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In contrast to adult mammalian hearts, the adult zebrafish heart efficiently replaces cardiomyocytes lost after injury. Here we reveal shared and species-specific injury response pathways and a correlation between Hmga1, an architectural non-histone protein, and regenerative capacity, as Hmga1 is required and sufficient to induce cardiomyocyte proliferation and required for heart regeneration. In addition, Hmga1 was shown to reactivate developmentally silenced genes, likely through modulation of H3K27me3 levels, poising them for a pro-regenerative gene program. Furthermore, AAV-mediated Hmga1 expression in injured adult mouse hearts led to controlled cardiomyocyte proliferation in the border zone and enhanced heart function, without cardiomegaly and adverse remodeling. Histone modification mapping in mouse border zone cardiomyocytes revealed a similar modulation of H3K27me3 marks, consistent with findings in zebrafish. Our study demonstrates that Hmga1 mediates chromatin remodeling and drives a regenerative program, positioning it as a promising therapeutic target to enhance cardiac regeneration after injury., Competing Interests: Competing interests: D.E.M.d.B. and J.B. are co-inventors on a patent application associated with this study: patent no. PCT/NL2022/050553, ‘Mammalian heart regeneration’. J.B. is co-founder of Myogene Therapeutics. The other authors declare no competing interests., (© 2025. The Author(s).)

Chemobehavioural phenotyping presents unique opportunities for analyzing neurotoxicants and discovering behavior-modifying neuroceuticals in small aquatic model organisms such as zebrafish (Danio rerio). A recently popularized approach in this field involves the utilization of zebrafish embryos for a photo-motor response (PMR) bioassay. The PMR bioassay entails stimulating zebrafish embryos between 24 and 36 h post fertilization (hpf) with a high-intensity light stimulus, inducing a transient increase in the frequency of photo-induced embryo body flexions. These flexions can be computationally analyzed to derive behavioral signatures, enabling the categorization of neuromodulating chemicals. Despite the significant advantages of the PMR bioassay, its widespread implementation is hindered by lack of well described and straightforward high-throughput bioinformatic analysis of behavioral data. In this methods article, we present an easily implementable bioinformatics protocol specifically designed for rapid behavioral analysis of large cohorts of zebrafish specimens in PMR bioassays. We also address common pitfalls encountered during PMR analysis, discuss its limitations, and propose future directions for developing next-generation biometric analysis techniques in chemobehavioural assays utilizing zebrafish embryos., 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 Inc. All rights reserved.)

Assessment of animals' sensory-motor functions requires precise and electronically controlled stimuli to induce and quantify specific behavioral phenotypes. However, accessible and inexpensive tools for conducting diverse sensory-motor biotests with fish are lacking. In this work, we present an open-source software and hardware interface that enables automated delivery of three independent and fully programmable stimuli for behavioral bioassays. We demonstrate the proof-of-concept application of this low-cost technology in establishing reproducible fear responses using a mechanical tap-startle stimulus in larval zebrafish. This response is characterized by a sudden burst of motion in response to a nondirectional mechanical stimulus delivered to the fish chamber. We propose that the simplicity and flexibility of this interface offer innovative opportunities for studying sensory-motor functions in various fields, including neurobiology, neuropharmacology, neurotoxicology, and aquatic ecotoxicology.

Unlike mammals, adult and larval zebrafish exhibit robust regeneration following traumatic spinal cord injury. This remarkable regenerative capacity, combined with exquisite imaging capabilities and an abundance of powerful genetic techniques, has established the zebrafish as an important vertebrate model for the study of neural regeneration. Here, we describe a protocol for the complete mechanical ablation of the larval zebrafish spinal cord. With practice, this protocol can be used to reproducibly injure upward of 100 samples per hour, facilitating the high-throughput screening of factors involved in spinal cord regeneration and repair., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Aquatic eco-neurotoxicology is an emerging field that requires new analytical systems to study the effects of pollutants on animal behaviors. This is especially true if we are to gain insights into one of the least studied aspects: the potential perturbations that neurotoxicants can have on cognitive behaviors. The paucity of experimental data is partly caused by a lack of low-cost technologies for the analysis of higher-level neurological functions (e.g., associative learning) in small aquatic organisms. Here, we present a proof-of-concept prototype that utilizes a new real-time animal tracking software for on-the-fly video analysis and closed-loop, external hardware communications to deliver stimuli based on specific behaviors in aquatic organisms, spanning three animal phyla: chordates (fish, frog), platyhelminthes (flatworm), and arthropods (crustacean). The system's open-source software features an intuitive graphical user interface and advanced adaptive threshold-based image segmentation for precise animal detection. We demonstrate the precision of animal tracking across multiple aquatic species with varying modes of locomotion. The presented technology interfaces easily with low-cost and open-source hardware such as the Arduino microcontroller family for closed-loop stimuli control. The new system has potential future applications in eco-neurotoxicology, where it could enable new opportunities for cognitive research in diverse small aquatic model organisms.

Due to technical limitations, there have been minimal studies performed on thermal preferences and thermotactic behaviors of aquatic ectotherm species commonly used in ecotoxicity testing. In this work, we demonstrate an innovative, purpose-built and miniaturized electrothermal array for rapid thermal preference behavioral tests. We applied the novel platform to define thermal preferences in multiple invertebrate and vertebrate species. Specifically, Dugesia notogaea (freshwater planarians), Chironomus tepperi (nonbiting midge larvae), Ostracoda (seed shrimp), Artemia franciscana (brine shrimp), Daphnia carinata (water flea), Austrochiltonia subtenuis (freshwater amphipod), Physa acuta (freshwater snail), Potamopyrgus antipodarum (New Zealand mud snail) and larval stage of Danio rerio (zebrafish) were tested. The Australian freshwater water fleas, amphipods, snail Physa acuta as well as zebrafish exhibited the most consistent preference to cool zones and clear avoidance of zones >27 °C out of nine species tested. Our results indicate the larval stage of zebrafish as the most responsive species highly suitable for prospective development of multidimensional behavioral test batteries. We also showcase preliminary data that environmentally relevant concentrations of pharmaceutical pollutants such as non-steroidal anti-inflammatory drug (NSAID) ibuprofen (9800 ng/L) and insecticide imidacloprid (4600 ng/L) but not anti-depressant venlafaxine (2200 ng/L) and (iv) anticonvulsant medications gabapentin (400 ng/L) can perturb thermal preference behavior of larval zebrafish. Collectively our results demonstrate the utility of simple and inexpensive thermoelectric technology in rapid exploration of thermal preference in diverse species of aquatic animals. We postulate that more broadly such technologies can also have added value in ecotoxicity testing of emerging contaminants., 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 © 2022 Elsevier Ltd. All rights reserved.)

Many targeted natural product isolation approaches rely on the use of pre-existing bioactivity information to inform the strategy used for the isolation of new bioactive compounds. Bioactivity information can be available either in the form of prior assay data or via Structure Activity Relationship (SAR) information which can indicate a potential chemotype that exhibits a desired bioactivity. The work described herein utilizes a unique method of targeted isolation using structure-based virtual screening to identify potential antibacterial compounds active against MRSA within the marine sponge order Verongiida. This is coupled with molecular networking-guided, targeted isolation to provide a novel drug discovery procedure. A total of 12 previously reported bromotyrosine-derived alkaloids were isolated from the marine sponge species Pseudoceratina durissima, and the compound, (+)-aeroplysinin-1 (1) displayed activity against the MRSA pathogen (MIC: <32 µg/mL). The compounds (1−3, 6 and 9) were assessed for their central nervous system (CNS) interaction and behavioral toxicity to zebrafish (Danio rerio) larvae, whereby several of the compounds were shown to induce significant hyperactivity. Anthelmintic activity against the parasitic nematode Haemonchus contorutus was also evaluated (2−4, 6−8)., Competing Interests: The authors declare no conflict of interest.

Due to increasing numbers of anthropogenic chemicals with unknown neurotoxic properties, there is an increasing need for a paradigm shift toward rapid and higher throughput behavioral bioassays. In this work, we demonstrate application of a purpose-built high throughput multidimensional behavioral test battery on larval stages of Danio rerio (zebrafish) at 5 days post fertilization (dpf). The automated battery comprised of the established spontaneous swimming (SS), simulated predator response (SPR), larval photomotor response (LPR) assays as well as a new thermotaxis (TX) assay. We applied the novel system to characterize environmentally relevant concentrations of emerging pharmaceutical micropollutants including anticonvulsants (gabapentin: 400 ng/L; carbamazepine: 3000 ng/L), inflammatory drugs (ibuprofen: 9800 ng/L), and antidepressants (fluoxetine: 300 ng/L; venlafaxine: 2200 ng/L). The successful integration of the thermal preference assay into a multidimensional behavioral test battery provided means to reveal ibuprofen-induced perturbations of thermal preference behaviors upon exposure during embryogenesis. Moreover, we discovered that photomotor responses in larval stages of fish are also altered by the as yet understudied anticonvulsant gabapentin. Collectively our results demonstrate the utility of high-throughput multidimensional behavioral ecotoxicity test batteries in prioritizing emerging risks associated with neuroactive drugs that can perturb neurodevelopment. Moreover, we showcase the added value of thermotaxis bioassays for preliminary screening of emerging contaminants.

Cell adhesion molecules are membrane-bound proteins predominantly expressed in the central nervous system along principal axonal pathways with key roles in nervous system development, neural cell differentiation and migration, axonal growth and guidance, myelination, and synapse formation. Here, we describe ten affected individuals with bi-allelic variants in the neuronal cell adhesion molecule NRCAM that lead to a neurodevelopmental syndrome of varying severity; the individuals are from eight families. This syndrome is characterized by developmental delay/intellectual disability, hypotonia, peripheral neuropathy, and/or spasticity. Computational analyses of NRCAM variants, many of which cluster in the third fibronectin type III (Fn-III) domain, strongly suggest a deleterious effect on NRCAM structure and function, including possible disruption of its interactions with other proteins. These findings are corroborated by previous in vitro studies of murine Nrcam-deficient cells, revealing abnormal neurite outgrowth, synaptogenesis, and formation of nodes of Ranvier on myelinated axons. Our studies on zebrafish nrcama Δ mutants lacking the third Fn-III domain revealed that mutant larvae displayed significantly altered swimming behavior compared to wild-type larvae (p < 0.03). Moreover, nrcama Δ mutants displayed a trend toward increased amounts of α-tubulin fibers in the dorsal telencephalon, demonstrating an alteration in white matter tracts and projections. Taken together, our study provides evidence that NRCAM disruption causes a variable form of a neurodevelopmental disorder and broadens the knowledge on the growing role of the cell adhesion molecule family in the nervous system., Competing Interests: Declaration of interests C.G.-J. is a full-time employee of the Regeneron Genetics Center and receives stock options as part of compensation. All other authors have no conflicts to declare., (Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Cyclin-dependent kinase-like-5 (CDKL5) deficiency disorder (CDD) is a severe X-linked neurodegenerative disease characterised by early-onset epileptic seizures, low muscle tone, progressive intellectual disability and severe motor function. CDD affects ∼1 in 60,000 live births, with many patients experiencing a reduced quality of life due to the severity of their neurological symptoms and functional impairment. There are no effective therapies for CDD, with current treatments focusing on improving symptoms rather than addressing the underlying causes of the disorder. Zebrafish offer many unique advantages for high-throughput preclinical evaluation of potential therapies for neurological diseases, including CDD. In particular, the large number of offspring produced, together with the possibilities for in vivo imaging and genetic manipulation, allows for the detailed assessment of disease pathogenesis and therapeutic discovery. We have characterised a loss-of-function zebrafish model for CDD, containing a nonsense mutation in cdkl5. cdkl5 mutant zebrafish display defects in neuronal patterning, seizures, microcephaly, and reduced muscle function caused by impaired muscle innervation. This study provides a powerful vertebrate model for investigating CDD disease pathophysiology and allowing high-throughput screening for effective therapies. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Large-scale chemobehavioral phenotyping with zebrafish embryos is a promising avenue for accelerated neurotoxicity testing and discovery of behavior-modifying neuroceuticals. These strategies are hampered by lack of effective embryo in-test positioning, wide-field imaging, and high-throughput bioinformatic analytics. In this study, we demonstrate advantages of using custom large-density embryo arrays in conjunction with an open-source ultra-high-definition video imaging system. Moreover, we present a high-throughput bioinformatics workflow for rapid behavioral analysis of large cohorts of specimens in photomotor response bioassays. The system validation was showcased in a proof-of-concept neurotoxicity analysis.

Spermatogenesis-associated 5 like 1 (SPATA5L1) represents an orphan gene encoding a protein of unknown function. We report 28 bi-allelic variants in SPATA5L1 associated with sensorineural hearing loss in 47 individuals from 28 (26 unrelated) families. In addition, 25/47 affected individuals (53%) presented with microcephaly, developmental delay/intellectual disability, cerebral palsy, and/or epilepsy. Modeling indicated damaging effect of variants on the protein, largely via destabilizing effects on protein domains. Brain imaging revealed diminished cerebral volume, thin corpus callosum, and periventricular leukomalacia, and quantitative volumetry demonstrated significantly diminished white matter volumes in several individuals. Immunofluorescent imaging in rat hippocampal neurons revealed localization of Spata5l1 in neuronal and glial cell nuclei and more prominent expression in neurons. In the rodent inner ear, Spata5l1 is expressed in the neurosensory hair cells and inner ear supporting cells. Transcriptomic analysis performed with fibroblasts from affected individuals was able to distinguish affected from controls by principal components. Analysis of differentially expressed genes and networks suggested a role for SPATA5L1 in cell surface adhesion receptor function, intracellular focal adhesions, and DNA replication and mitosis. Collectively, our results indicate that bi-allelic SPATA5L1 variants lead to a human disease characterized by sensorineural hearing loss (SNHL) with or without a nonprogressive mixed neurodevelopmental phenotype., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

LRRK2 gain-of-function is considered a major cause of Parkinson's disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD., Competing Interests: The authors have declared that no competing interests exist.

Amyloid aggregation is a ubiquitous form of protein misfolding underlying the pathologies of Alzheimer's disease (AD), Parkinson's disease (PD) and type 2 diabetes (T2D), three primary forms of human amyloid diseases. While much has been learned about the origin, diagnosis and management of these neurological and metabolic disorders, no cure is currently available due in part to the dynamic and heterogeneous nature of the toxic oligomers induced by amyloid aggregation. Here we synthesized beta casein-coated iron oxide nanoparticles (βCas IONPs) via a BPA-P(OEGA-b-DBM) block copolymer linker. Using a thioflavin T kinetic assay, transmission electron microscopy, Fourier transform infrared spectroscopy, discrete molecular dynamics simulations and cell viability assays, we examined the Janus characteristics and the inhibition potential of βCas IONPs against the aggregation of amyloid beta (Aβ), alpha synuclein (αS) and human islet amyloid polypeptide (IAPP) which are implicated in the pathologies of AD, PD and T2D. Incubation of zebrafish embryos with the amyloid proteins largely inhibited hatching and elicited reactive oxygen species, which were effectively rescued by the inhibitor. Furthermore, Aβ-induced damage to mouse brain was mitigated in vivo with the inhibitor. This study revealed the potential of Janus nanoparticles as a new nanomedicine against a diverse range of amyloid diseases., Competing Interests: Competing financial interests The authors declare no conflicting financial interests.

Tissue regeneration and functional restoration after injury are considered as stem- and progenitor-cell-driven processes. In the central nervous system, stem cell-driven repair is slow and problematic because function needs to be restored rapidly for vital tasks. In highly regenerative vertebrates, such as zebrafish, functional recovery is rapid, suggesting a capability for fast cell production and functional integration. Surprisingly, we found that migration of dormant "precursor neurons" to the injury site pioneers functional circuit regeneration after spinal cord injury and controls the subsequent stem-cell-driven repair response. Thus, the precursor neurons make do before the stem cells make new. Furthermore, RNA released from the dying or damaged cells at the site of injury acts as a signal to attract precursor neurons for repair. Taken together, our data demonstrate an unanticipated role of neuronal migration and RNA as drivers of neural repair., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N -sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgsh Δex5-6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgsh Δex5-6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgsh Δex5-6 zebrafish is largely dependent on interleukin-1β and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgsh Δex5-6 mutant larvae in a context-dependent manner. We expect the sgsh Δex5-6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.

The Zebrafish Embryo Genotyper (ZEG) device provides a promising tool for genotyping live embryos. Although the gross morphology and survival of embryos after the use of ZEG are unaffected, the cellular and molecular effects of the ZEG protocol remain unknown. To address this, we have examined the integrity of specific tissues, and evaluated the expression of stress-responsive genes to determine the impact of the ZEG protocol. Our analyses reveal that although ZEG results in a low-level acute stress response, no long-lasting effects are evident, supporting its utilization for a variety of downstream assays.

Identifying endogenous tissue stem cells remains a key challenge in developmental and regenerative biology. To distinguish and molecularly characterise stem cell populations in large heterogeneous tissues, the combination of cytochemical cell markers with ultrastructural morphology is highly beneficial. Here, we realise this through workflows of multi-resolution immuno-correlative light and electron microscopy (iCLEM) methodologies. Taking advantage of the antigenicity preservation of the Tokuyasu technique, we have established robust protocols and workflows and provide a side-by-side comparison of iCLEM used in combination with scanning EM (SEM), scanning TEM (STEM), or transmission EM (TEM). Evaluation of the applications and advantages of each method highlights their practicality for the identification, quantification, and characterization of heterogeneous cell populations in small organisms, organs, or tissues in healthy and diseased states. The iCLEM techniques are broadly applicable and can use either genetically encoded or cytochemical markers on plant, animal and human tissues. We demonstrate how these protocols are particularly suited for investigating neural stem and progenitor cell populations of the vertebrate nervous system.

The vertebrate inner ear employs sensory hair cells and neurons to mediate hearing and balance. In mammals, damaged hair cells and neurons are not regenerated. In contrast, hair cells in the inner ear of zebrafish are produced throughout life and regenerate after trauma. However, it is unknown whether new sensory neurons are also formed in the adult zebrafish statoacoustic ganglion (SAG), the sensory ganglion connecting the inner ear to the brain. Using transgenic lines and marker analysis, we identify distinct cell populations and anatomical landmarks in the juvenile and adult SAG. In particular, we analyze a Neurod/Nestin-positive progenitor pool that produces large amounts of new neurons at juvenile stages, which transitions to a quiescent state in the adult SAG. Moreover, BrdU pulse chase experiments reveal the existence of a proliferative but otherwise marker-negative cell population that replenishes the Neurod/Nestin-positive progenitor pool at adult stages. Taken together, our study represents the first comprehensive characterization of the adult zebrafish SAG showing that zebrafish, in sharp contrast to mammals, display continued neurogenesis in the SAG well beyond embryonic and larval stages., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

An innovative drug delivery vehicle based on polynorepinephrine (PNE) with controllable size modification, high delivery efficacy and low cytotoxicity is presented. Highly monodisperse PNE nanoparticles are fabricated by the autoxidation of norepinephrine monomers in an alkaline water/ethanol mixture via stirring at room temperature. We demonstrated the facile optimization of particle size to enhance particle stability and biocompatibility by varying solvent and monomer dosage. To demonstrate the suitability and potential application of PNE particles in cancer therapy, we show that these particles are biocompatible in vitro with HeLa cells and in vivo in zebrafish embryos. After loading the anti-cancer chemotherapy drug doxorubicin (DOX) into the PNE nanoparticles, a consistent and pH responsive drug release profile of DOX was achieved in different environmental conditions. It was found that DOX loaded PNE nanoparticles (PNE/DOX) exhibit much higher pharmaceutical cytotoxicity than free DOX on HeLa cells. Furthermore, the amount of drug released was significantly enhanced in acidic environments that mimic the pH of extracellular tumour microenvironments. Taken together, the PNE nanoparticles represent a new class of melanin particles with promising potential in drug delivery and as a therapeutic platform for cancer treatment.

Background: The molecular changes involved in Alzheimer's disease (AD) progression remain unclear since we cannot easily access antemortem human brains. Some non-mammalian vertebrates such as the zebrafish preserve AD-relevant transcript isoforms of the PRESENILIN genes lost from mice and rats. One example is PS2V, the alternative transcript isoform of the PSEN2 gene. PS2V is induced by hypoxia/oxidative stress and shows increased expression in late onset, sporadic AD brains. A unique, early onset familial AD mutation of PSEN2, K115fs, mimics the PS2V coding sequence suggesting that forced, early expression of PS2V-like isoforms may contribute to AD pathogenesis. Here we use zebrafish to model the K115fs mutation to investigate the effects of forced PS2V-like expression on the transcriptomes of young adult and aged adult brains., Methods: We edited the zebrafish genome to model the K115fs mutation. To explore its effects at the molecular level, we analysed the brain transcriptome and proteome of young (6-month-old) and aged (24-month-old) wild type and heterozygous mutant female sibling zebrafish. Finally, we used gene co-expression network analysis (WGCNA) to compare molecular changes in the brains of these fish to human AD., Results: Young heterozygous mutant fish show transcriptional changes suggesting accelerated brain aging and increased glucocorticoid signalling. These early changes precede a transcriptional 'inversion' that leads to glucocorticoid resistance and other likely pathological changes in aged heterozygous mutant fish. Notably, microglia-associated immune responses regulated by the ETS transcription factor family are altered in both our zebrafish mutant model and in human AD. The molecular changes we observe in aged heterozygous mutant fish occur without obvious histopathology and possibly in the absence of Aβ., Conclusions: Our results suggest that forced expression of a PS2V-like isoform contributes to immune and stress responses favouring AD pathogenesis. This highlights the value of our zebrafish genetic model for exploring molecular mechanisms involved in AD pathogenesis., Competing Interests: The authors have declared that no competing interests exist.

Human amyloids and plaques uncovered post mortem are highly heterogeneous in structure and composition, yet literature concerning the heteroaggregation of amyloid proteins is extremely scarce. This knowledge deficiency is further exacerbated by the fact that peptide delivery is a major therapeutic strategy for targeting their full-length counterparts associated with the pathologies of a range of human diseases, including dementia and type 2 diabetes (T2D). Accordingly, here we examined the coaggregation of full-length human islet amyloid polypeptide (IAPP), a peptide associated with type 2 diabetes, with its primary and secondary amyloidogenic fragments 19-29 S20G and 8-20. Single-molecular aggregation dynamics was obtained by high-speed atomic force microscopy, augmented by transmission electron microscopy, X-ray diffraction, and super-resolution stimulated emission depletion microscopy. The coaggregation significantly prolonged the pause phase of fibril elongation, increasing its dwell time by 3-fold. Surprisingly, unidirectional elongation of mature fibrils, instead of protofilaments, was observed for the coaggregation, indicating a new form of tertiary protein aggregation unknown to existing theoretical models. Further in vivo zebrafish embryonic assay indicated improved survival and hatching, as well as decreased frequency and severity of developmental abnormalities for embryos treated with the heteroaggregates of IAPP with 19-29 S20G, but not with 8-20, compared to the control, indicating the therapeutic potential of 19-29 S20G against T2D.

The environmental impact of exposure to 3D-printed plastics as well as potential migration of toxic chemicals from 3D-printed plastics remains largely unexplored. In this work we applied leachates from plastics fabricated using a stereolithography (SLA) process to early developmental stages of zebrafish (Danio rerio) to investigate developmental toxicity and neurotoxicity. Migration of unpolymerized photoinitiator, 1-hydroxycyclohexyl phenyl ketone (1-HCHPK) from a plastic solid phase to aqueous media at up to 200 mg/L in the first 24 h was detected using gas chromatography-mass spectrometry. Both plastic extracts (LC 50 22.25% v/v) and 1-HCHPK (LC 50 60 mg/L) induced mortality and teratogenicity within 48 h of exposure. Developmental toxicity correlated with in situ generation of reactive oxygen species (ROS), an increase in lipid peroxidation and protein carbonylation markers and enhanced activity of superoxide dismutase (SOD) and glutathione-S-transferase (GST) in embryos exposed to concentrations as low as 20% v/v for plastic extracts and 16 mg/L for 1-HCHPK. ROS-induced cellular damage led to induction of caspase-dependent apoptosis which could be pharmacologically inhibited with both antioxidant ascorbic acid and a pan-caspase inhibitor. Neuro-behavioral analysis showed that exposure to plastic leachates reduced spontaneous embryonic movement in 24-36 hpf embryos. Plastic extracts in concentrations above 20% v/v induced rapid retardation of locomotion, changes in photomotor response and habituation to photic stimuli with progressive paralysis in 120 hpf larvae. Significantly decreased acetylcholinesterase (AChE) activity with lack of any CNS-specific apoptotic phenotypes as well as lack of changes in motor neuron density, axonal growth, muscle segment integrity or presence of myoseptal defects were detected upon exposure to plastic extracts during embryogenesis. Considering implications of the results for environmental risk assessment and the growing usage of 3D-printing technologies, we speculate that some 3D-printed plastic waste may represent a significant and yet very poorly uncharacterized environmental hazard that merits further investigation on a range of aquatic and terrestrial species., (Copyright © 2019 Elsevier B.V. All rights reserved.)

How diverse adult stem and progenitor populations regenerate tissue following damage to the brain is poorly understood. In highly regenerative vertebrates, such as zebrafish, radial-glia (RG) and neuro-epithelial-like (NE) stem/progenitor cells contribute to neuronal repair after injury. However, not all RG act as neural stem/progenitor cells during homeostasis in the zebrafish brain, questioning the role of quiescent RG (qRG) post-injury. To understand the function of qRG during regeneration, we performed a stab lesion in the adult midbrain tectum to target a population of homeostatic qRG, and investigated their proliferative behaviour, differentiation potential, and Wnt/β-catenin signalling. EdU-labelling showed a small number of proliferating qRG after injury (pRG) but that progeny are restricted to RG. However, injury promoted proliferation of NE progenitors in the internal tectal marginal zone (TMZi) resulting in amplified regenerative neurogenesis. Increased Wnt/β-catenin signalling was detected in TMZi after injury whereas homeostatic levels of Wnt/β-catenin signalling persisted in qRG/pRG. Attenuation of Wnt signalling suggested that the proliferative response post-injury was Wnt/β-catenin-independent. Our results demonstrate that qRG in the tectum have restricted capability in neuronal repair, highlighting that RG have diverse functions in the zebrafish brain. Furthermore, these findings suggest that endogenous stem cell compartments compensate lost tissue by amplifying homeostatic growth.

Maternal hyperglycaemia has a profound impact on the developing foetus and increases the risk of developing abnormalities like obesity, impaired glucose tolerance and insulin secretory defects in the post-natal life. Increased levels of glucose in the blood stream due to diabetes causes visual disorders like retinopathy. However, the impact of maternal hyperglycaemia due to pre-existing or gestational diabetes on the developing foetal retina is unknown. The aim of this work was to study the effect of hyperglycaemia on the developing retina using zebrafish as a vertebrate model. Wild-type and transgenic zebrafish embryos were exposed to 0, 4 and 5% D-Glucose in a pulsatile manner to mimic the fluctuations in glycaemia experienced by the developing foetus in pregnant women with diabetes. The zebrafish embryos displayed numerous ocular defects associated with altered retinal cell layer thickness, increased presence of macrophages, and decreased number of Müeller glial and retinal ganglion cells following high-glucose exposure. We have developed a model of gestational hyperglycaemia using the zebrafish embryo to study the effect of hyperglycaemia on the developing embryonic retina. The data suggests that glucose exposure is detrimental to the development of embryonic retina and the legacy of this exposure may extend into adulthood. These data suggest merit in retinal assessment in infants born to mothers with pre-existing and gestational diabetes both in early and adult life.

Zebrafish larvae are suitable in vivo models for toxicological and pharmacological screens due to their transparency, small size, ex utero development, and genetic and physiological similarity to humans. Using modern imaging techniques, cells and tissues can be dynamically visualized over several days in multiple zebrafish larvae. However, precise specimen immobilization and maintenance of homeostatic conditions remain a challenge for longitudinal studies. A highly customizable mounting configuration with inbuilt means of controlling temperature and media flow would therefore be a valuable tool to facilitate long-term imaging of a large number of specimens. Using three-dimensional printing, we have developed a millifluidic, modular homeostatic imaging plate (HIP), which consists of a customizable sample insert and a temperature-controlled incubation chamber that is continuously perfused, providing an ideal environment for long-term experiments where homeostatic conditions are desired. The HIP is cheap to produce, has a standard microtiter well plate format, and can be fitted to most microscopes. We used the device to image dynamic regeneration of spinal cord neurons. The flexibility and adaptability of the HIP facilitate long-term in vivo imaging of many samples, and can be easily adapted to suit a broad range of specimens.

Gene editing using clustered regularly interspaced short palindromic repeats (CRISPR) is widely used throughout the zebrafish community for the generation of knockouts and knockins. One of the bottlenecks that exists during the process is the laborious screening of injected embryos for F0 founder fish or CRISPants, weeks after the injection date. In this study we show that the use of fluorescently tagged tracrRNA and sorting for fluorescent embryos as early as the 512-cell stage using stereomicroscope significantly improve yield of fish with successfully CRISPR/Cas9-edited genomes. This is a cost-effective strategy that significantly improves workflow and efficacy in genome editing in particular for less experienced researchers.

Background: Despite conserved developmental processes and organization of the vertebrate central nervous system, only some vertebrates including zebrafish can efficiently regenerate neural damage including after spinal cord injury. The mammalian spinal cord shows very limited regeneration and neurogenesis, resulting in permanent life-long functional impairment. Therefore, there is an urgent need to identify the cellular and molecular mechanisms that can drive efficient vertebrate neurogenesis following injury. A key pathway implicated in zebrafish neurogenesis is fibroblast growth factor signaling., Methods: In the present study we investigated the roles of distinct fibroblast growth factor members and their receptors in facilitating different aspects of neural development and regeneration at different timepoints following spinal cord injury. After spinal cord injury in adults and during larval development, loss and/or gain of Fgf signaling was combined with immunohistochemistry, in situ hybridization and transgenes marking motor neuron populations in in vivo zebrafish and in vitro mammalian PC12 cell culture models., Results: Fgf3 drives neurogenesis of Islet1 expressing motor neuron subtypes and mediate axonogenesis in cMet expressing motor neuron subtypes. We also demonstrate that the role of Fgf members are not necessarily simple recapitulating development. During development Fgf2, Fgf3 and Fgf8 mediate neurogenesis of Islet1 expressing neurons and neuronal sprouting of both, Islet1 and cMet expressing motor neurons. Strikingly in mammalian PC12 cells, all three Fgfs increased cell proliferation, however, only Fgf2 and to some extent Fgf8, but not Fgf3 facilitated neurite outgrowth., Conclusions: This study demonstrates differential Fgf member roles during neural development and adult regeneration, including in driving neural proliferation and neurite outgrowth of distinct spinal cord neuron populations, suggesting that factors including Fgf type, age of the organism, timing of expression, requirements for different neuronal populations could be tailored to best drive all of the required regenerative processes.