An adult zebrafish heart possesses a high capacity of regeneration. However, it has been unclear whether and how myocyte hyperplasia contributes to cardiac remodeling in response to biomechanical stress and whether myocyte hypertrophy exists in the zebrafish. To address these questions, we characterized the zebrafish mutant tr265/tr265, whose Band 3 mutation disrupts erythrocyte formation and results in anemia. Although Band 3 does not express and function in the heart, the chronic anemia imposes a sequential biomechanical stress towards the heart.

Light Sheet Fluorescence Microscopy (LSFM) enables multi-dimensional and multi-scale imaging via illuminating specimens with a separate thin sheet of laser. It allows rapid plane illumination for reduced photo-damage and superior axial resolution and contrast. We hereby demonstrate cardiac LSFM (c-LSFM) imaging to assess the functional architecture of zebrafish embryos with a retrospective cardiac synchronization algorithm for four-dimensional reconstruction (3-D space + time). By combining our approach with tissue clearing techniques, we reveal the entire cardiac structures and hypertrabeculation of adult zebrafish hearts in response to doxorubicin treatment. By integrating the resolution enhancement technique with c-LSFM to increase the resolving power under a large field-of-view, we demonstrate the use of low power objective to resolve the entire architecture of large-scale neonatal mouse hearts, revealing the helical orientation of individual myocardial fibers. Therefore, our c-LSFM imaging approach provides multi-scale visualization of architecture and function to drive cardiovascular research with translational implication in congenital heart diseases.

TTN-encoded titin, CSRP3-encoded muscle LIM protein, and TCAP-encoded telethonin are Z-disc proteins essential for the structural organization of the cardiac sarcomere and the cardiomyocyte's stretch sensor. All three genes have been established as cardiomyopathy-associated genes for both dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). Here, we sought to characterize the frequency, spectrum, and phenotype associated with HCM-associated mutations in these three genes in a large cohort of unrelated patients evaluated at a single tertiary outpatient center.

The adult zebrafish is an emerging vertebrate model for studying human cardiomyopathies; however, whether the simple zebrafish heart can model different subtypes of cardiomyopathies, such as dilated cardiomyopathy (DCM), remains elusive. Here, we generated and characterized an inherited DCM model in adult zebrafish and used this model to search for therapeutic strategies. We employed transcription activator-like effector nuclease (TALEN) genome editing technology to generate frame-shift mutants for the zebrafish ortholog of human BCL2-associated athanogene 3 (BAG3), an established DCM-causative gene. As in mammals, the zebrafish bag3 homozygous mutant (bag3e2/e2 ) exhibited aberrant proteostasis, as indicated by impaired autophagy flux and elevated ubiquitinated protein aggregation. Through comprehensive phenotyping analysis of the mutant, we identified phenotypic traits that resembled DCM phenotypes in mammals, including cardiac chamber enlargement, reduced ejection fraction characterized by increased end-systolic volume/body weight (ESV/BW), and reduced contractile myofibril activation kinetics. Nonbiased transcriptome analysis identified the hyperactivation of the mechanistic target of rapamycin (mTOR) signaling in bag3e2/e2 mutant hearts. Further genetic studies showed that mtorxu015/+ , an mTOR haploinsufficiency mutant, repaired abnormal proteostasis, improved cardiac function and rescued the survival of the bag3e2/e2 mutant. This study established the bag3e2/e2 mutant as a DCM model in adult zebrafish and suggested mtor as a candidate therapeutic target gene for BAG3 cardiomyopathy.

As a common injury almost all cells face, DNA damage in oocytes-especially double-strand breaks (DSBs), which occur naturally during the first meiosis phase (meiosis I) due to synaptic complex separation-affects the fertilization ability of oocytes, instead of causing cancer (as in somatic cells). The mechanism of oocytes to effectively repair DSB damage has not yet been clearly studied, especially considering medically induced DSBs superimposed on naturally occurring DSBs in meiosis I. It was found that maturation rates decreased or increased, respectively corresponding with overexpression or interference of p21 in bovine oocytes. At the same time, the maturation rate of bovine oocytes decreased with a gradual increase in Zeocin dose, and the p21 expression in those immature oocytes changed significantly with the gradual increase in Zeocin dose (same as increased DSB intensity). Same as p21, the variation trend of ATM expression was consistent with the gradual increase in Zeocin dose. Furthermore, the oocytes demonstrated tolerance to DSBs during meiosis I, while the maturation rates decreased when the damage exceeded a certain threshold; according to which, it may be that ATM regulates the p53-p21 pathway to affect the completion of meiosis. In addition, nonhomologous recombination and cumulus cells are potentially involved in the process by which oocytes respond to DSB damage.

One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b, fras1, tnnt2a, edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease.

Inhibitory control hierarchically regulates cognitive and emotional systems in the service of adaptive goal-directed behavior across changing task demands and environments. While previous studies convergently determined the contribution of prefrontal-striatal systems to general inhibitory control, findings on the specific circuits that mediate emotional context-specific impact on inhibitory control remained inconclusive. Against this background we combined an evaluated emotional Go/No Go task with fMRI in a large cohort of subjects (N=250) to segregate brain systems and circuits that mediate domain-general from emotion-specific inhibitory control. Particularly during a positive emotional context, behavioral results showed a lower accuracy for No Go trials and a faster response time for Go trials. While the dorsal striatum and lateral frontal regions were involved in inhibitory control irrespective of emotional context, activity in the ventral striatum (VS) and medial orbitofrontal cortex (mOFC) varied as a function of emotional context. On the voxel-wise whole-brain network level, limbic and striatal systems generally exhibited highest changes in global brain connectivity during inhibitory control, while global brain connectivity of the left mOFC was less decreased during emotional contexts. Functional connectivity analyses moreover revealed that negative coupling between the VS with inferior frontal gyrus (IFG)/insula and mOFC varied as a function of emotional context. Together these findings indicate separable domain- general as well as emotional context-specific inhibitory brain systems which specifically encompass the VS and its connections with frontal regions.

Major Depressive Disorder (MDD) and Generalized Anxiety Disorder (GAD) are highly debilitating and often co-morbid disorders. The disorders exhibit partly overlapping dysregulations on the behavioral and neurofunctional level. The determination of disorder-specific behavioral and neurofunctional dysregulations may therefore promote neuro-mechanistic and diagnostic specificity. In order to determine disorder-specific alterations in the domain of emotion-cognition interactions the present study examined emotional context-specific inhibitory control in treatment-naïve MDD (n = 37) and GAD (n = 35) patients and healthy controls (n = 35). On the behavioral level MDD but not GAD exhibited impaired inhibitory control irrespective of emotional context. On the neural level, MDD-specific attenuated recruitment of inferior/medial parietal, posterior frontal, and mid-cingulate regions during inhibitory control were found during the negative context. GAD exhibited a stronger engagement of the left dorsolateral prefrontal cortex relative to MDD. Overall the findings from the present study suggest disorder- and emotional context-specific behavioral and neurofunctional inhibitory control dysregulations in major depression and may point to a depression-specific neuropathological and diagnostic marker.

MicroRNA (miRNA) is vital to the progression of hepatocellular carcinoma (HCC). Thereinto, miR-369-5p could yield assorted effects on various cancers, but there are few reports concerning the effect of miR-369-5p on HCC. Thus this study aimed to investigate the effect and mechanism of miR-369-5p in HCC. The data of miR-369-5p and HOXA13 expressions in liver hepatocellular carcinoma (LIHC) were analyzed by starBase, and then the miR-369-5p expression in HCC tissues and cells was detected by quantitative real-time PCR. Subsequently, miR-369-5p mimic was transfected into HCC cells and then its effects on cell activities were evaluated by cell counting kit-8, colony formation, wound healing, transwell assays, respectively. Expressions of epithelial-mesenchymal transition (EMT)-related genes were determined by western blot. The targeting relationship between miR-369-5p and HOXA13 was predicted by Targetscan and verified by dual-luciferase reporter assay. Pearson correlation test was used to analyze the correlation between HOXA13 and miR-369-5p. The above assays were experimented again to investigate the effects of HOXA13 on biological activity and EMT of HCC cells. MiR-369-5p expression was down-regulated and HOXA13 expression was up-regulated in LIHC. MiR-369-5p targeted HOXA13 and the expression of miR-369-5p was negatively correlated with the HOXA13 expression. MiR-369-5p inhibited the viability, proliferation, migration and invasion of HCC cells, increased E-cadherin level and decreased N-cadherin and Vimentin expressions. Concurrently, HOXA13 overexpression could counteract the effects of miR-369-5p on biological activity and EMT-related biomarkers of HCC cells. To conclude, miR-369-5p inhibits the viability, proliferation, migration and invasion of HCC cells by repressing the expression of HOXA13.

Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37-gene set and overall genomic structure, ranging from 16,596 base pairs (bp) in the teleost zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most prevalent inherited diseases, affecting roughly 1 in every 5000 individuals. Currently, few effective treatments exist for those with mitochondrial ailments, representing a major unmet patient need. Mitochondrial dysfunction is also a common component of a wide variety of other human illnesses, ranging from neurodegenerative disorders such as Huntington's disease and Parkinson's disease to autoimmune illnesses such as multiple sclerosis and rheumatoid arthritis. The electron transport chain (ETC) component of mitochondria is critical for mitochondrial biology and defects can lead to many mitochondrial disease symptoms. Here, we present a publicly available collection of genetic mutants created in highly conserved, nuclear-encoded mitochondrial genes in Danio rerio. The zebrafish system represents a potentially powerful new opportunity for the study of mitochondrial biology and disease due to the large number of orthologous genes shared with humans and the many advanced features of this model system, from genetics to imaging. This collection includes 15 mutant lines in 13 different genes created through locus-specific gene editing to induce frameshift or splice acceptor mutations, leading to predicted protein truncation during translation. Additionally, included are 11 lines created by the random insertion of the gene-breaking transposon (GBT) protein trap cassette. All these targeted mutant alleles truncate conserved domains of genes critical to the proper function of the ETC or genes that have been implicated in human mitochondrial disease. This collection is designed to accelerate the use of zebrafish to study many different aspects of mitochondrial function to widen our understanding of their role in biology and human disease.

Real-time functional magnetic resonance imaging (rtfMRI)-assisted neurofeedback (NF) training allows subjects to acquire volitional control over regional brain activity. Emerging evidence suggests its potential clinical utility as an effective non-invasive treatment approach in mental disorders. The therapeutic potential of rtfMRI-NF training depends critically upon whether: (1) acquired self-regulation produces functionally relevant changes at behavioral and brain network levels and (2) training effects can be maintained in the absence of feedback. To address these key questions, the present study combined rtfMRI-NF training for acquiring volitional anterior insula (AI) regulation with a sham-controlled between-subject design. The functional relevance of acquired AI control was assessed using both behavioral (pain empathy) and neural (activity, functional connectivity) indices. Maintenance of training effects in the absence of feedback was assessed two days later. During successful acquisition of volitional AI up-regulation subjects exhibited stronger empathic responses, increased AI-prefrontal coupling in circuits involved in learning and emotion regulation and increased resting state connectivity within AI-centered empathy networks. At follow-up both self-regulation and increased connectivity in empathy networks were fully maintained, although without further increases in empathy ratings. Overall these findings support the potential clinical application of rtfMRI-NF for inducing functionally relevant and lasting changes in emotional brain circuitry.

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure with high morbidity and mortality. Although more than 40 genes have been reported to cause DCM, the role of genetic testing in clinical practice is not well defined. Mutations in the troponin T (TNNT2) gene represent an important subset of known disease-causing mutations associated with DCM. Therefore, the aim of the present study was to determine the genetic variations in TNNT2 and the associations of those variations with DCM in Chinese patients.

The Wnt/β-catenin pathway is implicated in left-right (LR) axis determination; however, the underlying mechanism remains elusive. Prompted by our recent discovery that Wnt signaling regulates ciliogenesis in the zebrafish Kupffer's vesicle (KV) via Foxj1a, a ciliogenic transcription factor, we decided to elucidate functions of Foxj1a in Wnt-regulated LR pattern formation. We showed that targeted injection of wnt8a mRNA into a single cell at the 128-cell stage is sufficient to induce ectopic foxj1a expression and ectopic cilia. By interrogating the transcription circuit of foxj1a regulation, we found that both Lef1 and Tcf7 bind to a consensus element in the foxj1a promoter region. Depletion of Lef1 and Tcf7 inhibits foxj1a transcription in the dorsal forerunner cells, downregulates cilia length and number in KV, and randomizes LR asymmetry. Targeted overexpression of a constitutively active form of Lef1 also induced an ectopic protrusion that contains ectopic transcripts for sox17, foxj1a, and charon, and ectopic monocilia. Further genetic studies using this ectopic expression platform revealed two distinct functions of Foxj1a; mediating Wnt-governed monocilia length elongation as well as charon transcription. The novel Foxj1a-charon regulation is conserved in KV, and importantly, it is independent of the canonical role of Foxj1a in the biosynthesis of motile cilia. Together with the known function of motile cilia movement in generating asymmetric expression of charon, our data put forward a hypothesis that Foxj1a confers both ciliary and non-ciliary functions of Wnt signaling, which converge on charon to regulate LR pattern formation.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), one of the crucial pro-angiogenic factors, functions as a potent inhibitor of endothelial cell (EC) apoptosis. Previous progress has been made towards delineating the VPF/VEGF survival signaling downstream of the activation of VEGFR-2. Here, we seek to define the function of NRP-1 in VPF/VEGF-induced survival signaling in EC and to elucidate the concomitant molecular signaling events that are pivotal for our understanding of the signaling of VPF/VEGF. Utilizing two different in vitro cell culture systems and an in vivo zebrafish model, we demonstrate that NRP-1 mediates VPF/VEGF-induced EC survival independent of VEGFR-2. Furthermore, we show here a novel mechanism for NRP-1-specific control of the anti-apoptotic pathway in EC through involvement of the NRP-1-interacting protein (NIP/GIPC) in the activation of PI-3K/Akt and subsequent inactivation of p53 pathways and FoxOs, as well as activation of p21. This study, by elucidating the mechanisms that govern VPF/VEGF-induced EC survival signaling via NRP-1, contributes to a better understanding of molecular mechanisms of cardiovascular development and disease and widens the possibilities for better therapeutic targets.

The zebrafish ventricular myosin heavy chain (vmhc) gene exhibits restricted expression in the ventricle. However, the molecular mechanism underlying this chamber-specific expression is unclear. Here, we exploited both transient and transgenic technologies to dissect the zebrafish vmhc promoter. We demonstrated that a combination of two transient assays in this animal model quickly identified chamber-specific cis-elements, isolating a 2.2 kb fragment upstream from the vmhc gene that can drive ventricle-specific expression. Furthermore, deletion analysis identified multiple cis-elements that exhibited cardiac-specific expression. To achieve chamber specificity, a distal element was required to coordinate with and suppress a proximal enhancer element. Finally, we discovered that Nkx2.5-binding sites (NKE) were essential for this repressive function. In summary, our study of the zebrafish vmhc promoter suggests that ventricle-specific expression is achieved through an inhibitory mechanism that suppresses expression in the atrium. Developmental Dynamics 238:1564-1573, 2009. (c) 2009 Wiley-Liss, Inc.

To uncover the genetic basis of anthracycline-induced cardiotoxicity (AIC), we recently established a genetic suppressor screening strategy in zebrafish. Here, we report the molecular and cellular nature of GBT0419, a salutary modifier mutant that affects retinoid x receptor alpha a (rxraa). We showed that endothelial, but not myocardial or epicardial, RXRA activation confers AIC protection. We then identified isotretinoin and bexarotene, two FDA-approved RXRA agonists, which exert cardioprotective effects. The therapeutic effects of these drugs only occur when administered during early, but not late, phase of AIC or as pretreatment. Mechanistically, these spatially- and temporally-predominant benefits of RXRA activation can be ascribed to repair of damaged endothelial cell-barrier via regulating tight-junction protein Zonula occludens-1. Together, our study provides the first in vivo genetic evidence supporting RXRA as the therapeutic target for AIC, and uncovers a previously unrecognized spatiotemporally-predominant mechanism that shall inform future translational efforts.

Dibutyl phthalate (DBP) is recognized as an environmental endocrine disruptor that has been detected in fetal and postnatal samples. Recent evidence found that in utero DBP exposure was associated with an increase of adipose tissue weight and serum lipids in offspring, but the precise mechanism is unknown. Here we aimed to study the effects of in utero DBP exposure on obesity in offspring and examine possible mechanisms. SPF C57BL/6J pregnant mice were gavaged with either DBP (5 mg /kg/day) or corn oil, from gestational day 12 until postnatal day 7. After the offspring were weaned, the mice were fed a standard diet for 21 weeks, and in the last 2 weeks 20 mice were selected for TUDCA treatment. Intrauterine exposure to low-dose DBP promoted obesity in offspring, with evidence of glucose and lipid metabolic disorders and a decreased metabolic rate. Compared to controls, the DBP exposed mice had lower expression of UCP1 and significantly higher expression of Bip and Chop, known markers of endoplasmic reticulum (ER) stress. However, TUDCA treatment of DBP exposed mice returned these parameters nearly to the levels of the controls, with increased expression of UCP1, lower expression of Bip and Chop and ameliorated obesity. Intrauterine exposure of mice to low-dose DBP appears to promote obesity in offspring by inhibiting UCP1 via ER stress, a process that was largely reversed by treatment with TUDCA.

Hepatic cysts are fluid-filled lesions in the liver that are estimated to occur in 5% of the population. They may cause hepatomegaly and abdominal pain. Progression to secondary fibrosis, cirrhosis, or cholangiocarcinoma can lead to morbidity and mortality. Previous studies of patients and rodent models have associated hepatic cyst formation with increased proliferation and fluid secretion in cholangiocytes, which are partially due to impaired primary cilia. Congenital hepatic cysts are thought to originate from faulty bile duct development, but the underlying mechanisms are not fully understood. In a forward genetic screen, we identified a zebrafish mutant that developed hepatic cysts during larval stages. The cyst formation was not due to changes in biliary cell proliferation, bile secretion, or impairment of primary cilia. Instead, time-lapse live imaging data showed that the mutant biliary cells failed to form interconnecting bile ducts because of defects in motility and protrusive activity. Accordingly, immunostaining revealed a disorganized actin and microtubule cytoskeleton in the mutant biliary cells. By whole-genome sequencing, we determined that the cystic phenotype in the mutant was caused by a missense mutation in the furinb gene, which encodes a proprotein convertase. The mutation altered Furinb localization and caused endoplasmic reticulum (ER) stress. The cystic phenotype could be suppressed by treatment with the ER stress inhibitor 4-phenylbutyric acid and exacerbated by treatment with the ER stress inducer tunicamycin. The mutant liver also exhibited increased mammalian target of rapamycin (mTOR) signaling. Treatment with mTOR inhibitors halted cyst formation at least partially through reducing ER stress. Conclusion: Our study has established a vertebrate model for studying hepatic cystogenesis and illustrated the contribution of ER stress in the disease pathogenesis.

The respective roles of the neuropeptides arginine vasopressin (AVP) and oxytocin (OXT) in modulating social cognition and for therapeutic intervention in autism spectrum disorder have not been fully established. In particular, while numerous studies have demonstrated effects of oxytocin in promoting social attention the role of AVP has not been examined. The present study employed a randomized, double-blind, placebo (PLC)-controlled between-subject design to explore the social- and emotion-specific effects of AVP on both bottom-up and top-down attention processing with a validated emotional anti-saccade eye-tracking paradigm in 80 healthy male subjects (PLC = 40, AVP = 40). Our findings showed that AVP increased the error rate for social (angry, fearful, happy, neutral and sad faces) but not non-social (oval shapes) stimuli during the anti-saccade condition and reduced error rates in the pro-saccade condition. Comparison of these findings with a previous study (sample size: PLC = 33, OXT = 33) using intranasal oxytocin revealed similar effects of the two peptides on anti-saccade errors, although with some difference in effects of specific face emotions, but a significantly greater effect of AVP on pro-saccades. Both peptides also produced a post-task anxiolytic effect by reducing state anxiety. Together these findings suggested that both AVP and OXT decrease goal-directed top-down attention control to social salient stimuli but that AVP more potently increased bottom-up social attentional processing.

Previously we showed the generation of a protein trap library made with the gene-break transposon (GBT) in zebrafish (Danio rerio) that could be used to facilitate novel functional genome annotation towards understanding molecular underpinnings of human diseases (Ichino et al, 2020). Here, we report a significant application of this library for discovering essential genes for heart rhythm disorders such as sick sinus syndrome (SSS). SSS is a group of heart rhythm disorders caused by malfunction of the sinus node, the heart's primary pacemaker. Partially owing to its aging-associated phenotypic manifestation and low expressivity, molecular mechanisms of SSS remain difficult to decipher. From 609 GBT lines screened, we generated a collection of 35 zebrafish insertional cardiac (ZIC) mutants in which each mutant traps a gene with cardiac expression. We further employed electrocardiographic measurements to screen these 35 ZIC lines and identified three GBT mutants with SSS-like phenotypes. More detailed functional studies on one of the arrhythmogenic mutants, GBT411, in both zebrafish and mouse models unveiled Dnajb6 as a novel SSS causative gene with a unique expression pattern within the subpopulation of sinus node pacemaker cells that partially overlaps with the expression of hyperpolarization activated cyclic nucleotide gated channel 4 (HCN4), supporting heterogeneity of the cardiac pacemaker cells.