Neuroprotective gap-junction-mediated bystander transformations in the adult zebrafish spinal cord after injury.

The adult zebrafish spinal cord displays an impressive innate ability to regenerate after traumatic insults, yet the underlying adaptive cellular mechanisms remain elusive. Here, we show that while the cellular and tissue responses after injury are largely conserved among vertebrates, the large-size fast spinal zebrafish motoneurons are remarkably resilient by remaining viable and functional. We also reveal the dynamic changes in motoneuron glutamatergic input, excitability, and calcium signaling, and we underscore the critical role of calretinin (CR) in binding and buffering the intracellular calcium after injury. Importantly, we demonstrate the presence and the dynamics of a neuron-to-neuron bystander neuroprotective biochemical cooperation mediated through gap junction channels. Our findings support a model in which the intimate and dynamic interplay between glutamate signaling, calcium buffering, gap junction channels, and intercellular cooperation upholds cell survival and promotes the initiation of regeneration.

The physical and cellular mechanism of structural color change in zebrafish.

Many animals exhibit remarkable colors that are produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These animals can fine-tune their crystal-based structural colors to communicate with each other, regulate body temperature, and create camouflage. While it is known that these changes in color are caused by changes in the angle of the crystal arrays relative to incident light, the cellular machinery that drives color change is not understood. Here, using a combination of 3D focused ion beam scanning electron microscopy (FIB-SEM), micro-focused X-ray diffraction, superresolution fluorescence light microscopy, and pharmacological perturbations, we characterized the dynamics and 3D cellular reorganization of crystal arrays within zebrafish iridophores during norepinephrine (NE)-induced color change. We found that color change results from a coordinated 20° tilting of the intracellular crystals, which alters both crystal packing and the angle at which impinging light hits the crystals. Importantly, addition of the dynein inhibitor dynapyrazole-a completely blocked this NE-induced red shift by hindering crystal dynamics upon NE addition. FIB-SEM and microtubule organizing center (MTOC) mapping showed that microtubules arise from two MTOCs located near the poles of the iridophore and run parallel to, and in between, individual crystals. This suggests that dynein drives crystal angle change in response to NE by binding to the limiting membrane surrounding individual crystals and walking toward microtubule minus ends. Finally, we found that intracellular cAMP regulates the color change process. Together, our results provide mechanistic insight into the cellular machinery that drives structural color change.

ADA2 regulates inflammation and hematopoietic stem cell emergence via the A2bR pathway in zebrafish.

Deficiency of adenosine deaminase 2 (DADA2) is an inborn error of immunity caused by loss-of-function mutations in the adenosine deaminase 2 (ADA2) gene. Clinical manifestations of DADA2 include vasculopathy and immuno-hematological abnormalities, culminating in bone marrow failure. A major gap exists in our knowledge of the regulatory functions of ADA2 during inflammation and hematopoiesis, mainly due to the absence of an ADA2 orthologue in rodents. Exploring these mechanisms is essential for understanding disease pathology and developing new treatments. Zebrafish possess two ADA2 orthologues, cecr1a and cecr1b, with the latter showing functional conservation with human ADA2. We establish a cecr1b-loss-of-function zebrafish model that recapitulates the immuno-hematological and vascular manifestations observed in humans. Loss of Cecr1b disrupts hematopoietic stem cell specification, resulting in defective hematopoiesis. This defect is caused by induced inflammation in the vascular endothelium. Blocking inflammation, pharmacological modulation of the A2r pathway, or the administration of the recombinant human ADA2 corrects these defects, providing insights into the mechanistic link between ADA2 deficiency, inflammation and immuno-hematological abnormalities. Our findings open up potential therapeutic avenues for DADA2 patients.

mTOR mutation disrupts larval zebrafish tail fin regeneration via regulating proliferation of blastema cells and mitochondrial functions.

BACKGROUND: The larval zebrafish tail fin can completely regenerate in 3 days post amputation. mTOR, the main regulator of cell growth and metabolism, plays an essential role in regeneration. Lots of studies have documented the role of mTOR in regeneration. However, the mechanisms involved are still not fully elucidated. MATERIALS AND RESULTS: This study aimed to explore the role and mechanism of mTOR in the regeneration of larval zebrafish tail fins. Initially, the spatial and temporal expression of mTOR signaling in the larval fin was examined, revealing its activation following tail fin amputation. Subsequently, a mTOR knockout (mTOR-KO) zebrafish line was created using CRISPR/Cas9 gene editing technology. The investigation demonstrated that mTOR depletion diminished the proliferative capacity of epithelial and mesenchymal cells during fin regeneration, with no discernible impact on cell apoptosis. Insight from SMART-seq analysis uncovered alterations in the cell cycle, mitochondrial functions and metabolic pathways when mTOR signaling was suppressed during fin regeneration. Furthermore, mTOR was confirmed to enhance mitochondrial functions and Ca2 + activation following fin amputation. These findings suggest a potential role for mTOR in promoting mitochondrial fission to facilitate tail fin regeneration. CONCLUSION: In summary, our results demonstrated that mTOR played a key role in larval zebrafish tail fin regeneration, via promoting mitochondrial fission and proliferation of blastema cells.

egr3 is a mechanosensitive transcription factor gene required for cardiac valve morphogenesis.

Biomechanical forces, and their molecular transducers, including key mechanosensitive transcription factor genes, such as KLF2, are required for cardiac valve morphogenesis. However, klf2 mutants fail to completely recapitulate the valveless phenotype observed under no-flow conditions. Here, we identify the transcription factor EGR3 as a conserved biomechanical force transducer critical for cardiac valve formation. We first show that egr3 null zebrafish display a complete and highly penetrant loss of valve leaflets, leading to severe blood regurgitation. Using tissue-specific loss- and gain-of-function tools, we find that during cardiac valve formation, Egr3 functions cell-autonomously in endothelial cells, and identify one of its effectors, the nuclear receptor Nr4a2b. We further find that mechanical forces up-regulate egr3/EGR3 expression in the developing zebrafish heart and in porcine valvular endothelial cells, as well as during human aortic valve remodeling. Altogether, these findings reveal that EGR3 is necessary to transduce the biomechanical cues required for zebrafish cardiac valve morphogenesis, and potentially for pathological aortic valve remodeling in humans.

Developmental Delay and Male-Biased Sex Ratio in esr2b Knockout Zebrafish.

The estrogen receptor signaling pathway plays an important role in vertebrate embryonic development and sexual differentiation. There are four major estrogen receptors in zebrafish: esr1, esr2a, esr2b and gper. However, the specific role of different estrogen receptors in zebrafish is not clear. To investigate the role of esr2b in zebrafish development and reproduction, this study utilized TALENs technology to generate an esr2b knockout homozygous zebrafish line. The number of eggs laid by esr2b knockout female zebrafish did not differ significantly from that of wild zebrafish. The embryonic development process of wild-type and esr2b knockout zebrafish was observed, revealing a significant developmental delay in the esr2b knockout zebrafish. Additionally, mortality rates were significantly higher in esr2b knockout zebrafish than in their wild-type counterparts at 24 hpf. The reciprocal cross experiment between esr2b knockout zebrafish and wild-type zebrafish revealed that the absence of esr2b resulted in a decline in the quality of zebrafish oocytes, while having no impact on sperm cells. The knockout of esr2b also led to an abnormal sex ratio in the adult zebrafish population, with a female-to-male ratio of approximately 1:7. The quantitative PCR (qPCR) and in situ hybridization results demonstrated a significant downregulation of cyp19ab1b expression in esr2b knockout embryos compared to wild-type embryos throughout development (at 2 dpf, 3 dpf and 4 dpf). Additionally, the estrogen-mediated induction expression of cyp19ab1b was attenuated, while the estradiol-induced upregulated expression of vtg1 was disrupted. These results suggest that esr2b is involved in regulating zebrafish oocyte development and sex differentiation.

Expression of Insl3 Protein in Adult Danio rerio.

Insulin-like peptide 3 (INSL3) is a biomarker for Leydig cells in the testes of vertebrates, and it is principally involved in spermatogenesis through specific binding with the RXFP2 receptor. This study reports the insl3 gene transcript and the Insl3 prepropeptide expression in both non-reproductive and reproductive tissues of Danio rerio. An immunohistochemistry analysis shows that the hormone is present at a low level in the Leydig cells and germ cells at all stages of Danio rerio testis differentiation. Considering that the insl3 gene is transcribed in Leydig cells, our results highlight an autocrine and paracrine function of this hormone in the Danio rerio testis, adding new information on the Insl3 mode of action in reproduction. We also show that Insl3 and Rxfp2 belonging to Danio rerio and other vertebrate species share most of the amino acid residues involved in the ligand-receptor interaction and activation, suggesting a conserved mechanism of action during vertebrate evolution.

Knockdown of vitamin D receptor affects early stages of pectoral fin development in zebrafish.

The vitamin D receptor (VDR) signalling has been implicated in vertebrate limb or fin formation. However, the involvement of VDR signalling in the early stages of limb/fin development remains to be elucidated. In this study, the role of VDR signalling in pectoral fin development was investigated in zebrafish embryos. Knockdown of vdr induced the severe impairment of pectoral fin development. The zebrafish larvae lacking vdr exhibited reduced pectoral fins with no skeletal elements. In situ hybridization revealed depletion of vdr downregulated fibroblast growth factor 24 (fgf24), a marker of early pectoral fin bud mesenchyme, in the presumptive fin field even before fin buds were visible. Moreover, a perturbed expression pattern of bone morphogenetic protein 4 (bmp4), a marker of the pectoral fin fold, was observed in the developing fin buds of zebrafish embryos that lost the vdr function. These findings suggest that VDR signalling is crucial in the early stages of fin development, potentially influencing the process by regulating other signalling molecules such as Fgf24 and Bmp4.

Multiple cis-regulatory elements control prox1a expression in distinct lymphatic vascular beds.

During embryonic development, lymphatic endothelial cell (LEC) precursors are distinguished from blood endothelial cells by the expression of Prospero-related homeobox 1 (Prox1), which is essential for lymphatic vasculature formation in mouse and zebrafish. Prox1 expression initiation precedes LEC sprouting and migration, serving as the marker of specified LECs. Despite its crucial role in lymphatic development, Prox1 upstream regulation in LECs remains to be uncovered. SOX18 and COUP-TFII are thought to regulate Prox1 in mice by binding its promoter region. However, the specific regulation of Prox1 expression in LECs remains to be studied in detail. Here, we used evolutionary conservation and chromatin accessibility to identify enhancers located in the proximity of zebrafish prox1a active in developing LECs. We confirmed the functional role of the identified sequences through CRISPR/Cas9 mutagenesis of a lymphatic valve enhancer. The deletion of this region results in impaired valve morphology and function. Overall, our results reveal an intricate control of prox1a expression through a collection of enhancers. Ray-finned fish-specific distal enhancers drive pan-lymphatic expression, whereas vertebrate-conserved proximal enhancers refine expression in functionally distinct subsets of lymphatic endothelium.

Angpt1 binding to Tie1 regulates the signaling required for lymphatic vessel development in zebrafish.

Development of the vascular system is regulated by multiple signaling pathways mediated by receptor tyrosine kinases. Among them, angiopoietin (Ang)/Tie signaling regulates lymphatic and blood vessel development in mammals. Of the two Tie receptors, Tie2 is well known as a key mediator of Ang/Tie signaling, but, unexpectedly, recent studies have revealed that the Tie2 locus has been lost in many vertebrate species, whereas the Tie1 gene is more commonly present. However, Tie1-driven signaling pathways, including ligands and cellular functions, are not well understood. Here, we performed comprehensive mutant analyses of angiopoietins and Tie receptors in zebrafish and found that only angpt1 and tie1 mutants show defects in trunk lymphatic vessel development. Among zebrafish angiopoietins, only Angpt1 binds to Tie1 as a ligand. We indirectly monitored Ang1/Tie1 signaling and detected Tie1 activation in sprouting endothelial cells, where Tie1 inhibits nuclear import of EGFP-Foxo1a. Angpt1/Tie1 signaling functions in endothelial cell migration and proliferation, and in lymphatic specification during early lymphangiogenesis, at least in part by modulating Vegfc/Vegfr3 signaling. Thus, we show that Angpt1/Tie1 signaling constitutes an essential signaling pathway for lymphatic development in zebrafish.

Macrophage transplantation rescues RNASET2-deficient leukodystrophy by replacing deficient microglia in a zebrafish model.

RNASET2-deficient leukodystrophy is a rare infantile white matter disorder mimicking a viral infection and resulting in severe psychomotor impairments. Despite its severity, there is little understanding of cellular mechanisms of pathogenesis and no treatments. Recent research using the rnaset2 mutant zebrafish model has suggested that microglia may be the drivers of the neuropathology, due to their failure to digest apoptotic debris during neurodevelopment. Therefore, we developed a strategy for microglial replacement through transplantation of adult whole kidney marrow-derived macrophages into embryonic hosts. Using live imaging, we revealed that transplant-derived macrophages can engraft within host brains and express microglia-specific markers, suggesting the adoption of a microglial phenotype. Tissue-clearing strategies revealed the persistence of transplanted cells in host brains beyond embryonic stages. We demonstrated that transplanted cells clear apoptotic cells within the brain, as well as rescue overactivation of the antiviral response otherwise seen in mutant larvae. RNA sequencing at the point of peak transplant-derived cell engraftment confirms that transplantation can reduce the brain-wide immune response and particularly, the antiviral response, in rnaset2-deficient brains. Crucially, this reduction in neuroinflammation resulted in behavioral rescue-restoring rnaset2 mutant motor activity to wild-type (WT) levels in embryonic and juvenile stages. Together, these findings demonstrate the role of microglia as the cellular drivers of neuropathology in rnaset2 mutants and that macrophage transplantation is a viable strategy for microglial replacement in the zebrafish. Therefore, microglia-targeted interventions may have therapeutic benefits in RNASET2-deficient leukodystrophy.

Genetically defined nucleus incertus neurons differ in connectivity and function.

The nucleus incertus (NI), a conserved hindbrain structure implicated in the stress response, arousal, and memory, is a major site for production of the neuropeptide relaxin-3. On the basis of goosecoid homeobox 2 (gsc2) expression, we identified a neuronal cluster that lies adjacent to relaxin 3a (rln3a) neurons in the zebrafish analogue of the NI. To delineate the characteristics of the gsc2 and rln3a NI neurons, we used CRISPR/Cas9 targeted integration to drive gene expression specifically in each neuronal group, and found that they differ in their efferent and afferent connectivity, spontaneous activity, and functional properties. gsc2 and rln3a NI neurons have widely divergent projection patterns and innervate distinct subregions of the midbrain interpeduncular nucleus (IPN). Whereas gsc2 neurons are activated more robustly by electric shock, rln3a neurons exhibit spontaneous fluctuations in calcium signaling and regulate locomotor activity. Our findings define heterogeneous neurons in the NI and provide new tools to probe its diverse functions.

Pharmacological reprogramming of zebrafish lateral line supporting cells to a migratory progenitor state.

In the zebrafish lateral line, non-sensory supporting cells readily re-enter the cell cycle to generate new hair cells and supporting cells during homeostatic maintenance and following damage to hair cells. This contrasts with supporting cells from mammalian vestibular and auditory sensory epithelia which rarely re-enter the cell cycle, and hence loss of hair cells results in permanent sensory deficit. Lateral line supporting cells are derived from multipotent progenitor cells that migrate down the trunk midline as a primordium and are deposited to differentiate into a neuromast. We have found that we can revert zebrafish support cells back to a migratory progenitor state by pharmacologically altering the signaling environment to mimic that of the migratory primordium, with active Wnt signaling and repressed FGF signaling. The reverted supporting cells migrate anteriorly and posteriorly along the horizontal myoseptum and will re-epithelialize to form an increased number of neuromasts along the midline when the pharmacological agents are removed. These data demonstrate that supporting cells can be readily reprogrammed to a migratory multipotent progenitor state that can form new sensory neuromasts, which has important implications for our understanding of how the lateral line system matures and expands in fish and also suggest avenues for returning mammalian supporting cells back to a proliferative state.

Forward genetics combined with unsupervised classifications identified zebrafish mutants affecting biliary system formation.

Impaired formation of the biliary network can lead to congenital cholestatic liver diseases; however, the genes responsible for proper biliary system formation and maintenance have not been fully identified. Combining computational network structure analysis algorithms with a zebrafish forward genetic screen, we identified 24 new zebrafish mutants that display impaired intrahepatic biliary network formation. Complementation tests suggested these 24 mutations affect 24 different genes. We applied unsupervised clustering algorithms to unbiasedly classify the recovered mutants into three classes. Further computational analysis revealed that each of the recovered mutations in these three classes has a unique phenotype on node-subtype composition and distribution within the intrahepatic biliary network. In addition, we found most of the recovered mutations are viable. In those mutant fish, which are already good animal models to study chronic cholestatic liver diseases, the biliary network phenotypes persist into adulthood. Altogether, this study provides unique genetic and computational toolsets that advance our understanding of the molecular pathways leading to biliary system malformation and cholestatic liver diseases.

GABA attenuates neurotoxicity of zinc oxide nanoparticles due to oxidative stress via DAF-16/FoxO and SKN-1/Nrf2 pathways.

Zinc oxide nanoparticles (ZnO-NPs) are one of the most widely used metal oxide nanomaterials. The increased use of ZnO-NPs has exacerbated environmental pollution and raised the risk of neurological disorders in organisms through food chains, and it is urgent to look for detoxification strategies. γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter that has been shown to have anxiolytic, anti-aging and inhibitory effects on nervous system excitability. However, there are few reports on the prevention and control of the toxicity of nano-metal ions by GABA. In zebrafish, ZnO-NPs exposure led to increased mortality and behavioral abnormalities of larva, which could be moderated by GABA intervention. Similar results were investigated in Caenorhabditis elegans, showing lifespan extension, abnormal locomotor frequency and behavior recovery when worms fed with GABA under ZnO-NPs exposure. Moreover, GABA enhanced antioxidant enzyme activities by upregulating the expression of antioxidant-related genes and thus scavenged excessive O2-. In the case of ZnO-NPs exposure, inhibition of nuclear translocation of DAF-16 and SKN-1 was restored by GABA. Meanwhile, the protective effect of GABA was blocked in daf-16 (-) and skn-1 (-) mutant, suggesting that DAF-16/FoxO and SKN-1/Nrf2 pathways is the key targets of GABA. This study provides a new solution for the application of GABA and mitigation of metal nanoparticle neurotoxicity.

Polystyrene nanoplastics exert cardiotoxicity through the Notch and Wnt pathways in zebrafish (Danio rerio).

The ubiquity of micro(nano)plastics has raised significant concerns among people. Their accumulation in the cardiovascular system necessitates attention to their cardiotoxicity. However, research on the cardiotoxicity of micro(nano)plastics remains scarce. Our study exposed zebrafish embryos to four different concentrations (0, 1, 10, 100 µg/mL) of polystyrene nanoplastics (PSNPs) for a period of 7 days. The results indicated that PSNPs noticeably decreased the hatching and survival rates of zebrafish and also induced cardiac developmental abnormalities. The mRNA level analysis revealed significant upregulations of heart development-related genes nkx2.5, cmlc-2, and myh-7 in response to PSNPs. Additionally, PSNPs significantly up-regulated the mRNA level associated with the Notch signaling pathway (notch-1a, jag-1a, and her-7) while remarkably suppressing the expression of the Wnt signaling pathway gene (wnt-3a). Further research showed that PSNPs significantly increased the expression of endoplasmic reticulum stress genes atf-6 and chop, while noticeably inhibiting mitochondrial copy numbers. Moreover, PSNPs were found to decrease calcium ion level and superoxide dismutase (SOD) activity in zebrafish larvae. Additionally, prolonged exposure to PSNPs for 7 days exacerbated abnormalities in various indicators compared to a 4-day exposure. In conclusion, our study demonstrates that PSNPs induce oxidative stress in zebrafish larvae, thereby activating endoplasmic reticulum stress and inhibiting mitochondrial activity, ultimately disrupting the Notch and Wnt signaling pathways. These disruptions result in abnormalities in cardiac developmental genes, ultimately leading to cardiac developmental abnormalities in zebrafish. The present research contributes to a novel understanding of the cardiotoxicity of PSNPs.

Radix Rehmanniae Praeparata promoted zebrafish fin regeneration through aryl hydrocarbon receptor-dependent autophagy.

HEADINGS ETHNOPHARMACOLOGICAL RELEVANCE: Rehmanniae Radix Praeparata (RRP), a staple in traditional Chinese medicine, is derived from Rehmannia glutinosa Libosch and is renowned for its wound-healing properties. Despite its clinical prevalence, the molecular mechanisms underlying RRP's wound-healing effects have not been fully elucidated. AIM OF THE STUDY: This research endeavored to delineate the molecular and cellular mechanisms underlying the beneficial effects of RRP on wound healing, utilizing a zebrafish model. MATERIALS AND METHODS: Zebrafish larvae at 3 days post-fertilization were amputated at the fin and subsequently treated with RRP. The pro-wound healing and regenerative effects of RRP were evaluated through morphological analysis, assessment of cell proliferation and apoptosis, Additionally, mechanistic insights were gained through a comprehensive approach encompassing network pharmacology analysis, cell tracing, RNA-sequencing, CRISPR/Cas9 gene editing, and pharmacological inhibition. RESULTS: Our findings demonstrate that RRP significantly accelerates caudal fin regeneration in zebrafish following injury by suppressing cell apoptosis, promoting cell proliferation, and upregulating the expression of regenerative-related genes. Furthermore, RRP triggers autophagy signals during the regenerative process, which is attenuated by the autophagy inhibitor chloroquine (CQ). Notably, the administration of RRP enhances the expression of ahr1 and ahr2 in the regenerating fin. Genetic knockout of ahr1a, ahr1b, or ahr2 using CRISPR/Cas9, or pharmacological blockade of AHR signals with the antagonist CH-223191, diminishes the regenerative potential of RRP. Remarkably, zebrafish lacking ahr2 completely lose their fin regeneration ability. Additionally, inhibition of AHR signaling suppresses autophagy signaling during fin regeneration. CONCLUSIONS: This study uncovers that RRP stimulates fin regeneration in zebrafish by inducing AHR signals and, at least partially, activating the autophagy process. These findings provide novel insights into the molecular mechanisms underlying the wound-healing effects of RRP and may pave the way for the development of novel therapeutic strategies.

Deciphering the function of the fifth class of Gα proteins: regulation of ionic homeostasis as unifying hypothesis.

Trimeric G proteins transduce signals from a superfamily of receptors and each G protein controls a wide range of cellular and systemic functions. Their highly conserved alpha subunits fall in five classes, four of which have been well investigated (Gs, Gi, G12, Gq). In contrast, the function of the fifth class, Gv is completely unknown, despite its broad occurrence and evolutionary ancient origin (older than metazoans). Here we show a dynamic presence of Gv mRNA in several organs during early development of zebrafish, including the hatching gland, the pronephros and several cartilage anlagen, employing in situ hybridisation. Next, we generated a Gv frameshift mutation in zebrafish and observed distinct phenotypes such as reduced oviposition, premature hatching and craniofacial abnormalities in bone and cartilage of larval zebrafish. These phenotypes could suggest a disturbance in ionic homeostasis as a common denominator. Indeed, we find reduced levels of calcium, magnesium and potassium in the larvae and changes in expression levels of the sodium potassium pump atp1a1a.5 and the sodium/calcium exchanger ncx1b in larvae and in the adult kidney, a major osmoregulatory organ. Additionally, expression of sodium chloride cotransporter slc12a3 and the anion exchanger slc26a4 is altered in complementary ways in adult kidney. It appears that Gv may modulate ionic homeostasis in zebrafish during development and in adults. Our results constitute the first insight into the function of the fifth class of G alpha proteins.

Cachd1 interacts with Wnt receptors and regulates neuronal asymmetry in the zebrafish brain.

Neurons on the left and right sides of the nervous system often show asymmetric properties, but how such differences arise is poorly understood. Genetic screening in zebrafish revealed that loss of function of the transmembrane protein Cachd1 resulted in right-sided habenula neurons adopting left-sided identity. Cachd1 is expressed in neuronal progenitors, functions downstream of asymmetric environmental signals, and influences timing of the normally asymmetric patterns of neurogenesis. Biochemical and structural analyses demonstrated that Cachd1 can bind simultaneously to Lrp6 and Frizzled family Wnt co-receptors. Consistent with this, lrp6 mutant zebrafish lose asymmetry in the habenulae, and epistasis experiments support a role for Cachd1 in modulating Wnt pathway activity in the brain. These studies identify Cachd1 as a conserved Wnt receptor-interacting protein that regulates lateralized neuronal identity in the zebrafish brain.