). In zebrafish, pharyngeal tooth formation depends on a proper physical contact between the embryonic endodermal pouch anterior to the site of tooth formation, and the adjacent ectodermal cleft, an RA-dependent process. Here, we test the hypothesis that a proper pouch-cleft contact is required for pharyngeal tooth formation in embryonic medaka, as it is in zebrafish. We used 4-[diethylamino]benzaldehyde (DEAB) to pharmacologically inhibit RA production, and thus pouch-cleft contacts, in experiments strictly controlled in time, and analyzed these using high-resolution imaging., Results: Pharyngeal teeth in medaka were present only when the corresponding anterior pouch had reached the ectoderm (i.e., a physical pouch-cleft contact established), similar to the situation in zebrafish. Oral teeth were present even when the treatment started approximately 4 days before normal oral tooth appearance., Conclusions: RA dependency for pharyngeal tooth formation is not different between zebrafish and medaka. We propose that the differential response to DEAB of oral versus pharyngeal teeth in medaka could be ascribed to the distinct germ layer origin of the epithelia involved in tooth formation in these two regions., (© 2024 American Association for Anatomy.)

Background: Mutations of human WNT10A are associated with odonto-ectodermal dysplasia syndromes. Here, we present analyses of wnt10a loss-of-function mutants in the zebrafish., Results: wnt10a mutant zebrafish embryos display impaired tooth development and a collapsing median fin fold (MFF). Rescue experiments show that wnt10a is essential for MFF maintenance both during embryogenesis and later metamorphosis. The MFF collapse could not be attributed to increased cell death or altered proliferation rates of MFF cell types. Rather, wnt10a mutants show reduced expression levels of dlx2a in distal-most MFF cells, followed by compromised expression of col1a1a and other extracellular matrix proteins encoding genes. Transmission electron microscopy analysis shows that although dermal MFF compartments of wnt10a mutants initially are of normal morphology, with regular collagenous actinotrichia, positioning of actinotrichia within the cleft of distal MFF cells becomes compromised, coinciding with actinotrichia shrinkage and MFF collapse., Conclusions: MFF collapse of wnt10a mutant zebrafish is likely caused by the loss of distal properties in the developing MFF, strikingly similar to the proposed molecular pathomechanisms underlying the teeth defects caused by the loss of Wnt10 in fish and mammals. In addition, it points to thus fur unknown mechanisms controlling the linear growth and stability of actinotrichia and their collagen fibrils., (© 2023 The Authors. Developmental Dynamics published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)