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Understanding the early stages of human congenital myopathies is critical for proposing strategies for improving musculoskeletal muscle performance, such as restoring the functional integrity of the cytoskeleton. SH3 and cysteine-rich domain 3 (STAC3) are proteins involved in nutrient regulation and are an essential component of the excitation-contraction (EC) coupling machinery for Ca 2+ releasing. A mutation in STAC3 causes debilitating Native American Myopathy (NAM) in humans, while loss of this gene in mice and zebrafish (ZF) results in premature death. Clinically, NAM patients demonstrated increased lipids in skeletal muscle, but it is unclear if neutral lipids are associated with altered muscle function in NAM. Using a CRISPR/Cas9 induced stac3 -/- knockout (KO) zebrafish model, we determined that loss of stac3 leads to delayed larval hatching which corresponds with muscle weakness and decreased whole-body Ca 2+ level during early skeletal development. Specifically, we observed defects in the cytoskeleton in F-actin and slow muscle fibers at 5 and 7 days post-fertilizations (dpf). Myogenesis regulators such as myoD and myf5, mstnb were significantly altered in stac3 -/- larvae. These muscle alterations were associated with elevated neutral lipid levels starting at 5 dpf and persisting beyond 7 dpf. Larva lacking stac3 had reduced viability with no larva knockouts surviving past 11 dpf. This data suggests that our stac3 -/- zebrafish serve as an alternative model to study the diminished muscle function seen in NAM patients. The data gathered from this new model over time supports a mechanistic view of lipotoxicity as a critical part of the pathology of NAM and the associated loss of function in muscle., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: DK has been supported by grant ISF-1121/19 from Israel Science Foundation and DK and HFZ have funded by grant ISF-2058/21 from China-Israel Cooperation. DK, HFZ, CLAB, and DR declare no potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In mammalian hematopoiesis, megakaryocytes mature and become polyploid in the bone marrow before releasing platelets into circulation. In contrast, fish produce thrombocytes in kidney marrow, where young thrombocytes undergo maturation in circulation, akin to platelets. Despite morphological differences, our single-cell sequencing revealed significant gene expression similarities between fish thrombocytes and mammalian megakaryocytes, including Nfe2, which is crucial in platelet production. In addition to nfe2 expression, we found four nfe2-related genes, nfe2I1a, nfe2I1b, nfe2I2a, and nfe2I3, were expressed in mature thrombocytes. However, only nfe2, nfe2l2a, and nfe2l3 are expressed in young thrombocytes. Thus, we hypothesized that Nfe2-related factors may also be involved in thrombocyte production. To address this, we knocked down the four novel nfe2-related genes by the piggyback method and found both young and mature thrombocytes reduced when nfe2, nfe2l1a, and nfe2l3 were knocked down. They also exhibited greater gill bleeding and prolonged time to occlusion (TTO) compared to controls. In summary, our study shows Nfe2I1a and Nfe2l3 as novel transcription factors that are positive regulators influencing adult zebrafish thrombopoiesis., Competing Interests: Declaration of competing interest The authors report no conflict of interest., (Published by Elsevier Inc.)

Though spinal cord injury (SCI) causes irreversible sensory and motor impairments in human, adult zebrafish retain the potent regenerative capacity by injury-induced proliferation of central nervous system (CNS)-resident progenitor cells to develop new functional neurons at the lesion site. The hallmark of SCI in zebrafish lies in a series of changes in the epigenetic landscape, specifically DNA methylation and histone modifications. Decoding the post-SCI epigenetic modifications is therefore critical for the development of therapeutic remedies that boost SCI recovery process. Here, we have studied on Sirtuin1 (Sirt1), a non-classical histone deacetylase that potentially plays a critical role in neural progenitor cells (NPC) proliferation and axonal regrowth following SCI in zebrafish. We investigated the role of Sirt1 in NPC proliferation and axonal regrowth in response to injury in the regenerating spinal cord and found that Sirt1 is involved in the induction of NPC proliferation along with glial bridging during spinal cord regeneration. We also demonstrate that Sirt1 plays a pivotal role in regulating the HIPPO pathway through deacetylation-mediated inactivation of Dnmt1 and subsequent hypomethylation of yap1 promoter, leading to the induction of ctgfa expression, which drives the NPC proliferation and axonal regrowth to complete the regenerative process. In conclusion, our study reveals a novel cross-talk between two important epigenetic effectors, Sirt1 and Dnmt1, in the context of spinal cord regeneration, establishing a previously undisclosed relation between Sirt1 and Yap1 which provides a deeper understanding of the underlying mechanisms governing injury-induced NPC proliferation and axonal regrowth. Therefore, we have identified Sirt1 as a novel, major epigenetic regulator of spinal cord regeneration by modulating the HIPPO pathway in zebrafish., Competing Interests: Declarations. Ethical Approval: The study was approved by the animal ethics committee (No. IAEC-04/BIOCHEM/CU/02–2022/03-SH) of Department of Biochemistry, University of Calcutta (Calcutta, India). Consent for Publication: All authors have read and approve the manuscript and gave consent for publication. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Adaptor proteins play crucial roles in signal transduction across diverse signaling pathways. Src-homology 2 domain-containing E (SH2E) is the adaptor protein highly expressed in vascular endothelial cells and myocardium during zebrafish embryogenesis. In this study we investigated the function and mechanisms of SH2E in cardiogenesis. We first analyzed the spatiotemporal expression of SH2E and then constructed zebrafish lines with SH2E deficiency using the CRISPR-Cas9 system. We showed that homozygous mutants developed progressive pericardial edema (PCE), dilated atrium, abnormal atrioventricular looping and thickened atrioventricular wall from 3 days post fertilization (dpf) until death; inducible overexpression of SH2E was able to partially rescue the PCE phenotype. Using transcriptome sequencing analysis, we demonstrated that the MAPK/ERK and NF-κB signaling pathways might be involved in SH2E-deficiency-caused PCE. This study underscores the pivotal role of SH2E in cardiogenesis, and might help to identify innovative diagnostic techniques and therapeutic strategies for congenital heart disease., Competing Interests: Competing interests: The authors declare no conflict of interest. Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences has applied for a patent, China Patent Application No. 202311014379.7 by inventors Qing Jing, Yang-xi Hu, Yu-lai Liang and Zhi-hua Liu for the usage of L-selenomethionine in antioxidant stress and alleviation of cardiac developmental defects caused by SH2E gene mutations described in this paper under the support of the Program of EnShi TuJia and Miao Autonomous Prefecture Bureau of Scientific and Technological Affairs., (© 2024. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

The role of immune cells is crucial in nerve regeneration following spinal cord injury. Kif15, a member of the kinesin family, has been shown to enhance macrophage phagocytosis. This study investigates the impact of Kif15 deficiency on immune cells in zebrafish with spinal cord injury. Using kif15 morphants in Tg(coro1a:EGFP) zebrafish, we observed increased recruitment of Coro1a + cells to the injury site, followed by a rapid decline in kif15 morphants. Transcriptome analysis revealed that inflammatory and phagocytic signals were significantly enhanced at 1-hour post-injury (hpi), while MAPK pathways indicated growth at 24 hpi. Enhanced phagocytosis was confirmed using neutral red particles, and the Kif15 inhibitor GW406108X further supported increased migration and phagocytosis in macrophages. Activation of Cdc42 and RhoA was significantly increased, contributing to cell motility and phagocytosis. Additionally, the number of apoptotic cells was reduced in kif15 morphants, suggesting that Kif15 depletion could activate immune cells and efficiently remove apoptotic cells. Our study provides in vivo evidence that Kif15 is involved in immune cell migration and phagocytosis and suggests potential therapeutic roles for Kif15 inhibitors in spinal cord injury treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The lateral line system in fish is crucial for detecting water flow, which facilitates various behaviors such as prey detection, predator avoidance, and rheotaxis. The cupula, a gelatinous structure overlaying the hair cells in neuromasts, plays a key role in transmitting mechanical stimuli to hair cells. However, the molecular composition of the cupula matrix remains poorly understood. In this study, we found that Mucin-5AC, a novel family of mucin proteins, composed of 2-27 cysteine-rich domains, presents in cartilaginous and bony fishes. Using in situ hybridization and transgenic reporter assays, we demonstrated that zebrafish muc5AC is specifically expressed in the support cells of neuromasts. Knockdown of muc5AC via antisense morpholino resulted in shorter cupulae in zebrafish lateral line. Additionally, we generated zebrafish muc5AC mutants using CRISPR/Cas9 and found that cupulae in muc5AC mutants were significantly shorter than that in wild-types, but the hair cell number in neuromasts was not changed obviously. Furthermore, muc5AC mutant zebrafish larvae displayed compromised sensitivity to vibration stimuli compared to wild-type larvae. This study provides the first evidence linking the muc5AC gene to cupula development and vibration detection in zebrafish. Our findings suggest that Mucin-5AC is likely a critical component of the cupula matrix, offering an important clue to the molecular composition of the lateral line cupula in fish.

Heterozygous variants in SOX10 cause congenital syndromes affecting pigmentation, digestion, hearing, and neural development, primarily attributable to failed differentiation or loss of non-skeletal neural crest derivatives. We report here an additional, previously undescribed requirement for Sox10 in bone mineralization. Neither crest- nor mesoderm-derived bones initiate mineralization on time in zebrafish sox10 mutants, despite normal osteoblast differentiation and matrix production. Mutants are deficient in the Trpv6+ ionocytes that take up calcium from the environment, resulting in severe calcium deficiency. As these ionocytes derive from ectoderm, not crest, we hypothesized that the primary defect resides in a separate organ that systemically regulates ionocyte numbers. RNA sequencing revealed significantly elevated stanniocalcin (Stc1a), an anti-hypercalcemic hormone, in sox10 mutants. Stc1a inhibits calcium uptake in fish by repressing trpv6 expression and Trpv6+ ionocyte proliferation. Epistasis assays confirm excess Stc1a as the proximate cause of the calcium deficit. The pronephros-derived glands that synthesize Stc1a interact with sox10+ cells, but these cells are missing in mutants. We conclude that sox10+ crest-derived cells non-autonomously limit Stc1a production to allow the inaugural wave of calcium uptake necessary to initiate bone mineralization., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2025. Published by The Company of Biologists.)

We established a zebrafish model of depression-like behaviour induced by exposure to artificial light at night (ALAN) and found that nobiletin (NOB) alleviated depression-like behaviour. Subsequently, based on the results of a 24-h free movement assay, clock gene expression and brain tissue transcriptome sequencing, the glycolysis signalling pathway was identified as a potential target through which NOB exerted antidepressant effects. Using the ALAN zebrafish model, we found that supplementation with exogenous L-lactic acid alleviated depressive-like behaviour. Molecular docking and molecular dynamics simulations revealed an inter-molecular interaction between NOB and the pyruvate kinase isozyme M1/M2 (PKM2) protein. We then used compound 3 k to construct a zebrafish model in which PKM2 was inhibited. Our analysis of this model suggested that NOB alleviated depression-like behaviour via inhibition of PKM2. In summary, NOB alleviated depressive-like behaviour induced by ALAN in zebrafish via targeting of PKM2 and activation of the glycolytic signalling pathway., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. This work has not been submitted/published or being submitted to another journal., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

G protein-coupled receptor 4 (GPR4) belongs to the subfamily of proton-sensing GPCRs (psGPCRs), which detect pH changes in extracellular environment and regulate diverse physiological responses. GPR4 was found to be overactivated in acidic tumor microenvironment as well as inflammation sites, with a triad of acidic residues within the transmembrane domain identified as crucial for proton sensing. However, the 3D structure remains unknown, and the roles of other conserved residues within psGPCRs are not well understood. Here we report cryo-electron microscopy (cryo-EM) structures of active zebrafish GPR4 at both pH 6.5 and 8.5, each highlighting a distribution of histidine and acidic residues at the extracellular region. Cell-based assays show that these ionizable residues moderately influence the proton-sensing capacity of zebrafish GPR4, compared to the more significant effects of the triad residues. Furthermore, we reveal a cluster of aromatic residues within the orthosteric pocket that may propagate the signaling to the intercellular region via repacking the aromatic patch at the central region. This study provides a framework for future signaling and functional investigation of psGPCRs., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Non-muscle myosin (NMII) motor proteins have diverse developmental functions due to their roles in cell shape changes, cell migration, and cell adhesion. Zebrafish are an ideal vertebrate model system to study the NMII encoding myh genes and proteins due to high sequence homology, established gene editing tools, and rapid ex utero development. In humans, mutations in the NMII encoding MYH genes can lead to abnormal developmental processes and disease. This study utilized zebrafish myh9a, myh9b, and myh10 null mutants to examine potential genetic interactions and roles for each gene in development. It was determined that the myh9b gene is the most critical NMII encoding gene, as myh9b mutants develop pericardial edema and have a partially penetrant lethal phenotype, which was not observed in the other myh mutants. This study also established that genetic interactions occur between the zebrafish myh9a, myh9b, and myh10 genes where myh9b is required for the expression of both myh9a and myh10, and myh10 is required for the expression of myh9b. Additionally, protein analyses suggested that enhanced NMII protein stability in some mutant backgrounds may play a role in compensation. Finally, double mutant studies revealed different and more severe phenotypes at earlier time points than single mutants, suggesting roles for tissue specific genetic redundancy, and in some genotypes, haploinsufficiency. These mutants are the first in vivo models allowing for the study of complete loss of the NMIIA and NMIIB proteins, establishing them as valuable tools to elucidate the role of NMII encoding myh genes in development and disease., Competing Interests: Conflicts of interest: The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechano-electrical transduction (MET) apparatus. These interactions have been proposed to be functionally relevant across mechanosensory organs and vertebrate species. Here, we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia., Competing Interests: AG, WW, KK, HC, JM, ER, AB, RA, JL, SW, MF, OZ, JH, SR, KC, MS, ZA No competing interests declared

Background/Objectives: Abnormal development of the second heart field significantly contributes to congenital heart defects, often caused by disruptions in tightly regulated molecular pathways. Smyd1 , a gene encoding a protein with SET and MYND domains, is essential for heart and skeletal muscle development. Mutations in SMYD1 result in severe cardiac malformations and misregulation of Hand2 expression in mammals. This study examines the role of Smyd1b in zebrafish cardiac morphogenesis to elucidate its function and the mechanisms underlying congenital heart defects. Methods: Smyd1b ( still heart ) mutant embryos were analyzed for cardiac defects, and changes in gene expression related to heart development using live imaging, in situ hybridization, quantitative PCR and immunofluorescent comparisons and analysis. Results: Smyd1b mutants displayed severe cardiac defects, including failure to loop, severe edema, and an expansion of cardiac jelly linked to increased has2 expression. Additionally, the expression of key cardiac transcription factors, such as gata4 , gata5 , and nkx2.5 , was notably reduced, indicating disrupted transcriptional regulation. The migration of cardiac progenitors was impaired and the absence of Islet-1-positive cells in the mutant hearts suggests a failed contribution of SHF progenitor cells. Conclusions: These findings underscore the essential role of Smyd1b in regulating cardiac morphogenesis and the development of the second heart field. This study highlights the potential of Smyd1b as a key factor in understanding the genetic and molecular mechanisms underlying congenital heart defects and cardiac development.

The TIRAP protein is an adaptor protein in TLR signaling which links TLR2 and TLR4 to the adaptor protein Myd88. The transcriptomic profiles of zebrafish larvae from a tirap , myd88 and tlr2 mutant and the corresponding wild type controls under unchallenged developmental conditions revealed a specific involvement of tirap in calcium homeostasis and myosin regulation. Metabolomic profiling showed that the tirap mutation results in lower glucose levels, whereas a tlr2 mutation leads to higher glucose levels. A tail-wounding zebrafish larval model was used to identify the role of tirap in leukocyte migration to tissue wounding. We found that more neutrophils were recruited to the wounded region in the tirap mutant larvae compared to the wild type controls, whereas there was no difference in macrophage recruitment. In contrast, published data show that tlr2 and myd88 mutants recruit fewer neutrophils and macrophages to the wounds. Based on cell tracking analysis, we demonstrate that the neutrophil migration speed is increased in the tirap mutant in contrast to neutrophil behavior in myd88 and tlr2 mutants. In conclusion, we show that tirap plays specialized roles distinct from tlr2 and myd88 in signaling, metabolic control, and in regulating neutrophil migration speed upon wounding., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Pattern recognition receptors (PRRs) such as C-type lectin receptors (CLRs) and Toll-like receptors (TLRs) are used by hosts to recognize pathogen-associated molecular patterns (PAMPs) in microorganisms and to initiate innate immune responses. While PRRs exist across invertebrate and vertebrate species, the functional homology of many of these receptors is still unclear. In this study, we investigate the innate immune response of zebrafish larvae to zymosan, a β-glucan-containing particle derived from fungal cell walls. Macrophages and neutrophils robustly respond to zymosan and are required for zymosan-induced activation of the NF-κB transcription factor. Full activation of NF-κB in response to zymosan depends on Card9/Syk and Myd88, conserved CLR and TLR adaptor proteins, respectively. Two putative CLRs, Clec4c and Sclra, are both required for maximal sensing of zymosan and NF-κB activation but not required for inflammatory gene expression. Altogether, we identify conserved PRRs and PRR signaling pathways in larval zebrafish that promote recognition of fungal PAMPs. These results inform modeling of human fungal infections in zebrafish and increase our knowledge of the evolution and conservation of PRR pathways in vertebrates., Competing Interests: Declaration of competing interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The liver is a key endoderm-derived multifunctional organ within the digestive system. Prospero homeobox 1 (Prox1) is an essential transcription factor for liver development, but its specific function is not well understood. Here, we show that hepatic development, including the formation of intrahepatic biliary and vascular networks, is severely disrupted in prox1a mutant zebrafish. We find that Prox1a is essential for liver growth and proper differentiation but not required for early hepatic cell fate specification. Intriguingly, prox1a depletion leads to ectopic initiation of a Cdx1b-mediated intestinal program and the formation of intestinal lumen-like structures within the liver. Morpholino knockdown of cdx1b alleviates liver defects in the prox1a mutant zebrafish. Finally, chromatin immunoprecipitation analysis reveals that Prox1a binds directly to the promoter region of cdx1b, thereby repressing its expression. Overall, our findings indicate that Prox1a is required to promote and protect hepatic development by repression of Cdx1b-mediated intestinal cell fate in zebrafish., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2024 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

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

One of the most highly induced genes in zebrafish (Danio rerio) larvae after infection with the nodavirus red-spotted grouper nervous necrosis virus (RGNNV) was a member of the immunoglobulin superfamily (IgSF), which has remained uncharacterized and erroneously annotated in zebrafish and other fish species as galectin 17 (lgals17). We characterized this gene and named it immunoglobulin (Ig)-like domain-containing protein (igldcp), a new member of the IgSF that does not possess orthologs in mammals. Igldcp expression is induced by viral infection and it belongs to the group of interferon-stimulated genes (ISGs). In vitro overexpression of igldcp decreased RGNNV replication, whereas in vivo knockdown of this gene had the opposite effect, resulting in increased larval mortality. RNA-Seq analyses of larvae overexpressing igldcp in the absence or presence of infection with RGNNV showed that the main processes affected by Igldcp could be directly involved in the regulation of various cellular processes associated with the modulation of the immune system., Competing Interests: Declaration of competing interests The authors declare that they have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Stargardt disease (STGD), the leading cause of inherited childhood blindness, is primarily caused by mutations in the ABCA4 gene; yet, the underlying mechanisms of photoreceptor degeneration remain elusive, partly due to limitations in existing animal disease models. To expand our understanding, we mutated the human ABCA4 paralogues abca4a and abca4b in zebrafish, which has a cone-rich retina. Our study unveiled striking dysmorphology and elongation of cone outer segments (COS) in abca4a;abca4b double mutants, alongside reduced phagocytosis by the retinal pigmented epithelium (RPE). We report that zebrafish Abca4 protein forms a distinctive stripe along the length of COS, suggesting a potential structural role. We further show that, in wild-type zebrafish, outer segments of cone cells constitutively present externalized phosphatidylserine, an apoptotic 'eat-me' signal, and that this pattern is disrupted in abca4a;abca4b double mutants, potentially contributing to reduced RPE phagocytic activity. More broadly, constitutive presentation of the 'eat-me' signal by COS - if conserved in humans - might have important implications for other retinal degenerative diseases, including age-related macular degeneration. Our zebrafish model provides novel insights into cone dysfunction and presents a promising platform for unraveling the mechanisms of STGD pathogenesis and advancing therapeutic interventions., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2025. Published by The Company of Biologists.)

The different spatiotemporal distribution of progenitor and neurogenic capacities permits that brain regions engage asynchronously in neurogenesis. In the hindbrain, rhombomere progenitor cells contribute to neurons during the first neurogenic phase, whereas boundary cells participate later. To analyze what maintains boundary cells as non-neurogenic progenitors, we addressed the role of Her9, a zebrafish Hes1-related protein. her9 expression is temporarily sustained in boundary cells independently of Notch at early embryonic stages, while they are non-neurogenic progenitors. Complementary functional approaches show that Her9 inhibits the onset of Notch signaling and the neurogenic program, keeping boundary cells as progenitors. Multicolor clonal analysis combined with genetic perturbations reveal that Her9 expands boundary progenitors by promoting symmetric proliferative and preventing neurogenic cell divisions. Her9 also regulates the proliferation of boundary cells by inhibiting the cell cycle arrest gene cdkn1ca and interplaying with Cyclin D1. Moreover, her9 is enriched in hindbrain radial glial cells at late embryonic stages independently of Notch. Together these data demonstrate that Her9 maintains the stemness properties of hindbrain boundary progenitors and late radial glial cells, ensuring the different temporal distribution of neurogenic capacities within the hindbrain., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2025. Published by The Company of Biologists.)

PLK4 plays a crucial role in centriole duplication, which is essential for maintaining cellular processes such as cell division, cytoskeletal stability, and cilia formation. However, the mechanisms of PLK4 remain incompletely understood, especially in the embryonic development of vertebrate species. In this study, we observed that Plk4 dysfunction led to abnormal embryonic development in zebrafish, characterized by symptoms such as dark and wrinkled skin, microphthalmia, and body axis curvature. In plk4 mutants, defects in centriole duplication led to abnormal cell division, apoptosis, and ciliogenesis defects. Moreover, overexpression of plk4 in zebrafish embryos caused excessive centrosome amplification, disrupting embryonic gastrulation through abnormal cell division and ultimately resulting in embryonic lethality. Furthermore, we identified the "cryptic" polo box (CPB) domain, consisting of two PBs (PB1 and PB2), as the critical centrosome localization domain of Plk4. Surprisingly, overexpression of these two PB domains alone was sufficient to induce embryonic lethality. Additionally, we discovered a truncated form of CPB that localizes to the centrosome without causing defects in embryonic development. Our results demonstrate that Plk4 tightly controls centriole duplication, which is essential for early embryonic development in zebrafish., (Copyright © 2024 Elsevier Inc. All rights reserved.)

As a signal molecule, nitric oxide (NO) has several physiological actions in fish. However, the action of NO on the brain/gut axis, a classic inter-organal axis that bridges the gastrointestinal tract and the CNS, still requires more understanding. The short-term in vivo action of a NO inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME), on mitochondrial energetic markers and the receptor expression of thyroid hormone (TH) and neuroendocrine hormones involved in stress/ease response was tested in the brain/gut axis of zebrafish exposed to either in non-stressed or air-exposed condition. L-NAME treatment decreased the NO content in brain and gut segments in non-stressed fish but rose upon L-NAME treatment in air-exposed fish that corresponded with the activation of inos, nnos, hif1a and hif1an transcript expressions. The brain/gut segments that showed spatial and differential sensitivity to L-NAME, modified the transcript expression patterns of stress (adra2da, adrb1, nr3c2)- and ease-responsive (htr2b, slc6a4a, mtnr1aa) hormone receptors. The expression pattern of the TH receptor/regulator system (thra, thrb, dio1, dio2, dio3) becomes more active in gut segments than brain segments upon L-NAME challenge in stressed zebrafish. The data provide evidence for a novel role of NO as an integrator of brain/gut axis segments in zebrafish, where the endogenously produced NO in mid-brain/posterior-gut axis aligns together upon air-exposure stress, providing a lead role to the posterior gut that activates and directs the neuroendocrine receptor expressions of stress/ease responsive genes. The data further invites studies exploring the therapeutic potential of L-NAME in this biomedical model to control the brain/gut axis segments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

During inner ear semicircular canal morphogenesis in zebrafish, patterned canal-genesis zones express genes for extracellular matrix component synthesis. These include hyaluronan and the hyaluronan-binding chondroitin sulfate proteoglycan Versican, which are abundant in the matrices of many developing organs. Charged hyaluronate polymers play a key role in canal morphogenesis through osmotic swelling. However, the developmental factor(s) that pattern the synthesis of the matrix components and regulation of hyaluronate density and swelling are unknown. Here, we identify the transcription factor Lmx1b as a positive transcriptional regulator of hyaluronan, Versican, and chondroitin synthesis genes crucial for canal morphogenesis. We show that Versican regulates hyaluronan density through its protein core, whereas the charged chondroitin side chains contribute to the hydration of hyaluronate-containing extracellular matrices. Versican-tuned properties of hyaluronate matrices may be a broadly used mechanism in morphogenesis with important implications for understanding diseases in which these matrices are impaired, and for hydrogel engineering for tissue regeneration., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2025. Published by The Company of Biologists.)

Stat3 is a transcription factor with a key role in cell proliferation and migration. Using the stat3 -/- zebrafish line we showed that the stat3 genetic ablation results in a marked decrease of tail fin regrowth, demonstrating that this transcription factor is fundamental in the regeneration process. Stat3 activity is finely modulated by post-translational modifications that occur in several residues of the protein (i.e., Y705 and S727 phosphorylation), with tissue-specific effects. Using the newly generated stat3 S→A751 zebrafish line, we demonstrated that the Stat3 phosphorylation in the non-canonical S751 site (homologous of mammalian serine 727) is required for the regeneration of tail fin in both larval and adult stage, even if this phosphorylation has largely been reported to have marginal roles in Stat3 activity. Our analysis showed that both stat3 -/- and stat3 S→A751 mutant zebrafish lines have alterations in the expression of genes involved in epithelial and bone tissue regeneration, including genes coding for the vitamin D signaling pathway. Interestingly, the reduced regeneration activity in zebrafish stat3 -/- and stat3 A751/A751 larvae is partially rescued by vitamin D treatment. Together, these results reveal a Stat3-vitamin D co-regulatory mechanism during zebrafish tail fin regeneration., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The low-density lipoprotein receptor (LDLr) is the first member of a closely related transmembrane protein family. It is known for its involvement in various physiological processes, mainly in the regulation of lipid metabolism, especially in the brains of mammals and zebrafish. In zebrafish, two ldlr genes (ldlra and b) have been identified and their distribution in the brain is not well documented. Recently, the roles of ldlr and its inhibitor pcsk9 in regenerative process after telencephalic brain injury have been discussed. In this study, we explored the expression patterns of these genes during zebrafish development. We found that ldlra expression was detected at the end of the pharyngula period (48 hpf) and increased during the larval stage. Conversely, ldlrb expression was observed from zygotic to larval stages. Using techniques like in situ hybridization and taking advantage of transgenic fish, we demonstrated the widespread distribution of ldlra, ldlrb and pcsk9 in the brain of adult zebrafish. Specifically, these genes were expressed in neurons and neural stem cells and also at lower levels in endothelial cells. As expected, intraperitoneal injection of fluorescent-labelled LDLs resulted in their uptake by cerebral endothelial cells in a homeostatic context, whereas they diffused within the brain parenchyma after telencephalic injury. However, after intracerebroventricular injections into animals, LDL particles were not taken up by neural stem cells. In conclusion, our results provide additional evidence for LDLr expression in the brain of adult zebrafish. These results raise the question of the role of LDLr in the cholesterol/lipid imbalance in cerebral complications., (© 2024 The Author(s). European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

The carbohydrate sulfotransferase 6 (chst6) gene is linked to macular corneal dystrophy (MCD), a rare disease that leads to bilateral blindness due to the accumulation of opaque aggregates in the corneal stroma. chst6 encodes for a keratan sulfate proteoglycan (KSPG) specific sulfotransferase. MCD patients lose sulfated KSPGs (cKS) in the cornea and the serum. The significance of serum cKS loss has not been understood. Zebrafish cornea structure is similar to that of humans and it contains high levels of sulfated cKS in the stroma. Here, zebrafish chst6 is shown to be expressed in the cornea and head structures of the embryos. An animal model of MCD is developed by generating chst6 mutant animals with CRISPR/Cas9-mediated gene editing. The dramatic decrease in cKS epitopes in the mutants was shown with ELISA and immunofluorescence. Morphological defects or alterations of jaw cartilage were detected in a minor fraction of the mutant larvae. Loss of cKS epitopes and morphological defects was fully rescued with wild-type chst6. Mutant adult zebrafish displayed all clinical manifestations of MCD, while a fraction also displayed jaw and skeleton defects. Opaque accumulations formed in the eye, which were alcian blue positive. Loss of cKS in the corneal stroma and a decrease in corneal thickness were shown. Interestingly, alteration of transforming growth factor beta-induced (BIGH3) expression which was not described in patients was also observed. This is the first report of an MCD model in a genetically tractable organism, providing a preclinical model and insight into the importance of KSPG sulfation for proper skeletal morphogenesis., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Fetal Alcohol Spectrum Disorders (FASD) describes a wide array of neurological defects and craniofacial malformations, associated with ethanol teratogenicity. While there is growing evidence for a genetic component to FASD, little is known of the genes underlying these ethanol-induced defects. Along with timing and dosage, genetic predispositions may help explain the variability within FASD. From a screen for gene-ethanol interactions, we found that mutants for Bmp signaling components are ethanol-sensitive leading to defects in the zebrafish palate. Loss of Bmp signaling results in reductions in gata3 expression in the maxillary domain of the neural crest in the 1st pharyngeal arch, leading to palate defects while upregulation of human GATA3 rescues these defects. Here, we show that ethanol-treated Bmp mutants exhibit misshaped and/or broken trabeculae. Surprisingly, up regulation of GATA3 does not rescue ethanol-induced palate defects and gata3 expression was not altered in ethanol-treated Bmp mutants or dorsomorphin-treated larvae. Timing of ethanol sensitivity shows that Bmp mutants are ethanol sensitive from 10 to 18 hours post-fertilization (hpf), prior to Bmp's regulation of gata3 in palate formation. This is consistent with our previous work with dorsomorphin-dependent knock down of Bmp signaling from 10 to 18 hpf disrupting endoderm formation and subsequent jaw development. Overall, this suggests that ethanol disrupts Bmp-dependent palate development independent of and earlier than the role of gata3 in palate formation by disrupting epithelial development. Ultimately, these data demonstrate that zebrafish is a useful model to identify and characterize gene-ethanol interactions and this work will directly inform our understanding of FASD., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. C. Ben Lovely reports financial support was provided by National Institute on Alcohol Abuse and Alcoholism. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Paired locomotion appendages are hypothesized to have redeployed the developmental program of median appendages, such as the dorsal and anal fins. Compared with paired fins, and limbs, median appendages remain surprisingly understudied. Here, we report that a dominant zebrafish mutant, smoothback (smb), fails to develop a dorsal fin. Moreover, the anal fin is reduced along the antero-posterior axis, and spine defects develop. Mechanistically, the smb mutation is caused by an insertion of a sox10:Gal4VP16 transgenic construct into a non-coding region. The first step in fin, and limb, induction is aggregation of undifferentiated mesenchyme at the appendage development site. In smb, this dorsal fin mesenchyme is absent. Lineage tracing demonstrates the previously unknown developmental origin of the mesenchyme, the sclerotome, which also gives rise to the spine. Strikingly, we find that there is significantly less sclerotome in smb than in wild type. Our results give insight into the origin and modularity of understudied median fins, which have changed position, number, size, and even disappeared, across evolutionary time., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

In the vertebrate nervous system, neurogenesis generally precedes gliogenesis. The mechanisms driving the switch in cell type production and generation of the correct proportion of cell types remain unclear. Here, we show that Fgf20 signalling patterns progenitors to induce the switch from neurogenesis to oligodendrogenesis in the zebrafish hindbrain. Fgf20 emanating from earlier-born neurons signals at a short range to downregulate proneural gene expression in the segment centre with high spatial precision along both anterior-posterior and dorsal-ventral axes. This signal induces oligodendrocytes in the segment centre by upregulating olig2 and sox10 expression in pre-patterned competent progenitors. We show that the magnitude of proneural gene downregulation and the quantity of oligodendrocyte precursor cells specified is dependent on the extent of Fgf20 signalling. Overexpression of fgf20a induces precocious specification and differentiation of oligodendrocytes among olig2+ progenitors, resulting in an increase in oligodendrocytes at the expense of neurogenesis. Thus, Fgf20 signalling defines the proportion of each cell type produced. Taken together, Fgf20 signalling from earlier-born neurons patterns hindbrain segments spatially and temporally to induce the neurogenesis-to-oligodendrogenesis switch., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Fertilization, the basis for sexual reproduction, culminates in the binding and fusion of sperm and egg. Although several proteins are known to be crucial for this process in vertebrates, the molecular mechanisms remain poorly understood. Using an AlphaFold-Multimer screen, we identified the protein Tmem81 as part of a conserved trimeric sperm complex with the essential fertilization factors Izumo1 and Spaca6. We demonstrate that Tmem81 is essential for male fertility in zebrafish and mice. In line with trimer formation, we show that Izumo1, Spaca6, and Tmem81 interact in zebrafish sperm and that the human orthologs interact in vitro. Notably, complex formation creates the binding site for the egg fertilization factor Bouncer in zebrafish. Together, our work presents a comprehensive model for fertilization across vertebrates, where a conserved sperm complex binds to divergent egg proteins-Bouncer in fish and JUNO in mammals-to mediate sperm-egg interaction., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mutations in human VPS4A are associated with neurodevelopmental defects, including motor delays and defective muscle tone. VPS4A encodes a AAA-ATPase required for membrane scission, but how mutations in VPS4A lead to impaired control of motor function is not known. Here we identified a mutation in zebrafish vps4a , T248I, that affects sensorimotor transformation. Biochemical analyses indicate that the T248I mutation reduces the ATPase activity of Vps4a and disassembly of ESCRT filaments, which mediate membrane scission. Consistent with the role for Vps4a in exosome biogenesis, vps4a T248I larvae have enlarged endosomal compartments in the CNS and decreased numbers of circulating exosomes in brain ventricles. Resembling the central form of hypotonia in VPS4A patients, motor neurons and muscle cells are functional in mutant zebrafish. Both somatosensory and vestibular inputs robustly evoke tail and eye movements, respectively. In contrast, optomotor responses, vestibulospinal, and acoustic startle reflexes are absent or strongly impaired in vps4a T248I larvae, indicating a greater sensitivity of these circuits to the T248I mutation. ERG recordings revealed intensity-dependent deficits in the retina, and in vivo calcium imaging of the auditory pathway identified a moderate reduction in afferent neuron activity, partially accounting for the severe motor impairments in mutant larvae. Further investigation of central pathways in vps4a T248I mutants showed that activation of descending vestibulospinal and midbrain motor command neurons by sensory cues is strongly reduced. Our results suggest that defects in sensorimotor transformation underlie the profound yet selective effects on motor reflexes resulting from the loss of membrane scission mediated by Vps4a., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Hemodynamic cues are thought to control blood vessel hierarchy through a shear stress set point, where flow increases lead to blood vessel diameter expansion, while decreases in blood flow cause blood vessel narrowing. Aberrations in blood vessel diameter control can cause congenital arteriovenous malformations (AVMs). We show in zebrafish embryos that while arteries behave according to the shear stress set point model, veins do not. This behavior is dependent on distinct arterial and venous endothelial cell (EC) shapes and sizes. We show that arterial ECs enlarge more strongly when experiencing higher flow, as compared to vein cells. Through the generation of chimeric embryos, we discover that this behavior of vein cells depends on the bone morphogenetic protein (BMP) pathway components Endoglin and Alk1. Endoglin (eng) or alk1 (acvrl1) mutant vein cells enlarge when in normal hemodynamic environments, while we do not observe a phenotype in either acvrl1 or eng mutant ECs in arteries. We further show that an increase in vein diameters initiates AVMs in eng mutants, secondarily leading to higher flow to arteries. These enlarge in response to higher flow through increasing arterial EC sizes, fueling the AVM. This study thus reveals a mechanism through which BMP signaling limits vein EC size increases in response to flow and provides a framework for our understanding of how a small number of mutant vein cells via flow-mediated secondary effects on wildtype arterial ECs can precipitate larger AVMs in disease conditions, such as hereditary hemorrhagic telangiectasia (HHT)., (© 2024. The Author(s).)

Tendons, which transmit force from muscles to bones, are highly prone to injury. Understanding the mechanisms driving tendon fate would impact efforts to improve tendon healing, yet this knowledge is limited. To find direct regulators of tendon progenitor emergence, we performed a zebrafish high-throughput chemical screen. We established forskolin as a tenogenic inducer across vertebrates, functioning through Creb1a, which is required and sufficient for tendon fate. Putative enhancers containing cyclic AMP (cAMP) response elements (CREs) in humans, mice, and fish drove specific expression in zebrafish cranial and fin tendons. Analysis of these genomic regions identified motifs for early B cell factor (Ebf/EBF) transcription factors. Mutation of CRE or Ebf/EBF motifs significantly disrupted enhancer activity and specificity in tendons. Zebrafish ebf1a/ebf3a mutants displayed defects in tendon formation. Notably, Creb1a/CREB1 and Ebf1a/Ebf3a/EBF1 overexpression facilitated tenogenic induction in zebrafish and human pluripotent stem cells. Together, our work identifies the functional conservation of two transcription factors in promoting tendon fate., Competing Interests: Declaration of interests L.I.Z. is a founder and stockholder of Fate Therapeutics, CAMP4 Therapeutics, Amagma Therapeutics, and Scholar Rock and is a consultant for Celularity., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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

The contamination of food and feed by mycotoxins, particularly zearalenone (ZEA) and deoxynivalenol (DON), is a global issue. Prenatal exposure to ZEA and DON can result in congenital cardiac malformations in fetuses. Addressing the prevention and mitigation of embryonic cardiotoxicity caused by these toxins is crucial. Citrus limonoid nomilin (NOM) is an extract known for its pathological properties in various diseases. This study investigated the potential mechanism of NOM in mitigating cardiotoxicity caused by ZEA and DON co-exposure in a zebrafish model. The findings indicated that NOM pretreatment alleviated cardiac developmental toxicity induced by ZEA and DON and normalized the expression of key genes involved in heart development, including gata4, vmhc, nkx2.5, and sox9b. Co-exposure to NOM, ZEA, and DON enhanced SOD and catalase activity, increased glutathione levels, and reduced ROS and malondialdehyde production. Furthermore, NOM reduced cardiac oxidative damage by activating the Keap1/Nrf2 signaling pathway. In summary, this study offers new insights for preventive interventions against congenital heart disease caused by mycotoxin exposure., Competing Interests: Declarations Competing Interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Telethonin/titin-cap (TCAP) encodes a Z-disc protein that plays important roles in sarcomere/T-tubule interactions, stretch-sensing and signaling. Mutations in TCAP are associated with muscular dystrophy and cardiomyopathy; however, the complete etiology and its roles in myocardial infarction and regeneration are not fully understood. Here, we generated tcap gene knockout zebrafish with CRISPR/Cas9 technology and observed muscular dystrophy-like phenotypes and abnormal mitochondria in skeletal muscles. The stretch-sensing ability was inhibited in tcap - / - mutants. Moreover, Tcap deficiency led to alterations in cardiac morphology and function as well as increases in reactive oxygen species (ROS) and mitophagy. In addition, the cardiac regeneration and cardiomyocyte proliferation ability of tcap - / - mutants were impaired, but these impairments could be rescued by supplementation with ROS scavengers or autophagy inhibitors. Overall, our study demonstrates the essential roles of Tcap in striated muscle function and heart regeneration. Additionally, elevations in ROS and autophagy may account for the phenotypes resulting from Tcap deficiency and could serve as novel therapeutic targets for muscular dystrophy and cardiomyopathy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mutations in calcium-dependent papain-like protease CALPAIN3 (CAPN3) cause Limb-Girdle Muscular Dystrophy Recessive Type 1 (LGMDR1), the most common limb-girdle muscular dystrophy in humans. In addition to progressive muscle weakness, persistent inflammatory infiltration is also a feature of LGMDR1. Despite the underlying mechanism remaining poorly understood, we consider that it may relate to the newly defined role of CAPN3/Capn3b in the nucleolus. Here, we report that the loss of function of zebrafish capn3b, the counterpart of human CAPN3, induces an autoimmune response akin to that in LGMDR1 patients. capn3b mutant larvae are more susceptible to Listeria monocytogenes injection, characterized by recruiting more macrophages. Under germ-free conditions, transcriptome analysis of the capn3b mutant muscle reveals a significant upregulation of the chemokine-production-related genes. Coincidently, more neutrophils are recruited to the injury site imposed by either muscle stabbing or tail fin amputation. Nucleolar proteomic analysis and enzymatic assays reveal NKAP, an activating factor of the NF-κB pathway, to be a target of CAPN3. We conclude that the accumulation of Nkap and other factors in the capn3b mutant may be involved in the over-activation of innate immunity. Our studies indicate that the zebrafish capn3b mutant is a powerful model for studying the immunity-related progression of human LGMDR1., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2024 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Androgens are classically thought to act through intracellular androgen receptors (AR/NR3C4), but they can also trigger non-genomic effects via membrane proteins. Although several membrane androgen receptors have been characterized in vitro, their functions in vivo remain unclear. Using a chemical-genetic screen in zebrafish, we found that GPRC6A, a G-protein-coupled receptor, mediates non-genomic androgen actions during embryonic development. Exposure to androgens (androstanedione, DHT and testosterone) caused cardiac edema or tail curvature in wild-type embryos, as well as in ar mutants, suggesting AR-independent pathways. We then mutated putative membrane androgen receptors [gprc6a, hcar1-4 and zip9 (slc39a9)] and found that only gprc6a mutants exhibited a significant reduction in cardiac edema after testosterone exposure. Additionally, co-treatment of wild-type embryos with testosterone and GPRC6A antagonists significantly suppressed the cardiac edema phenotype. Using RNA-seq and RNA rescue approaches, we found that testosterone and GPRC6A cause cardiac phenotypes by reducing Pak1 signaling. Our results indicate that testosterone induces cardiac edema in zebrafish embryos through GPRC6A, independent of nuclear androgen receptors, highlighting a previously unappreciated non-genomic androgen signaling pathway in embryonic development., Competing Interests: Competing interests No authors have competing interests to declare, except that D.A.G. is Editor-in-Chief of Biology Open, which is also published by The Company of Biologists. Development is editorially independent of Biology Open., (© 2024. Published by The Company of Biologists Ltd.)

The Alzheimer's disease and Parkinson's disease risk locus FYN kinase is implicated in neurodegeneration and inflammatory signaling. To investigate in vivo mechanisms of Fyn-driven neurodegeneration, we built a zebrafish neural-specific Gal4:UAS model of constitutively active FynY531F signaling. Using in vivo live imaging, we demonstrated that neural FynY531F expression leads to dopaminergic neuron loss and mitochondrial aggregation in 5 day larval brain. Dopaminergic loss coincided with microglia activation and induction of tnfa, il1b and il12a inflammatory cytokine expression. Transcriptome analysis revealed Stat3 signaling as a potential Fyn target. Chemical inhibition experiments confirmed Fyn-driven dopaminergic neuron loss, and the inflammatory response was dependent upon activation of Stat3 and NF-κB pathways. Dual chemical inhibition demonstrated that Stat3 acts synergistically with NF-κB in dopaminergic neuron degeneration. These results identify Stat3 as a novel downstream effector of Fyn signaling in neurodegeneration and inflammation., Competing Interests: Competing interests M.M. has competing interests with Recombinetics Inc., Immusoft Inc., LifEngine and LifEngine Animal Health., (© 2024. Published by The Company of Biologists Ltd.)

In zebrafish, maternally deposited yolk is the source of nutrients for embryogenesis prior to digestive system maturation. Yolk nutrients are processed and secreted to the growing organism by an extra-embryonic tissue, the yolk syncytial layer (YSL). The export of lipids from the YSL occurs through the production of triacylglycerol-rich lipoproteins. Here we report that mutations in the triacylglycerol synthesis enzyme, diacylglycerol acyltransferase-2 (Dgat2), cause yolk sac opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. Although triacylglycerol synthesis continues, it is not properly coupled to lipoprotein production as dgat2 mutants produce fewer, smaller, ApoB-containing lipoproteins. Unlike DGAT2-null mice, which are lipopenic and die soon after birth, zebrafish dgat2 mutants are viable, fertile, and exhibit normal mass and adiposity. Residual Dgat activity cannot be explained by the activity of other known Dgat isoenzymes, as dgat1a;dgat1b;dgat2 triple mutants continue to produce YSL lipid droplets and remain viable as adults. Further, the newly identified diacylglycerol acyltransferase, Tmem68, is also not responsible for the residual triacylglycerol synthesis activity. Unlike overexpression of Dgat1a and Dgat1b, monoacylglycerol acyltransferase-3 (Mogat3b) overexpression does not rescue yolk opacity, suggesting it does not possess Dgat activity in the YSL. However, mogat3b;dgat2 double mutants exhibit increased yolk opacity and often have structural alterations of the yolk extension. Quadruple mogat3b;dgat1a;dgat1b;dgat2 mutants either have severely reduced viability and stunted growth or do not survive past 3 days post fertilization, depending on the dgat2 mutant allele present. Our study highlights the remarkable ability of vertebrates to synthesize triacylglycerol through multiple biosynthetic pathways., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

VEGFA administration has been explored as a pro-angiogenic therapy for cardiovascular diseases including heart failure for several years, but with little success. Here, we investigate a different approach to augment VEGFA bioavailability: by deleting the VEGFA decoy receptor VEGFR1 (also known as FLT1), one can achieve more physiological VEGFA concentrations. We find that after cryoinjury, zebrafish flt1 mutant hearts display enhanced coronary revascularization and endocardial expansion, increased cardiomyocyte dedifferentiation and proliferation, and decreased scarring. Suppressing Vegfa signaling in flt1 mutants abrogates these beneficial effects of flt1 deletion. Transcriptomic analyses of cryoinjured flt1 mutant hearts reveal enhanced endothelial MAPK/ERK signaling and downregulation of the transcription factor gene egr3. Using newly generated genetic tools, we observe egr3 upregulation in the regenerating endocardium, and find that Egr3 promotes myofibroblast differentiation. These data indicate that with enhanced Vegfa bioavailability, the endocardium limits myofibroblast differentiation via egr3 downregulation, thereby providing a more permissive microenvironment for cardiomyocyte replenishment after injury., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: Endothelial cell (EC)-pericyte interactions are known to remodel in response to hemodynamic forces; yet there is a lack of mechanistic understanding of the signaling pathways that underlie these events. Here, we have identified a novel signaling network regulated by blood flow in ECs-the chemokine receptor CXCR3 (CXC motif chemokine receptor 3) and one of its ligands, CXCL11 (CXC motif chemokine ligand 11)-that delimits EC angiogenic potential and promotes pericyte recruitment to ECs during development., Methods: We investigated the role of CXCR3 on vascular development using both 2- and 3-dimensional in vitro assays, to study EC-pericyte interactions and EC behavioral responses to blood flow. Additionally, genetic mutants and pharmacological modulators were used in zebrafish in vivo to study the impacts of CXCR3 loss and gain of function on vascular development., Results: In vitro modeling of EC-pericyte interactions demonstrates that suppression of EC-specific CXCR3 signaling leads to loss of pericyte association with EC tubes. In vivo, phenotypic defects are particularly noted in the cranial vasculature, where we see a loss of pericyte association with ECs and expansion of the vasculature in zebrafish treated with the Cxcr3 inhibitor AMG487 or in homozygous cxcr3.1/3.2/3.3 triple mutants. We also demonstrate that CXCR3-deficient ECs are more elongated, move more slowly, and have impaired EC-EC junctions compared with their control counterparts., Conclusions: Our results suggest that CXCR3 signaling in ECs helps promote vascular stabilization events during development by preventing EC overgrowth and promoting pericyte recruitment., Competing Interests: None.

The chromodomain helicase DNA binding domain 2 (CHD2) gene is an ATPase and a member of the SNF2-like family of helicase-related enzymes. CHD2 plays critical roles in human brain development and function, and homozygous mutation of Chd2 in mice results in perinatal lethality. To further elucidate the effects of chd2, we used CRISPR/Cas9 to create two chd2-knockout strains (fdu901, 11,979-11982delGGGT, and fdu902, 27350delG) in zebrafish. We found that the deformity and mortality rates of fdu901 and fdu902 were higher than those of the wild type. Developmental delay was more obvious and embryo mortality was higher in fdu901 than in fdu902. However, the embryo deformity rate in fdu902 was higher than that in fdu901. Although there were no significant differences in behavior between the two knockout zebrafish and wild-type zebrafish at 7 days post fertilization (dpf), fdu901 and fdu902 zebrafish showed different alterations. The excitability of fdu902 was higher than that of fdu901. Overall, our data demonstrate that two homozygous chd2 knockout mutations were survivable and could be stably inherited and that fdu901 and fdu902 zebrafish differed in behavior and morphology. These two models might be good tools for understanding the functions of the different domains of chd2., Competing Interests: Declarations. Ethical approval: All procedures are approved by the institutional animal care committee of Children’s Hospital of Fudan University, China. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Treacher Collins syndrome (TCS) is a genetic disorder affecting facial development, primarily caused by mutations in the TCOF1 gene. TCOF1, along with NOLC1, play important roles in ribosomal RNA transcription and processing. Previously, a zebrafish model of TCS successfully recapitulated the main characteristics of the syndrome by knocking down the expression of a gene on chromosome 13 (coding for Uniprot ID B8JIY2), which was identified as the TCOF1 orthologue. However, database updates renamed this gene as nolc1 and the zebrafish database (ZFIN) identified a different gene on chromosome 14 as the TCOF1 orthologue (coding for Uniprot ID E7F9D9). NOLC1 and TCOF1 are large proteins with unstructured regions and repetitive sequences that complicate alignments and comparisons. Also, the additional whole genome duplication of teleosts sets further difficulty. In this study, we present evidence that endorses that NOLC1 and TCOF1 are paralogs, and that the zebrafish gene on chromosome 14 is a low-complexity LisH domain-containing factor that displays homology to NOLC1 but lacks essential sequence features to accomplish TCOF1 nucleolar functions. Our analysis also supports the idea that zebrafish, as has been suggested for other non-tetrapod vertebrates, lack the TCOF1 gene that is associated with tripartite nucleolus. Using BLAST searches in a group of teleost genomes, we identified fish-specific sequences similar to E7F9D9 zebrafish protein. We propose naming them "LisH-containing Low Complexity Proteins" (LLCP). Interestingly, the gene on chromosome 13 (nolc1) displays the sequence features, developmental expression patterns, and phenotypic impact of depletion that are characteristic of TCOF1 functions. These findings suggest that in teleost fish, the nucleolar functions described for both NOLC1 and TCOF1 mediated by their repeated motifs, are carried out by a single gene, nolc1. Our study, which is mainly based on computational tools available as free web-based algorithms, could help to solve similar conflicts regarding gene orthology in zebrafish., Competing Interests: Declarations. Conflict of interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Ethical Approval: Not applicable. Consent to Participate: Not applicable. Consent for Publication: Not applicable., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Neurofibromatosis type 2 (NF-2) is a dominantly inherited genetic disorder that results from variants in the tumor suppressor gene, neurofibromin 2 (NF2). Here, we report the generation of a conditional zebrafish model of neurofibromatosis established by inducible genetic knockout of nf2a/b, the zebrafish homologs of human NF2. Analysis of nf2a and nf2b expression revealed ubiquitous expression of nf2b in the early embryo, with overlapping expression in the neural crest and its derivatives and in the cranial mesenchyme. In contrast, nf2a displayed lower expression levels. Induction of nf2a/b knockout at early stages increased the proliferation of larval Schwann cells and meningeal fibroblasts. Subsequently, in adult zebrafish, nf2a/b knockout triggered the development of a spectrum of tumors, including vestibular Schwannomas, spinal Schwannomas, meningiomas and retinal hamartomas, mirroring the tumor manifestations observed in patients with NF-2. Collectively, these findings highlight the generation of a novel zebrafish model that mimics the complexities of the human NF-2 disorder. Consequently, this model holds significant potential for facilitating therapeutic screening and elucidating key driver genes implicated in NF-2 onset., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The structure of the zebrafish retina appears to be very similar to that of mammals, that is why it is used as a model for studying the eye. Indeed, the zebrafish retina can regenerate itself through mechanisms of Müller cell reprogramming. In this research, adult zebrafish were exposed to aluminum to cause damage in the retina and thus evaluate the regenerative capacity of the damaged tissue. Histological and histochemical analyses assessed the retinal structure and the neurodegenerative process, respectively. An expression analysis of PARPs was carried out to verify whether a potential oxidative DNA damage happens. In addition, some genes involved in the regeneration process (pax6a, pax2a, ngn1, and notch1a) were analyzed. The data confirmed the toxicity of aluminum which caused retinal neurodegeneration, but also highlighted the ability of zebrafish to regenerate the retinal structure, repairing the damage and confirming its use as a good model for translational studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) play complex roles in liver health, influencing processes such as fibrosis, cancer development, and regeneration. WW domain binding protein-2 (WBP2) primarily enhances the co-translational activity of YAP/TAZ, which is crucial for the progression of liver diseases. Despite existing knowledge, the specific functions of WBP2 and its interactions with YAP remain inadequately understood. This study investigates the expression levels of WBP2 in zebrafish embryos and its molecular interaction with YAP. We employed morpholino-mediated knockdown of wbp2 and yap, followed by assessments of liver histology, immunofluorescence, and co-immunoprecipitation. Subsequently, RNA sequencing analyses were conducted to elucidate the signaling pathways and mechanisms underlying the interplay between YAP and WBP2 in liver injury. Our findings highlight the significant interaction between WBP2 and YAP, emphasizing their potential as therapeutic targets for liver diseases., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Retinal markers with high quality and specificity are important for the observation of pathologic changes of retinal cells during retinal development, degeneration, and regeneration. The zpr-3 antibody is widely used to label rods in zebrafish, but the exact antigen is still unknown. In this study, we provided evidence to demonstrate that the antigen gene of zpr-3 is rho, which encodes the rod opsin, and the exact epitope of zpr-3 is the 320-354 region of Rho protein. More importantly, our immunofluorescence assays indicated that zpr-3 labels both the outer segments of rods and green cones on zebrafish retinal sections, probably due to the cross-reaction with the green-cone opsin. Our work is valuable for the scientific community to interpret the experimental data involving the zpr-3 antibody.

Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in promoting arterial accumulation of vascular smooth muscle cells (vSMCs) during zebrafish development. Using a series of small molecule antagonists or agonists to temporally regulate Piezo1 activity, we identified a role for the Piezo1 channel in regulating klf2a, a blood flow responsive transcription factor, expression levels and altered targeting of vSMCs between arteries and veins. Increasing Piezo1 activity suppressed klf2a and increased vSMC association with the cardinal vein, while inhibition of Piezo1 activity increased klf2a levels and decreased vSMC association with arteries. We supported the small molecule findings with in vivo genetic suppression of piezo1 and 2 in zebrafish, resulting in loss of transgelin+ vSMCs on the dorsal aorta. Further, endothelial cell (EC)-specific Piezo1 knockout in mice was sufficient to decrease vSMC accumulation along the descending dorsal aorta during development, thus phenocopying our zebrafish data, and supporting functional conservation of Piezo1 in mammals. To determine the underlying mechanism, we used in vitro modeling assays to demonstrate that differential sensing of pulsatile versus laminar flow forces across endothelial cells changes the expression of mural cell differentiation genes. Together, our findings suggest a crucial role for EC Piezo1 in sensing force within large arteries to mediate mural cell differentiation and stabilization of the arterial vasculature., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: It is widely acknowledged that the p53 gene can be expressed as multiple isoforms with different functions, however the transcriptional mechanism of p53 still needs further investigation. Here we identified an elevated transcription signal about 3.6 kb upstream of the p53 promoter in cold acclimated zebrafish ZF4 cells., Methods and Results: Through rapid amplification of cDNA ends (RACE), an unreported p53 transcript was cloned, which is transcribed from a novel upstream promoter about 3.6 kb from the canonical p53 promoter. This Novel p53 transcript includes a novel 5'untranslated region (5'UTR) transcribed from the - 3.6 kb region, which is followed by the coding sequences (CDS) encoding wild type (WT) p53 protein. This Novel p53 transcript showed remarkably enhanced stability than WT p53 and Δ113p53 mRNAs, when its novel 5'UTR showed the lowest translation efficiency in luciferase assay. Novel p53 transcript is differentially expressed in various tissues and during different stages of embryonic development of zebrafish. Novel p53 transcript also showed different responses to different stimuli., Conclusions: A novel upstream promoter about 3.6 kb from the canonical P1 promoter of zebrafish p53 gene was found, which transcribes a novel p53 transcript that contains a new 5'UTR and the CDS encoding WT p53 protein. The findings of our study will enhance the current knowledge on the regulation and functionality of the p53 gene in fish., Competing Interests: Declarations. Ethical approval: This study was approved by the Animal Ethics committee of Shanghai Ocean University and abides by the Guidelines on Ethical Treatment of Experimental Animals established by the Ministry of Science and Technology, China. All authors have read and agreed to the published version of the manuscript. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Wnt signaling plays crucial roles in embryonic patterning including the regulation of convergent extension (CE) during gastrulation, the establishment of the dorsal axis, and later, craniofacial morphogenesis. Further, Wnt signaling is a crucial regulator of craniofacial morphogenesis. The adapter proteins Dact1 and Dact2 modulate the Wnt signaling pathway through binding to Disheveled. However, the distinct relative functions of Dact1 and Dact2 during embryogenesis remain unclear. We found that dact1 and dact2 genes have dynamic spatiotemporal expression domains that are reciprocal to one another suggesting distinct functions during zebrafish embryogenesis. Both dact1 and dact2 contribute to axis extension, with compound mutants exhibiting a similar CE defect and craniofacial phenotype to the wnt11f2 mutant. Utilizing single-cell RNAseq and an established noncanonical Wnt pathway mutant with a shortened axis ( gpc4 ), we identified dact1/2 -specific roles during early development. Comparative whole transcriptome analysis between wildtype and gpc4 and wildtype and dact1/2 compound mutants revealed a novel role for dact1/2 in regulating the mRNA expression of the classical calpain capn8 . Overexpression of capn8 phenocopies dact1/2 craniofacial dysmorphology. These results identify a previously unappreciated role of capn8 and calcium-dependent proteolysis during embryogenesis. Taken together, our findings highlight the distinct and overlapping roles of dact1 and dact2 in embryonic craniofacial development, providing new insights into the multifaceted regulation of Wnt signaling., Competing Interests: SC, SS, KK, CT, AJ, SH, EL, EL No competing interests declared, (© 2024, Carroll et al.)