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Behavioral larval zebrafish screens leverage a high-throughput small molecule discovery format to find neuroactive molecules relevant to mammalian physiology. We screen a library of 650 central nervous system active compounds in high replicate to train deep metric learning models on zebrafish behavioral profiles. The machine learning initially exploited subtle artifacts in the phenotypic screen, necessitating a complete experimental re-run with rigorous physical well-wise randomization. These large matched phenotypic screening datasets (initial and well-randomized) provide a unique opportunity to quantify and understand shortcut learning in a full-scale, real-world drug discovery dataset. The final deep metric learning model substantially outperforms correlation distance-the canonical way of computing distances between profiles-and generalizes to an orthogonal dataset of diverse drug-like compounds. We validate predictions by prospective in vitro radio-ligand binding assays against human protein targets, achieving a hit rate of 58% despite crossing species and chemical scaffold boundaries. These neuroactive compounds exhibit diverse chemical scaffolds, demonstrating that zebrafish phenotypic screens combined with metric learning achieve robust scaffold hopping capabilities.

The circadian clock is a cell-autonomous process that regulates daily internal rhythms by interacting with environmental signals. Reports across species show that infection can alter the expression of circadian genes; however, in teleosts, these effects are influenced by light exposure. Currently, no reports analyze the direct effects of bacterial exposure on the zebrafish clock. Using zebrafish Z3 cells, we demonstrate that exposure to heat-killed Streptococcus pneumoniae (HK-Spn) augments the expression of core repressive factors in a light- and time-dependent manner. In constant darkness, HK-Spn highly upregulated cry1a, per3, and per1b expression. In the presence of light, HK-Spn exposure rapidly and strongly upregulated per2 and cry1a, and this was proportionally increased with light intensity. The combinatorial effect of light and HK-Spn on per2 and cry1a was not duplicated with H2O2, a known byproduct of light exposure. However, the ROS inhibitor N-acetyl cysteine was sufficient to block HK-Spn augmentation of per2, cry1a, and per3. These findings demonstrate that exposure to an inactive bacteria influences the expression of zebrafish clock genes under different light conditions.

Pericardial edema - fluid accumulation within the pericardium - is a frequently observed malformation in zebrafish embryo-based chemical toxicity screens. We recently discovered that the severity of triphenyl phosphate (TPHP)-induced pericardial edema was dependent on the ionic strength of exposure media. TPHP is an aryl phosphate ester (APE) widely used as a plasticizer and flame retardant. APEs are characterized by having one or more aryl groups bound to a phosphate center, with TPHP containing only unsubstituted aryl groups. Therefore, the objective of this study was to begin investigating whether, similar to TPHP, pericardial edema induced by other structurally related APEs is dependent on the ionic composition of exposure media. We first mined the peer-reviewed literature to identify other APEs that 1) induced pericardial edema in zebrafish embryos within a minimum of three peer-reviewed publications, and 2) demonstrated a statistically significant induction of pericardial edema in at least 70 % of the studies evaluated. Based on this meta-analysis, we identified four other APEs that caused pericardial edema in zebrafish embryos: isopropylated triphenyl phosphate (IPTPP), cresyl diphenyl phosphate (CDP), tricresyl phosphate (TMPP), and 2-ethylhexyl diphenyl phosphate (EDHPHP). Using TPHP as a positive control and pericardial edema as a readout, we developed concentration-response curves for all four APEs based on static exposure from 24 to 72 h post-fertilization (hpf). We then conducted co-exposures with D-Mannitol (an osmotic diuretic) and exposures within reverse osmosis (RO) water determine whether the ionic composition of exposure media mitigated APE-induced pericardial edema at 72 hpf. Using pericardial edema as an endpoint, the approximate EC50s for TPHP (positive control), IPTPP, CDP, TMPP, and EDHPHP were 6.25, 3.125, 3.125, 25, and 100 µM, respectively, based on exposure from 24 to 72 hpf. Interestingly, similar to our findings with TPHP, co-exposure with D-Mannitol and exposure within ion-deficient water significantly mitigated IPTPP- CDP-, TMPP-, and EDHPHP-induced pericardial edema in zebrafish embryos, suggesting that chemically-induced pericardial edema may be 1) dependent on the ionic composition of exposure media and 2) driven by a disruption in osmoregulation across the embryonic epidermis. Therefore, similar to other assay parameters, our findings underscore the need to standardize the osmolarity of exposure media in order to minimize the potential for false positive/negative hits in zebrafish embryo-based chemical toxicity screens conducted around the world.

The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen21-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG21-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG211 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG21-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein localization. Our approach should be broadly useful for a wide range of applications.

Lipoprotein lipase (LPL) and multiple regulators of LPL activity (e.g., APOC2 and ANGPTL4) are present in all vertebrates, but GPIHBP1-the endothelial cell (EC) protein that captures LPL within the subendothelial spaces and transports it to its site of action in the capillary lumen-is present in mammals but in not chickens or other lower vertebrates. In mammals, GPIHBP1 deficiency causes severe hypertriglyceridemia, but chickens maintain low triglyceride levels despite the absence of GPIHBP1. To understand intravascular lipolysis in lower vertebrates, we examined LPL expression in mouse and chicken hearts. In both species, LPL was abundant on capillaries, but the distribution of Lpl transcripts was strikingly different. In mouse hearts, Lpl transcripts were extremely abundant in cardiomyocytes but were barely detectable in capillary ECs. In chicken hearts, Lpl transcripts were absent in cardiomyocytes but abundant in capillary ECs. In zebrafish hearts, lpl transcripts were also in capillary ECs but not cardiomyocytes. In both mouse and chicken hearts, LPL was present, as judged by immunogold electron microscopy, in the glycocalyx of capillary ECs. Thus, mammals produce LPL in cardiomyocytes and rely on GPIHBP1 to transport the LPL into capillaries, whereas lower vertebrates produce LPL directly in capillary ECs, rendering an LPL transporter unnecessary.

PURPOSE: Optical development of the zebrafish eye relies on the movement of the highly refractive lens nucleus from an anterior to a central location in the optical axis during development. We have shown that this mechanism in turn depends on the function of Aquaporin 0a (Aqp0a), a multifunctional and extremely abundant protein in lens fiber cell membranes. Here, we probe the specific cellular functions necessary for rescuing lens nucleus centralization defects in aqp0a-/- null mutants by stable overexpression of an Aqp0 orthologue from a killifish, MIPfun. METHODS: We test in vivo requirements for lens transparency and nucleus centralization of MIPfun for auto-adhesion, water permeability (Pf), and Pf sensitivity to regulation by Ca2+ or pH by overexpression of MIPfun mutants previously shown to have defects in these functions in vitro or in silico. RESULTS: Water permeability of MIPfun is essential for rescuing lens transparency and nucleus centralization defects, whereas auto-adhesion is not. Furthermore, water permeability regulation by Ca2+ and pH appear residue-dependent, because some Ca2+-insensitive mutants fail to rescue, and pH-insensitive mutants only partially rescue defects. MIPfun lacking Pf sensitivity to both, Ca2+ and pH, also fails to rescue lens nucleus centralization. CONCLUSION: This study shows that regulation of water permeability by Aqp0 plays a key role in the centralization of the zebrafish lens nucleus, providing the first direct evidence for water transport in this aspect of optical development.

Cranial sutures separate neighboring skull bones and are sites of bone growth. A key question is how osteogenic activity is controlled to promote bone growth while preventing aberrant bone fusions during skull expansion. Using single-cell transcriptomics, lineage tracing, and mutant analysis in zebrafish, we uncover key developmental transitions regulating bone formation at sutures during skull expansion. In particular, we identify a subpopulation of mesenchyme cells in the mid-suture region that upregulate a suite of genes including BMP antagonists (e.g. grem1a) and pro-angiogenic factors. Lineage tracing with grem1a:nlsEOS reveals that this mid-suture subpopulation is largely non-osteogenic. Moreover, combinatorial mutation of BMP antagonists enriched in this mid-suture subpopulation results in increased BMP signaling in the suture, misregulated bone formation, and abnormal suture morphology. These data reveal establishment of a non-osteogenic mesenchyme population in the mid-suture region that restricts bone formation through local BMP antagonism, thus ensuring proper suture morphology.

During Hedgehog (Hh) signal transduction in development and disease, the atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors by binding the protein kinase A catalytic subunit (PKA-C) and physically blocking its enzymatic activity. Here, we show that GPCR kinase 2 (GRK2) orchestrates this process during endogenous mouse and zebrafish Hh pathway activation in the primary cilium. Upon SMO activation, GRK2 rapidly relocalizes from the ciliary base to the shaft, triggering SMO phosphorylation and PKA-C interaction. Reconstitution studies reveal that GRK2 phosphorylation enables active SMO to bind PKA-C directly. Lastly, the SMO-GRK2-PKA pathway underlies Hh signal transduction in a range of cellular and in vivo models. Thus, GRK2 phosphorylation of ciliary SMO and the ensuing PKA-C binding and inactivation are critical initiating events for the intracellular steps in Hh signaling. More broadly, our study suggests an expanded role for GRKs in enabling direct GPCR interactions with diverse intracellular effectors.

Capture of a photon by an opsin visual pigment isomerizes its 11-cis-retinaldehyde (11cRAL) chromophore to all-trans-retinaldehyde (atRAL), which subsequently dissociates. To restore light sensitivity, the unliganded apo-opsin combines with another 11cRAL to make a new visual pigment. Two enzyme pathways supply chromophore to photoreceptors. The canonical visual cycle in retinal pigment epithelial cells supplies 11cRAL at low rates. The photic visual cycle in Müller cells supplies cones with 11-cis-retinol (11cROL) chromophore precursor at high rates. Although rods can only use 11cRAL to regenerate rhodopsin, cones can use 11cRAL or 11cROL to regenerate cone visual pigments. We performed a screen in zebrafish retinas and identified ZCRDH as a candidate for the enzyme that converts 11cROL to 11cRAL in cone inner segments. Retinoid analysis of eyes from Zcrdh-mutant zebrafish showed reduced 11cRAL and increased 11cROL levels, suggesting impaired conversion of 11cROL to 11cRAL. By microspectrophotometry, isolated Zcrdh-mutant cones lost the capacity to regenerate visual pigments from 11cROL. ZCRDH therefore possesses all predicted properties of the cone 11cROL dehydrogenase. The human protein most similar to ZCRDH is RDH12. By immunocytochemistry, ZCRDH was abundantly present in cone inner segments, similar to the reported distribution of RDH12. Finally, RDH12 was the only mammalian candidate protein to exhibit 11cROL-oxidase catalytic activity. These observations suggest that RDH12 in mammals is the functional ortholog of ZCRDH, which allows cones, but not rods, to regenerate visual pigments from 11cROL provided by Müller cells. This capacity permits cones to escape competition from rods for visual chromophore in daylight-exposed retinas.

Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries and play a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular disease and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating dedifferentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that arterial specification of CoW endothelial cells (ECs) occurs after their migration from cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors after they were recruited to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity and wall shear stress. Furthermore, pulsatile flow induces differentiation of human brain PDGFRB+ mural cells into VSMCs, and blood flow is required for VSMC differentiation on zebrafish CoW arteries. Consistently, flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight blood flow activation of endothelial klf2a as a mechanism regulating initial VSMC differentiation on vertebrate brain arteries.

Reproductive success relies on proper establishment and maintenance of biological sex. In many animals, including mammals, the primary gonad is initially ovary biased. We previously showed the RNA binding protein (RNAbp), Rbpms2, is required for ovary fate in zebrafish. Here, we identified Rbpms2 targets in oocytes (Rbpms2-bound oocyte RNAs; rboRNAs). We identify Rbpms2 as a translational regulator of rboRNAs, which include testis factors and ribosome biogenesis factors. Further, genetic analyses indicate that Rbpms2 promotes nucleolar amplification via the mTorc1 signaling pathway, specifically through the mTorc1-activating Gap activity towards Rags 2 (Gator2) component, Missing oocyte (Mios). Cumulatively, our findings indicate that early gonocytes are in a dual poised, bipotential state in which Rbpms2 acts as a binary fate-switch. Specifically, Rbpms2 represses testis factors and promotes oocyte factors to promote oocyte progression through an essential Gator2-mediated checkpoint, thereby integrating regulation of sexual differentiation factors and nutritional availability pathways in zebrafish oogenesis.

The Alliance of Genome Resources (Alliance) is an extensible coalition of knowledge bases focused on the genetics and genomics of intensively studied model organisms. The Alliance is organized as individual knowledge centers with strong connections to their research communities and a centralized software infrastructure, discussed here. Model organisms currently represented in the Alliance are budding yeast, Caenorhabditis elegans, Drosophila, zebrafish, frog, laboratory mouse, laboratory rat, and the Gene Ontology Consortium. The project is in a rapid development phase to harmonize knowledge, store it, analyze it, and present it to the community through a web portal, direct downloads, and application programming interfaces (APIs). Here, we focus on developments over the last 2 years. Specifically, we added and enhanced tools for browsing the genome (JBrowse), downloading sequences, mining complex data (AllianceMine), visualizing pathways, full-text searching of the literature (Textpresso), and sequence similarity searching (SequenceServer). We enhanced existing interactive data tables and added an interactive table of paralogs to complement our representation of orthology. To support individual model organism communities, we implemented species-specific landing pages and will add disease-specific portals soon; in addition, we support a common community forum implemented in Discourse software. We describe our progress toward a central persistent database to support curation, the data modeling that underpins harmonization, and progress toward a state-of-the-art literature curation system with integrated artificial intelligence and machine learning (AI/ML).

Craniosynostosis, defined by premature fusion of one or multiple cranial sutures, is a common congenital defect affecting more than 1/2000 infants and results in restricted brain expansion. Single gene mutations account for 15%-20% of cases, largely as part of a syndrome, but the majority are nonsyndromic with complex underlying genetics. We hypothesized that the two noncoding genomic regions identified by a GWAS for craniosynostosis contain distal regulatory elements for the risk genes BMPER and BMP2. To identify such regulatory elements, we surveyed conserved noncoding sequences from both risk loci for enhancer activity in transgenic Danio rerio. We identified enhancers from both regions that direct expression to skeletal tissues, consistent with the endogenous expression of bmper and bmp2. For each locus, we also found a skeletal enhancer that also contains a sequence variant associated with craniosynostosis risk. We examined the activity of each enhancer during craniofacial development and found that the BMPER-associated enhancer is active in the restricted region of cartilage closely associated with frontal bone initiation. The same enhancer is active in mouse skeletal tissues, demonstrating evolutionarily conserved activity. Using enhanced yeast one-hybrid assays, we identified transcription factors that bind each enhancer and observed differential binding between alleles, implicating multiple signaling pathways. Our findings help unveil the genetic mechanism of the two craniosynostosis risk loci. More broadly, our combined in vivo approach is applicable to many complex genetic diseases to build a link between association studies and specific genetic mechanisms.

How do vessels find optimal radii? Capillaries are known to adapt their radii to maintain the shear stress of blood flow at the vessel wall at a set point, yet models of adaptation purely based on average shear stress have not been able to produce complex loopy networks that resemble real microvascular systems. For narrow vessels where red blood cells travel in a single file, the shear stress on vessel endothelium peaks sharply when a red blood cell passes through. We show that stable shear-stress-based adaptation is possible if vessel shear stress set points are cued to the stress peaks. Model networks that respond to peak stresses alone can quantitatively reproduce the observed zebrafish trunk microcirculation, including its adaptive trajectory when hematocrit changes or parts of the network are amputated. Our work reveals the potential for mechanotransduction alone to generate stable hydraulically tuned microvascular networks.

The transcription factor and cell cycle regulator p53 is marked for degradation by the ubiquitin ligase MDM2. The interaction between these 2 proteins is mediated by a conserved binding motif in the disordered p53 transactivation domain (p53TAD) and the folded SWIB domain in MDM2. The conserved motif in p53TAD from zebrafish displays a 20-fold weaker interaction with MDM2, compared to the interaction in human and chicken. To investigate this apparent difference, we tracked the molecular evolution of the p53TAD/MDM2 interaction among ray-finned fishes (Actinopterygii), the largest vertebrate clade. Intriguingly, phylogenetic analyses, ancestral sequence reconstructions, and binding experiments showed that different loss-of-affinity changes in the canonical binding motif within p53TAD have occurred repeatedly and convergently in different fish lineages, resulting in relatively low extant affinities (KD = 0.5 to 5 μM). However, for 11 different fish p53TAD/MDM2 interactions, nonconserved regions flanking the canonical motif increased the affinity 4- to 73-fold to be on par with the human interaction. Our findings suggest that compensating changes at conserved and nonconserved positions within the motif, as well as in flanking regions of low conservation, underlie a stabilizing selection of functional affinity in the p53TAD/MDM2 interaction. Such interplay complicates bioinformatic prediction of binding and calls for experimental validation. Motif-mediated protein-protein interactions involving short binding motifs and folded interaction domains are very common across multicellular life. It is likely that the evolution of affinity in motif-mediated interactions often involves an interplay between specific interactions made by conserved motif residues and nonspecific interactions by nonconserved disordered regions.

Intercellular communication is indispensable across multicellular organisms, and any aberration in this process can give rise to significant anomalies in developmental and homeostatic processes. Thus, a comprehensive understanding of its mechanisms is imperative for addressing human health-related concerns. Recent advances have expanded our understanding of intercellular communication by elucidating additional signaling modalities alongside established mechanisms. Notably, cellular protrusion-mediated long-range communication, characterized by physical contact through thin and elongated cellular protrusions between cells involved in signal transmission and reception, has emerged as a significant intercellular signaling paradigm. This chapter delves into the exploration of a signaling cellular protrusion termed 'airinemes,' discovered in the zebrafish skin. It covers their identified signaling roles and the cellular and molecular mechanisms that underpin their functionality.

Cells interpret a variety of signals through G-protein-coupled receptors (GPCRs) and stimulate the generation of second messengers such as cyclic adenosine monophosphate (cAMP). A long-standing puzzle is deciphering how GPCRs elicit different physiological responses despite generating similar levels of cAMP. We previously showed that some GPCRs generate cAMP from both the plasma membrane and the Golgi apparatus. Here we demonstrate that cardiomyocytes distinguish between subcellular cAMP inputs to elicit different physiological outputs. We show that generating cAMP from the Golgi leads to the regulation of a specific protein kinase A (PKA) target that increases the rate of cardiomyocyte relaxation. In contrast, cAMP generation from the plasma membrane activates a different PKA target that increases contractile force. We further validated the physiological consequences of these observations in intact zebrafish and mice. Thus, we demonstrate that the same GPCR acting through the same second messenger regulates cardiac contraction and relaxation dependent on its subcellular location.

Aortic aneurysms, which may dissect or rupture acutely and be lethal, can be a part of multisystem disorders that have a heritable basis. We report four patients with deficiency of selenocysteine-containing proteins due to selenocysteine Insertion Sequence Binding Protein 2 (SECISBP2) mutations who show early-onset, progressive, aneurysmal dilatation of the ascending aorta due to cystic medial necrosis. Zebrafish and male mice with global or vascular smooth muscle cell (VSMC)-targeted disruption of Secisbp2 respectively show similar aortopathy. Aortas from patients and animal models exhibit raised cellular reactive oxygen species, oxidative DNA damage and VSMC apoptosis. Antioxidant exposure or chelation of iron prevents oxidative damage in patients cells and aortopathy in the zebrafish model. Our observations suggest a key role for oxidative stress and cell death, including via ferroptosis, in mediating aortic degeneration.

The longevity of sperm in teleost such as zebrafish and medaka is short when isolated even in saline-balanced solution at a physiological temperature. In contrast, some internal fertilizers exhibit the long-term storage of sperm, >10 months, in the female reproductive tract. This evidence implies that sperm in teleost possesses the ability to survive for a long time under suitable conditions; however, these conditions are not well understood. In this study, we show that the sperm of zebrafish can survive and maintain fertility in L-15-based storage medium supplemented with bovine serum albumin, fetal bovine serum, glucose, and lactic acid for 28 days at room temperature. The fertilized embryos developed to normal fertile adults. This storage medium was effective in medaka sperm stored for 7 days at room temperature. These results suggest that sperm from external fertilizer zebrafish and medaka has the ability to survive for at least 4 and 1 week, respectively, in the body fluid-like medium at a physiological temperature. This sperm storage method allows researchers to ship sperm by low-cost methods and to investigate key factors for motility and fertile ability in those sperm.

Mammals have limited capacity for heart regeneration, whereas zebrafish have extraordinary regeneration abilities. During zebrafish heart regeneration, endothelial cells promote cardiomyocyte cell cycle reentry and myocardial repair, but the mechanisms responsible for promoting an injury microenvironment conducive to regeneration remain incompletely defined. Here, we identify the matrix metalloproteinase Mmp14b as an essential regulator of heart regeneration. We identify a TEAD-dependent mmp14b endothelial enhancer induced by heart injury in zebrafish and mice, and we show that the enhancer is required for regeneration, supporting a role for Hippo signaling upstream of mmp14b. Last, we show that MMP-14 function in mice is important for the accumulation of Agrin, an essential regulator of neonatal mouse heart regeneration. These findings reveal mechanisms for extracellular matrix remodeling that promote heart regeneration.

Background & aimsBiliary atresia (BA) is poorly understood and leads to liver transplantation (LT), with the requirement for and associated risks of lifelong immunosuppression, in most children. We performed a genome-wide association study (GWAS) to determine the genetic basis of BA.MethodsWe performed a GWAS in 811 European BA cases treated with LT in US, Canadian and UK centers, and 4,654 genetically matched controls. Whole-genome sequencing of 100 cases evaluated synthetic association with rare variants. Functional studies included whole liver transcriptome analysis of 64 BA cases and perturbations in experimental models.ResultsA GWAS of common single nucleotide polymorphisms (SNPs), i.e. allele frequencies >1%, identified intronic SNPs rs6446628 in AFAP1 with genome-wide significance (p = 3.93E-8) and rs34599046 in TUSC3 at sub-threshold genome-wide significance (p = 1.34E-7), both supported by credible peaks of neighboring SNPs. Like other previously reported BA-associated genes, AFAP1 and TUSC3 are ciliogenesis and planar polarity effectors (CPLANE). In gene-set-based GWAS, BA was associated with 6,005 SNPs in 102 CPLANE genes (p = 5.84E-15). Compared with non-CPLANE genes, more CPLANE genes harbored rare variants (allele frequency

Ambient air pollutants, including PM2.5 (aerodynamic diameter d ~2.5 μm), PM10 (d ~10 μm), and ultrafine particles (UFP: d

BMP15 is a conserved regulator of ovarian development and maintenance in vertebrates. In humans, premature ovarian insufficiency is caused by autoimmunity and genetic factors, including mutation of BMP15. The cellular mechanisms underlying ovarian failure caused by BMP15 mutation and immune contributions are not understood. Using zebrafish, we established a causal link between macrophage activation and ovarian failure, which, in zebrafish, causes sex reversal. We define a germline-soma signaling axis that activates macrophages and drives ovarian failure and female-to-male sex reversal. Germline loss of zebrafish Bmp15 impairs oogenesis and initiates this cascade. Single-cell RNA sequencing and genetic analyses implicate ovarian somatic cells that express conserved macrophage-activating ligands as mediators of ovarian failure and sex reversal. Genetic ablation of macrophages or elimination of Csf1Rb ligands, Il34 or Csf1a, delays or blocks premature oocyte loss and sex reversal. The axis identified here provides insight into the cells and pathways governing oocyte and ovary maintenance and potential therapeutic targets to preserve female fertility.

Many neurons in the central nervous system produce a single primary cilium that serves as a specialized signaling organelle. Several neuromodulatory G-protein-coupled receptors (GPCRs) localize to primary cilia in neurons, although it is not understood how GPCR signaling from the cilium impacts circuit function and behavior. We find that the vertebrate ancient long opsin A (VALopA), a Gi-coupled GPCR extraretinal opsin, targets to cilia of zebrafish spinal neurons. In the developing 1-d-old zebrafish, brief light activation of VALopA in neurons of the central pattern generator circuit for locomotion leads to sustained inhibition of coiling, the earliest form of locomotion. We find that a related extraretinal opsin, VALopB, is also Gi-coupled, but is not targeted to cilia. Light-induced activation of VALopB also suppresses coiling, but with faster kinetics. We identify the ciliary targeting domains of VALopA. Retargeting of both opsins shows that the locomotory response is prolonged and amplified when signaling occurs in the cilium. We propose that ciliary localization provides a mechanism for enhancing GPCR signaling in central neurons.

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development.

Triphenyl phosphate (TPHP) - a widely used organophosphate-based flame retardant - blocks cardiac looping during zebrafish development in a concentration-dependent manner, a phenotype that is dependent on disruption of embryonic osmoregulation and pericardial edema formation. However, its currently unclear whether (1) TPHP-induced effects on osmoregulation are driven by direct TPHP-induced injury to the embryonic epidermis and (2) whether TPHP-induced pericardial edema is reversible or irreversible following cessation of exposure. Therefore, the objectives of this study were to determine whether TPHP-induced pericardial edema is reversible and whether TPHP causes injury to the embryonic epidermis by quantifying the number of DAPI-positive epidermal cells and analyzing the morphology of the yolk sac epithelium using scanning electron microscopy. First, we found that exposure to 5 μM TPHP from 24-72 h post-fertilization (hpf) did not increase prolactin - a hormone that regulates ions and water levels - in embryonic zebrafish, whereas high ionic strength exposure media was associated with elevated levels of prolactin. Second, we found that exposure to 5 μM TPHP from 24-72 hpf did not decrease DAPI-positive epidermal cells within the embryonic epithelium, and that co-exposure with 2.14 μM fenretinide - a synthetic retinoid that promotes epithelial wound repair - from 24-72 hpf did not mitigate the prevalence of TPHP-induced epidermal folds within the yolk sac epithelium when embryos were exposed within high ionic strength exposure media. Finally, we found that the pericardial area and body length of embryos exposed to 5 μM TPHP from 24-72 hpf were similar to vehicle-treated embryos at 120 hpf following transfer to clean water and depuration of TPHP from 72-120 hpf. Overall, our findings suggest that (1) the ionic strength of exposure media may influence the baseline physiology of zebrafish embryos; (2) TPHP does not cause direct injury to the embryonic epidermis; and (3) TPHP-induced effects on pericardial area and body length are reversible 48 h after transferring embryos to clean water.

Dae Seok Eom spoke with Cell Reports about his scientific journey, inspirations to become a scientist, and his work on cellular protrusions called "airinemes" that are involved in long-range intercellular signal transmission in zebrafish; in particular, he discussed recent work regarding characterization of a macrophage population that plays a critical role in this process.

Whole mount zebrafish antibody staining (ABS) is a common staining technique used to localize protein information in a zebrafish embryo or larva. Like most biological assays, the whole mount zebrafish ABS is still largely conducted manually through labor intensive and time-consuming steps which affect both consistency and throughput of the assay. In this work, we develop a milli fluidic device that can automatically trap and immobilize the fixed chorion-less zebrafish embryos for the whole mount ABS. With just a single loading step, the zebrafish embryos can be trapped by the milli fluidic device through a chaotic hydrodynamic trapping process. Moreover, a consistent body orientation (i.e., head point inward) for the trapped zebrafish embryos can be achieved without additional orientation adjustment device. Furthermore, we employed a consumer-grade SLA 3D printer assisted method for device prototyping which is ideal for labs with limited budgets. Notably, the milli fluidic device has enabled the optimization and successful implementation of whole mount zebrafish Caspase-3 ABS. We demonstrated our device can accelerate the overall procedure by reducing at least 50% of washing time in the standard well-plate-based manual procedure. Also, the consistency is improved, and manual steps are reduced using the milli fluidic device. This work fills the gap in the milli fluidic application for whole mount zebrafish immunohistochemistry. We hope the device can be accepted by the zebrafish community and be used for other types of whole mount zebrafish ABS procedures or expanded to more complicated in situ hybridization (ISH) procedure.

Definitive haematopoietic stem and progenitor cells (HSPCs) generate erythroid, lymphoid and myeloid lineages. HSPCs are produced in the embryo via transdifferentiation of haemogenic endothelial cells in the aorta-gonad-mesonephros (AGM). HSPCs in the AGM are heterogeneous in differentiation and proliferative output, but how these intrinsic differences are acquired remains unanswered. Here we discovered that loss of microRNA (miR)-128 in zebrafish leads to an expansion of HSPCs in the AGM with different cell cycle states and a skew towards erythroid and lymphoid progenitors. Manipulating miR-128 in differentiating haemogenic endothelial cells, before their transition to HSPCs, recapitulated the lineage skewing in both zebrafish and human pluripotent stem cells. miR-128 promotes Wnt and Notch signalling in the AGM via post-transcriptional repression of the Wnt inhibitor csnk1a1 and the Notch ligand jag1b. De-repression of cskn1a1 resulted in replicative and erythroid-biased HSPCs, whereas de-repression of jag1b resulted in G2/M and lymphoid-biased HSPCs with long-term consequence on the respective blood lineages. We propose that HSPC heterogeneity arises in the AGM endothelium and is programmed in part by Wnt and Notch signalling.

Animal sensory systems are tightly adapted to the demands of their environment. In the visual domain, research has shown that many species have circuits and systems that exploit statistical regularities in natural visual signals. The zebrafish is a popular model animal in visual neuroscience, but relatively little quantitative data is available about the visual properties of the aquatic habitats where zebrafish reside, as compared to terrestrial environments. Improving our understanding of the visual demands of the aquatic habitats of zebrafish can enhance the insights about sensory neuroscience yielded by this model system. We analyzed a video dataset of zebrafish habitats captured by a stationary camera and compared this dataset to videos of terrestrial scenes in the same geographic area. Our analysis of the spatiotemporal structure in these videos suggests that zebrafish habitats are characterized by low visual contrast and strong motion when compared to terrestrial environments. Similar to terrestrial environments, zebrafish habitats tended to be dominated by dark contrasts, particularly in the lower visual field. We discuss how these properties of the visual environment can inform the study of zebrafish visual behavior and neural processing and, by extension, can inform our understanding of the vertebrate brain.

BACKGROUND: Type IV collagen is an abundant component of basement membranes in all multicellular species and is essential for the extracellular scaffold supporting tissue architecture and function. Lower organisms typically have two type IV collagen genes, encoding α1 and α2 chains, in contrast with the six genes in humans, encoding α1-α6 chains. The α chains assemble into trimeric protomers, the building blocks of the type IV collagen network. The detailed evolutionary conservation of type IV collagen network remains to be studied. RESULTS: We report on the molecular evolution of type IV collagen genes. The zebrafish α4 non-collagenous (NC1) domain, in contrast with its human ortholog, contains an additional cysteine residue and lacks the M93 and K211 residues involved in sulfilimine bond formation between adjacent protomers. This may alter α4 chain interactions with other α chains, as supported by temporal and anatomic expression patterns of collagen IV chains during the zebrafish development. Despite the divergence between zebrafish and human α3 NC1 domain (endogenous angiogenesis inhibitor, Tumstatin), the zebrafish α3 NC1 domain exhibits conserved antiangiogenic activity in human endothelial cells. CONCLUSIONS: Our work supports type IV collagen is largely conserved between zebrafish and humans, with a possible difference involving the α4 chain.

Tissue-resident macrophages are heterogeneous and perform location-dependent functions. Skin resident macrophages play intriguing roles in long-distance intercellular signaling by mediating cellular protrusions called airinemes in zebrafish. These macrophages relay signaling molecules containing airineme vesicles between pigment cells, and their absence disrupts airineme-mediated signaling and pigment pattern formation. It is unknown if the same macrophages control both these signaling and typical immune functions or if a separate subpopulation functions in intercellular communication. With high-resolution imaging and genetic ablation approaches, we identify a macrophage subpopulation responsible for airineme-mediated signaling. These seem to be distinct from conventional skin-resident macrophages by their ameboid morphology and faster or expansive migratory behaviors. They resemble ectoderm-derived macrophages termed metaphocytes. Metaphocyte ablation markedly decreases airineme extension and signaling. In addition, these ameboid/metaphocytes require matrix metalloproteinase-9 for their migration and airineme-mediated signaling. These results reveal a macrophage subpopulation with specialized functions in airineme-mediated signaling, which may play roles in other aspects of intercellular communication.

Developmental exposure to environmental toxicants has been linked to the onset of neurological disorders and diseases. Despite substantial advances in the field of neurotoxicology, there remain significant knowledge gaps in our understanding of cellular targets and molecular mechanisms that mediate the neurotoxicological endpoints associated with exposure to both legacy contaminants and emerging contaminants of concern. Zebrafish are a powerful neurotoxicological model given their high degree sequence conservation with humans and the similarities they share with mammals in micro- and macro-level brain structures. Many zebrafish studies have effectively utilized behavioral assays to predict the neurotoxic potential of different compounds, but behavioral phenotypes are rarely able to predict the brain structures, cell types, or mechanisms affected by chemical exposures. Calcium-modulated photoactivatable ratiometric integrator (CaMPARI), a recently developed genetically-encoded calcium indicator, undergoes a permanent green to red switch in the presence of elevated intracellular Ca2+ concentrations and 405-nm light, which allows for a "snapshot" of brain activity in freely-swimming larvae. To determine whether behavioral results are predictive of patterns of neuronal activity, we assessed the effects of three common neurotoxicants, ethanol, 2,2',3,5',6-pentachlorobiphenyl (PCB 95), and monoethylhexyl phthalate (MEHP), on both brain activity and behavior by combining the behavioral light/dark assay with CaMPARI imaging. We demonstrate that brain activity profiles and behavioral phenotypes are not always concordant and, therefore, behavior alone is not sufficient to understand how toxicant exposure affects neural development and network dynamics. We conclude that pairing behavioral assays with functional neuroimaging tools such as CaMPARI provides a more comprehensive understanding of the neurotoxic endpoints of compounds while still offering a relatively high throughput approach to toxicity testing.

Neurodegenerative diseases are a major cause of disability in the world, but their etiologies largely remain elusive. Genetic factors can only account for a minority of risk for most of these disorders, suggesting environmental factors play a significant role in the development of these diseases. Prolonged exposure to air pollution has recently been identified to increase the risk of Alzheimer's and Parkinson's diseases, but the molecular mechanisms by which it acts are not well understood. Zebrafish embryos exposed to diesel exhaust particle extract (DEPe) lead to dysfunctional autophagy and neuronal loss. Here, we exposed zebrafish embryos to DEPe and performed high throughput proteomic and transcriptomic expression analyses from their brains to identify pathogenic pathways induced by air pollution. DEPe treatment altered several biological processes and signaling pathways relevant to neurodegenerative processes, including xenobiotic metabolism, phagosome maturation, and amyloid processing. The biggest induction of gene expression in brains was in Cyp1A (over 30-fold). The relevance of this expression change was confirmed by blocking induction using CRISPR/Cas9, which resulted in a dramatic increase in sensitivity to DEPe toxicity, confirming that Cyp1A induction was a compensatory protective mechanism. These studies identified disrupted molecular pathways that may contribute to the pathogenesis of neurodegenerative disorders. Ultimately, determining the molecular basis of how air pollution increases the risk of neurodegeneration will help in the development of disease-modifying therapies.

Neuronal wiring diagrams reconstructed by electron microscopy1,2,3,4,5 pose new questions about the organization of nervous systems following the time-honored tradition of cross-species comparisons.6,7 The C. elegans connectome has been conceptualized as a sensorimotor circuit that is approximately feedforward,8,9,10,11 starting from sensory neurons proceeding to interneurons and ending with motor neurons. Overrepresentation of a 3-cell motif often known as the "feedforward loop" has provided further evidence for feedforwardness.10,12 Here, we contrast with another sensorimotor wiring diagram that was recently reconstructed from a larval zebrafish brainstem.13 We show that the 3-cycle, another 3-cell motif, is highly overrepresented in the oculomotor module of this wiring diagram. This is a first for any neuronal wiring diagram reconstructed by electron microscopy, whether invertebrate12,14 or mammalian.15,16,17 The 3-cycle of cells is "aligned" with a 3-cycle of neuronal groups in a stochastic block model (SBM)18 of the oculomotor module. However, the cellular cycles exhibit more specificity than can be explained by the group cycles-recurrence to the same neuron is surprisingly common. Cyclic structure could be relevant for theories of oculomotor function that depend on recurrent connectivity. The cyclic structure coexists with the classic vestibulo-ocular reflex arc for horizontal eye movements,19 and could be relevant for recurrent network models of temporal integration by the oculomotor system.20,21.

Retinal membrane guanylyl cyclases (RetGCs) in vertebrate rod and cone photoreceptors are activated by a family of neuronal Ca2+ sensor proteins called guanylyl cyclase activating proteins (GCAP1-7). GCAP5 from zebrafish photoreceptors binds to RetGC and confers Ca2+/Fe2+-dependent regulation of RetGC enzymatic activity that promotes the recovery phase of visual phototransduction. We report NMR chemical shift assignments of GCAP5 with a R22A mutation (called GCAP5R22A) that abolishes protein dimerization and activates RetGC with 3-fold higher activity than that of wild type GCAP5 (BMRB No. 51,783).

Excessive erythrocytosis (EE) is a major hallmark of patients suffering from chronic mountain sickness (CMS, also known as Monge's disease) and is responsible for major morbidity and even mortality in early adulthood. We took advantage of unique populations, one living at high altitude (Peru) showing EE, with another population, at the same altitude and region, showing no evidence of EE (non-CMS). Through RNA-Seq, we identified and validated the function of a group of long noncoding RNAs (lncRNAs) that regulate erythropoiesis in Monge's disease, but not in the non-CMS population. Among these lncRNAs is hypoxia induced kinase-mediated erythropoietic regulator (HIKER)/LINC02228, which we showed plays a critical role in erythropoiesis in CMS cells. Under hypoxia, HIKER modulated CSNK2B (the regulatory subunit of casein kinase 2). A downregulation of HIKER downregulated CSNK2B, remarkably reducing erythropoiesis; furthermore, an upregulation of CSNK2B on the background of HIKER downregulation rescued erythropoiesis defects. Pharmacologic inhibition of CSNK2B drastically reduced erythroid colonies, and knockdown of CSNK2B in zebrafish led to a defect in hemoglobinization. We conclude that HIKER regulates erythropoiesis in Monge's disease and acts through at least one specific target, CSNK2B, a casein kinase.

BACKGROUND: Calcific aortic valve disease (CAVD) is characterized by a phenotypic switch of valvular interstitial cells to bone-forming cells. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors at the interface between innate immunity and tissue repair. Type I interferons (IFNs) are not only crucial for an adequate antiviral response but also implicated in bone formation. We hypothesized that the accumulation of endogenous TLR3 ligands in the valvular leaflets may promote the generation of osteoblast-like cells through enhanced type I IFN signaling. METHODS: Human valvular interstitial cells isolated from aortic valves were challenged with mechanical strain or synthetic TLR3 agonists and analyzed for bone formation, gene expression profiles, and IFN signaling pathways. Different inhibitors were used to delineate the engaged signaling pathways. Moreover, we screened a variety of potential lipids and proteoglycans known to accumulate in CAVD lesions as potential TLR3 ligands. Ligand-receptor interactions were characterized by in silico modeling and verified through immunoprecipitation experiments. Biglycan (Bgn), Tlr3, and IFN-α/β receptor alpha chain (Ifnar1)-deficient mice and a specific zebrafish model were used to study the implication of the biglycan (BGN)-TLR3-IFN axis in both CAVD and bone formation in vivo. Two large-scale cohorts (GERA [Genetic Epidemiology Research on Adult Health and Aging], n=55 192 with 3469 aortic stenosis cases; UK Biobank, n=257 231 with 2213 aortic stenosis cases) were examined for genetic variation at genes implicated in BGN-TLR3-IFN signaling associating with CAVD in humans. RESULTS: Here, we identify TLR3 as a central molecular regulator of calcification in valvular interstitial cells and unravel BGN as a new endogenous agonist of TLR3. Posttranslational BGN maturation by xylosyltransferase 1 (XYLT1) is required for TLR3 activation. Moreover, BGN induces the transdifferentiation of valvular interstitial cells into bone-forming osteoblasts through the TLR3-dependent induction of type I IFNs. It is intriguing that Bgn-/-, Tlr3-/-, and Ifnar1-/- mice are protected against CAVD and display impaired bone formation. Meta-analysis of 2 large-scale cohorts with >300 000 individuals reveals that genetic variation at loci relevant to the XYLT1-BGN-TLR3-interferon-α/β receptor alpha chain (IFNAR) 1 pathway is associated with CAVD in humans. CONCLUSIONS: This study identifies the BGN-TLR3-IFNAR1 axis as an evolutionarily conserved pathway governing calcification of the aortic valve and reveals a potential therapeutic target to prevent CAVD.

Central conducting lymphatic anomaly (CCLA) due to congenital maldevelopment of the lymphatics can result in debilitating and life-threatening disease with limited treatment options. We identified 4 individuals with CCLA, lymphedema, and microcystic lymphatic malformation due to pathogenic, mosaic variants in KRAS. To determine the functional impact of these variants and identify a targeted therapy for these individuals, we used primary human dermal lymphatic endothelial cells (HDLECs) and zebrafish larvae to model the lymphatic dysplasia. Expression of the p.Gly12Asp and p.Gly13Asp variants in HDLECs in a 2‑dimensional (2D) model and 3D organoid model led to increased ERK phosphorylation, demonstrating these variants activate the RAS/MAPK pathway. Expression of activating KRAS variants in the venous and lymphatic endothelium in zebrafish resulted in lymphatic dysplasia and edema similar to the individuals in the study. Treatment with MEK inhibition significantly reduced the phenotypes in both the organoid and the zebrafish model systems. In conclusion, we present the molecular characterization of the observed lymphatic anomalies due to pathogenic, somatic, activating KRAS variants in humans. Our preclinical studies suggest that MEK inhibition should be studied in future clinical trials for CCLA due to activating KRAS pathogenic variants.

Recent research has elucidated mechanochemical pathways of single cell polarization, but much less is known about collective motility initiation in adhesive cell groups. We used galvanotactic assays of zebrafish keratocyte cell groups, pharmacological perturbations, electric field switches, particle imaging velocimetry, and cell tracking to show that large cell groups initiate motility in minutes toward the cathode. Interestingly, while PI3K-inhibited single cells are biased toward the anode, inhibiting PI3K does not affect the cathode-directed cell group migration. We observed that control groups had the fastest cathode-migrating cell at the front, while the front cells in PI3K-inhibited groups were the slowest. Both control and PI3K-inhibited groups rapidly repolarized when the electric field direction was reversed, and the group migration continued after the electric field was switched off. Inhibiting myosin disrupted the cohesiveness of keratocyte groups and abolished the collective directionality and ability to switch direction when the electric field is reversed. Our data are consistent with a model according to which cells in the group sense the electric field individually and mechanical integration of the cells results in coherent group motility.

Hexahydro-1,3,5-trinitro-1,3,5-triazine, or Royal Demolition Explosive (RDX), is a major component of plastic explosives such as C-4. Acute exposures from intentional or accidental ingestion are a documented clinical concern, especially among young male U.S. service members in the armed forces. When ingested in large enough quantity, RDX causes tonic-clonic seizures. Previous in silico and in vitro experiments predict that RDX causes seizures by inhibiting α1β2γ2 γ-aminobutyric acid type A (GABAA) receptor-mediated chloride currents. To determine whether this mechanism translates in vivo, we established a larval zebrafish model of RDX-induced seizures. After a 3 h of exposure to 300 µM RDX, larval zebrafish exhibited a significant increase in motility in comparison to vehicle controls. Researchers blinded to experimental group manually scored a 20-min segment of video starting at 3.5 h post-exposure and found significant seizure behavior that correlated with automated seizure scores. Midazolam (MDZ), an nonselective GABAAR positive allosteric modulator (PAM), and a combination of Zolpidem (α1 selective PAM) and compound 2-261 (β2/3-selective PAM) were effective in mitigating RDX-triggered behavioral and electrographic seizures. These findings confirm that RDX induces seizure activity via inhibition of the α1β2γ2 GABAAR and support the use of GABAAR-targeted anti-seizure drugs for the treatment of RDX-induced seizures.

Skates are cartilaginous fish whose body plan features enlarged wing-like pectoral fins, enabling them to thrive in benthic environments1,2. However, the molecular underpinnings of this unique trait remain unclear. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins-including gene expression, chromatin occupancy and three-dimensional conformation-we find skate-specific genomic rearrangements that alter the three-dimensional regulatory landscape of genes that are involved in the planar cell polarity pathway. Functional inhibition of planar cell polarity signalling resulted in a reduction in anterior fin size, confirming that this pathway is a major contributor to batoid fin morphology. We also identified a fin-specific enhancer that interacts with several hoxa genes, consistent with the redeployment of hox gene expression in anterior pectoral fins, and confirmed its potential to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganization and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

Cardiovascular disease remains the leading cause of mortality worldwide. Cardiomyocytes are irreversibly lost due to cardiac ischemia secondary to disease. This leads to increased cardiac fibrosis, poor contractility, cardiac hypertrophy, and subsequent life-threatening heart failure. Adult mammalian hearts exhibit notoriously low regenerative potential, further compounding the calamities described above. Neonatal mammalian hearts, on the other hand, display robust regenerative capacities. Lower vertebrates such as zebrafish and salamanders retain the ability to replenish lost cardiomyocytes throughout life. It is critical to understand the varying mechanisms that are responsible for these differences in cardiac regeneration across phylogeny and ontogeny. Adult mammalian cardiomyocyte cell cycle arrest and polyploidization have been proposed as major barriers to heart regeneration. Here we review current models about why adult mammalian cardiac regenerative potential is lost including changes in environmental oxygen levels, acquisition of endothermy, complex immune system development, and possible cancer risk tradeoffs. We also discuss recent progress and highlight conflicting reports pertaining to extrinsic and intrinsic signaling pathways that control cardiomyocyte proliferation and polyploidization in growth and regeneration. Uncovering the physiological brakes of cardiac regeneration could illuminate novel molecular targets and offer promising therapeutic strategies to treat heart failure.

Aishwarya, R. et al. Visualizing subcellular localization of a protein in the heart using quantum dots-mediated immuno-labeling followed by transmission electron microscopy. Journal of Visualized Experiments. (187), e64085 (2022). Liu, B. et al. Visualizing mitophagy with fluorescent dyes for mitochondria and lysosome. Journal of Visualized Experiments. (189), e64647 (2022). Liu, C. Y., Zhu, J., Xie, Z. Visualizing yeast organelles with fluorescent protein markers. Journal of Visualized Experiments. (182), e63846 (2022). Zhao, X., Guo, S. Antibody uptake assay for tracking Notch/Delta endocytosis during the asymmetric division of zebrafish radial glia progenitors. Journal of Visualized Experiments. 65030 (2022). Park, S., Zhuang, Y., Shi, X. Label-retention expansion microscopy (LR-ExM) enables super-resolution imaging and high-efficiency labeling. Journal of Visualized Experiments. (188), e63793 (2022).

Bicuspid aortic valve (BAV), the most common cardiovascular malformation occurs in 0.5-1.2% of the population. Although highly heritable, few causal mutations have been identified in BAV patients. Here, we report the targeted sequencing of HOXA1 in a cohort of BAV patients and the identification of rare indel variants in the homopolymeric histidine tract of HOXA1. In vitro analysis shows that disruption of this motif leads to a significant reduction in protein half-life and defective transcriptional activity of HOXA1. In zebrafish, targeting hoxa1a ortholog results in aortic valve defects. In vivo assays indicates that these variants behave as dominant negatives leading abnormal valve development. In mice, deletion of Hoxa1 leads to BAV with a very small, rudimentary non-coronary leaflet. We also show that 17% of homozygous Hoxa1-1His knock-in mice present similar phenotype. Genetic lineage tracing in Hoxa1-/- mutant mice reveals an abnormal reduction of neural crest-derived cells in the valve leaflet, which is caused by a failure of early migration of these cells.

Hexafluoropropylene oxide-dimer acid (HFPO-DA) is one of the emerging replacements for the "forever" carcinogenic and toxic long-chain PFAS. HFPO-DA is a polymerization aid used for manufacturing fluoropolymers, whose global distribution and undetermined toxic properties are a concern regarding human and ecological health. To assess embryotoxic potential, zebrafish embryos were exposed to HFPO-DA at concentrations of 0.5-20,000 mg/L at 24-, 48-, and 72-h post-fertilization (hpf). Heart rate increased significantly in embryos exposed to 2 mg/L and 10 mg/L HFPO-DA across all time points. Spinal deformities and edema phenotypes were evident among embryos exposed to 1000-16,000 mg/L HFPO-DA at 72 hpf. A median lethal concentration (LC50) was derived as 7651 mg/L at 72 hpf. Shallow RNA sequencing analysis of 9465 transcripts identified 38 consistently differentially expressed genes at 0.5 mg/L, 1 mg/L, 2 mg/L, and 10 mg/L HFPO-DA exposures. Notably, seven downregulated genes were associated with visual response, and seven upregulated genes were expressed in or regulated the cardiovascular system. This study identifies biological targets and molecular pathways affected during animal development by an emerging, potentially problematic, and ubiquitous industrial chemical.

Direct contact between cells expressing either ephrin ligands or Eph receptor tyrosine kinase produces diverse developmental responses. Transmembrane ephrinB ligands play active roles in transducing bi-directional signals downstream of EphB/ephrinB interaction. However, it has not been well understood how ephrinB relays transcellular signals to neighboring cells and what intracellular effectors are involved. Here, we report that kindlin2 can mediate bi-directional ephrinB signaling through binding to a highly conserved NIYY motif in the ephrinB2 cytoplasmic tail. We show this interaction is important for EphB/ephrinB-mediated integrin activation in mammalian cells and for blood vessel morphogenesis during zebrafish development. A mixed two-cell population study revealed that kindlin2 (in ephrinB2-expressing cells) modulates transcellular EphB4 activation by promoting ephrinB2 clustering. This mechanism is also operative for EphB2/ephrinB1, suggesting that kindlin2-mediated regulation is conserved for EphB/ephrinB signaling pathways. Together, these findings show that kindlin2 enables EphB4/ephrinB2 bi-directional signal transmission.

In recent years, psychedelics have garnered significant interest as therapeutic agents for treating diverse neuropsychiatric disorders. However, the potential for these compounds to produce developmental neurotoxicity has not been rigorously assessed, and much of the available safety data is based on epidemiological studies with limited experimental testing in laboratory animal models. Moreover, the experimental safety data available thus far have focused on adult organisms, and the few studies conducted using developing organisms have tested a limited number of compounds, precluding direct comparisons between various chemical scaffolds. In the present study, 13 psychoactive compounds of different chemical or pharmacological classes were screened in a larval zebrafish model for teratological and behavioral abnormalities following acute and chronic developmental exposures. We found that the psychedelic tryptamines and ketamine were less neurotoxic to larval zebrafish than LSD and psychostimulants. Our work, which leverages the advantage of using zebrafish for higher throughput toxicity screening, provides a robust reference database for comparing the neurotoxicity profiles of novel psychedelics currently under development for therapeutic applications.

The integration of large-scale gene expression mapping into a multifaceted larval zebrafish brain atlas accelerates the characterization of neurons in behaviorally relevant circuits.

Zebrafish (Danio rerio) are aquatic vertebrates with significant homology to their terrestrial counterparts. While zebrafish have a centuries-long track record in developmental and regenerative biology, their utility has grown exponentially with the onset of modern genetics. This is exemplified in studies focused on skeletal development and repair. Herein, the numerous contributions of zebrafish to our understanding of the basic science of cartilage, bone, tendon/ligament, and other skeletal tissues are described, with a particular focus on applications to development and regeneration. We summarize the genetic strengths that have made the zebrafish a powerful model to understand skeletal biology. We also highlight the large body of existing tools and techniques available to understand skeletal development and repair in the zebrafish and introduce emerging methods that will aid in novel discoveries in skeletal biology. Finally, we review the unique contributions of zebrafish to our understanding of regeneration and highlight diverse routes of repair in different contexts of injury. We conclude that zebrafish will continue to fill a niche of increasing breadth and depth in the study of basic cellular mechanisms of skeletal biology.