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The spatial and temporal expression of steroidogenic genes in zebrafish has not been fully characterised. Because zebrafish are increasingly employed in endocrine and stress research, a better characterisation of steroidogenic pathways is required to target specific steps in the biosynthetic pathways. In the present study, we have systematically defined the temporal and spatial expression of steroidogenic enzymes involved in glucocorticoid biosynthesis (cyp21a2, cyp11c1, cyp11a1, cyp11a2, cyp17a1, cyp17a2, hsd3b1, hsd3b2), as well as the mitochondrial electron‐providing ferredoxin co‐factors (fdx1, fdx1b), during zebrafish development. Our studies showed an early expression of all these genes during embryogenesis. In larvae, expression of cyp11a2, cyp11c1, cyp17a2, cyp21a2, hsd3b1 and fdx1b can be detected in the interrenal gland, which is the zebrafish counterpart of the mammalian adrenal gland, whereas the fdx1 transcript is mainly found in the digestive system. Gene expression studies using quantitative reverse transcriptase‐PCR and whole‐mount in situ hybridisation in the adult zebrafish brain revealed a wide expression of these genes throughout the encephalon, including neurogenic regions. Using ultra‐high‐performance liquid chromatography tandem mass spectrometry, we were able to demonstrate the presence of the glucocorticoid cortisol in the adult zebrafish brain. Moreover, we demonstrate de novo biosynthesis of cortisol and the neurosteroid tetrahydrodeoxycorticosterone in the adult zebrafish brain from radiolabelled pregnenolone. Taken together, the present study comprises a comprehensive characterisation of the steroidogenic genes and the fdx co‐factors facilitating glucocorticoid biosynthesis in zebrafish. Furthermore, we provide additional evidence of de novo neurosteroid biosynthesising in the brain of adult zebrafish facilitated by enzymes involved in glucocorticoid biosynthesis. Our study provides a valuable source for establishing the zebrafish as a translational model with respect to understanding the roles of the genes for glucocorticoid biosynthesis and fdx co‐factors during embryonic development and stress, as well as in brain homeostasis and function. [ABSTRACT FROM AUTHOR]

Abstract: The cannabinoid receptor 1 (Cb1) mediates the psychoactive effect of marijuana. In mammals, there is abundant evidence advocating the importance of cannabinoid signaling; activation of Cb1 exerts diverse functions, chiefly by its ability to modulate neurotransmission. Thus, much attention has been devoted to understand its role in health and disease and to evaluate its therapeutic potential. Here, we have cloned zebrafish cb1 and investigated its expression in developing and adult zebrafish brain. Sequence analysis showed that there is a high degree of conservation, especially in residues demonstrated to be critical for function in mammals. In situ hybridization revealed that zebrafish cb1 appears first in the preoptic area at 24 hours post-fertilization. Subsequently, transcripts are detected in the dorsal telencephalon, hypothalamus, pretectum and torus longitudinalis. A similar pattern of expression is recapitulated in the adult brain. While cb1 is intensively stained in the medial zone of the dorsal telencephalon, expression elsewhere is weak by comparison. In particular, localization of cb1 in the telencephalic periventricular matrix is suggestive of the involvement of Cb1 in neurogenesis, bearing strong resemblance in terms of expression and function to the proliferative mammalian hippocampal formation. In addition, a gradient-like expression of cb1 is detected in the torus longitudinalis, a teleost specific neural tissue. In relation to dopaminergic neurons in the diencephalic posterior tuberculum (considered to be the teleostean homologue of the mammalian midbrain dopaminergic system), both cb1 and tyrosine hydroxylase-expressing cells occupy non-overlapping domains. However there is evidence that they are co-localized in the caudal zone of the hypothalamus, implying a direct modulation of dopamine release in this particular region. Collectively, our data indicate the propensity of zebrafish cb1 to participate in multiple neurological processes. [Copyright &y& Elsevier]

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Repair of lesions in the plasma membrane is key to sustaining cellular homeostasis. Cells maintain cytoplasmic as well as membrane-bound stores of repair proteins that can rapidly precipitate at the site of membrane lesions. However, little is known about the origins of lipids and proteins for resealing and repair of the plasma membrane. Here we study the dynamics of caveolar proteins after laser-induced lesioning of plasma membranes of mammalian C2C12 tissue culture cells and muscle cells of intact zebrafish embryos. Single-molecule diffusivity measurements indicate that caveolar clusters break up into smaller entities after wounding. Unlike Annexins and Dysferlin, caveolar proteins do not accumulate at the lesion patch. In caveolae-depleted cavin1a knockout zebrafish embryos, lesion patch formation is impaired, and injured cells show reduced survival. Our data suggest that caveolae disassembly releases surplus plasma membrane near the lesion to facilitate membrane repair after initial patch formation for emergency sealing., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

The efficient and biocompatible transfer of nucleic acids into mammalian cells for research applications or medical purposes is a long-standing, challenging task. Viral transduction is the most efficient transfer system, but often entails high safety levels for research and potential health impairments for patients in medical applications. Lipo- or polyplexes are commonly used transfer systems but result in comparably low transfer efficiencies. Moreover, inflammatory responses caused by cytotoxic side effects were reported for these transfer methods. Often accountable for these effects are various recognition mechanisms for transferred nucleic acids. Using commercially available fusogenic liposomes (Fuse-It-mRNA), we established highly efficient and fully biocompatible transfer of RNA molecules for in vitro as well as in vivo applications. We demonstrated bypassing of endosomal uptake routes and, therefore, of pattern recognition receptors that recognize nucleic acids with high efficiency. This may underlie the observed almost complete abolishment of inflammatory cytokine responses. RNA transfer experiments into zebrafish embryos and adult animals fully confirmed the functional mechanism and the wide range of applications from single cells to organisms.

A labeling strategy for in vivo 19 F-MRI (magnetic resonance imaging) based on highly fluorinated, short hydrophilic peptide probes, is developed. As dual-purpose probes, they are functionalized further by a fluorophore and an alkyne moiety for bioconjugation. High fluorination is achieved by three perfluoro-tert-butyl groups, introduced into asparagine analogues by chemically stable amide bond linkages. d-amino acids and β-alanine in the sequences endow the peptide probes with low cytotoxicity and high serum stability. This design also yielded unstructured peptides, rendering all 27 19 F substitutions chemically equivalent, giving rise to a single 19 F-NMR resonance with <10 Hz linewidth. The resulting performance in 19 F-MRI is demonstrated for six different peptide probes. Using fluorescence microscopy, these probes are found to exhibit high stability and long circulation times in living zebrafish embryos. Furthermore, the probes can be conjugated to bovine serum albumin with only amoderate increase in 19 F-NMR linewidth to ≈30 Hz. Overall, these peptide probes are hence suitable for in vivo 19 F-MRI applications., (© 2022 The Authors. Small published by Wiley-VCH GmbH.)

Primary cilia are key sensory organelles whose dysfunction leads to ciliopathy disorders such as Bardet-Biedl syndrome (BBS). Retinal degeneration is common in ciliopathies, since the outer segments (OSs) of photoreceptors are highly specialized primary cilia. BBS1, encoded by the most commonly mutated BBS-associated gene, is part of the BBSome protein complex. Using a bbs1 zebrafish mutant, we show that retinal development and photoreceptor differentiation are unaffected by Bbs1-loss, supported by an initially unaffected transcriptome. Quantitative proteomics and lipidomics on samples enriched for isolated OSs show that Bbs1 is required for BBSome-complex stability and that Bbs1-loss leads to accumulation of membrane-associated proteins in OSs, with enrichment in proteins involved in lipid homeostasis. Disruption of the tightly regulated OS lipid composition with increased OS cholesterol content are paralleled by early functional visual deficits, which precede progressive OS morphological anomalies. Our findings identify a role for Bbs1/BBSome in OS lipid homeostasis, suggesting a pathomechanism underlying retinal degeneration in BBS., (© 2022. The Author(s).)

Hearing impairment and deafness is frequently observed as one of the neurological signs in patients with Minamata disease caused by methylmercury (MeHg) poisoning. Loss of hair cells in humans and animals is a consequence of MeHg poisoning. However, it is still not clear how MeHg causes hearing deficits. We employed the hair cells of the lateral line system of zebrafish embryos as a model to explore this question. We exposed transgenic zebrafish embryos to MeHg (30-360 μg/L) at the different stages, and scored the numbers of hair cells. We find that MeHg-induced reduction of hair cells is in a concentration dependent manner. By employing antisense morpholino against to pu.1, we confirm that loss of hair cells involves the action of leukocytes. Moreover, hair cell loss is attenuated by co-treating MeHg-exposed embryos with pharmacological inhibitors of NADPH oxidases named diphenyleneiodonium (DPI) and VAS2870. In situ gene expression analysis showed that genes encoding the SQSTM1-Keap1-Nrf2 systems involved in combating oxidative stress and immune responses are highly expressed in the lateral line organs of embryos exposed to MeHg. This suggests that induction of hydrogen peroxide (H 2 O 2 ) is the primary effect of MeHg on the hair cells. Genes induced by MeHg are also involved in regeneration of the hair cells. These features are likely related to the capacity of the zebrafish to regenerate the lost hair cells., Competing Interests: Declaration of Competing Interest The authors declare no competing or financial interests., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In contrast to mammals, adult zebrafish display an extraordinary capacity to heal injuries and repair damage in the central nervous system. Pivotal for the regenerative capacity of the zebrafish brain at adult stages is the precise control of neural stem cell (NSC) behavior and the maintenance of the stem cell pool. The gene mdka , a member of a small family of heparin binding growth factors, was previously shown to be involved in regeneration in the zebrafish retina, heart, and fin. Here, we investigated the expression pattern of the gene mdka and its paralogue mdkb in the zebrafish adult telencephalon under constitutive and regenerative conditions. Our findings show that only mdka expression is specifically restricted to the telencephalic ventricle, a stem cell niche of the zebrafish telencephalon. In this brain region, mdka is particularly expressed in the quiescent stem cells. Interestingly, after brain injury, mdka expression remains restricted to the resting stem cell, which might suggest a role of mdka in regulating stem cell quiescence.

Direct control of protein interactions by chemically induced protein proximity holds great potential for both cell and synthetic biology as well as therapeutic applications. Low toxicity, orthogonality and excellent cell permeability are important criteria for chemical inducers of proximity (CIPs), in particular for in vivo applications. Here, we present the use of the agrochemical mandipropamid (Mandi) as a highly efficient CIP in cell culture systems and living organisms. Mandi specifically induces complex formation between a sixfold mutant of the plant hormone receptor pyrabactin resistance 1 (PYR1) and abscisic acid insensitive (ABI). It is orthogonal to other plant hormone-based CIPs and rapamycin-based CIP systems. We demonstrate the applicability of the Mandi system for rapid and efficient protein translocation in mammalian cells and zebrafish embryos, protein network shuttling and manipulation of endogenous proteins., (© 2021. The Author(s).)

The central nervous system of adult zebrafish displays an extraordinary neurogenic and regenerative capacity. In the zebrafish adult brain, this regenerative capacity relies on neural stem cells (NSCs) and the careful management of the NSC pool. However, the mechanisms controlling NSC pool maintenance are not yet fully understood. Recently, Bone Morphogenetic Proteins (BMPs) and their downstream effector Id1 (Inhibitor of differentiation 1) were suggested to act as key players in NSC maintenance under constitutive and regenerative conditions. Here, we further investigated the role of BMP/Id1 signaling in these processes, using different genetic and pharmacological approaches. Our data show that BMPs are mainly expressed by neurons in the adult telencephalon, while id1 is expressed in NSCs, suggesting a neuron-NSC communication via the BMP/Id1 signaling axis. Furthermore, manipulation of BMP signaling by conditionally inducing or repressing BMP signaling via heat-shock, lead to an increase or a decrease of id1 expression in the NSCs, respectively. Induction of id1 was followed by an increase in the number of quiescent NSCs, while knocking down id1 expression caused an increase in NSC proliferation. In agreement, genetic ablation of id1 function lead to increased proliferation of NSCs, followed by depletion of the stem cell pool with concomitant failure to heal injuries in repeatedly injured mutant telencephala. Moreover, pharmacological inhibition of BMP and Notch signaling suggests that the two signaling systems cooperate and converge onto the transcriptional regulator her4.1 . Interestingly, brain injury lead to a depletion of NSCs in animals lacking BMP/Id1 signaling despite an intact Notch pathway. Taken together, our data demonstrate how neurons feedback on NSC proliferation and that BMP1/Id1 signaling acts as a safeguard of the NSC pool under regenerative conditions.

Labeling biomolecules with fluorescent labels is an established tool for structural, biochemical, and biophysical studies; however, it remains underused for small peptides. In this work, an amino acid bearing a 3-hydroxychromone fluorophore, 2-amino-3-(2-(furan-2-yl)-3-hydroxy-4-oxo-4H-chromen-6-yl)propanoic acid (FHC), was incorporated in a known hexameric antimicrobial peptide, cyclo[RRRWFW] (cWFW), in place of aromatic residues. Circular dichroism spectropolarimetry and antibacterial activity measurements demonstrated that the FHC residue perturbs the peptide structure depending on labeling position but does not modify the activity of cWFW significantly. FHC thus can be considered an adequate label for studies of the parent peptide. Several analytical and imaging techniques were used to establish the activity of the obtained labeled cWFW analogues toward animal cells and to study the behavior of the peptides in a multicellular organism. The 3-hydroxychromone fluorophore can undergo excited-state intramolecular proton transfer (ESIPT), resulting in double-band emission from its two tautomeric forms. This feature allowed us to get insights into conformational equilibria of the labeled peptides, localize the cWFW analogues in human cells (HeLa and HEK293) and zebrafish embryos, and assess the polarity of the local environment around the label by confocal fluorescence microscopy. We found that the labeled peptides efficiently penetrated cancerous cells and localized mainly in lipid-containing and/or other nonpolar subcellular compartments. In the zebrafish embryo, the peptides remained in the bloodstream upon injection into the cardinal vein, presumably adhering to lipoproteins and/or microvesicles. They did not diffuse into any tissue to a significant extent during the first 3 h after administration. This study demonstrated the utility of fluorescent labeling by double-emission labels to evaluate biologically active peptides as potential drug candidates in vivo ., Competing Interests: The authors SK, AS, and IK, are employers of the company Enamine Co, Ltd. SA, OB, and IK are co-founders of the company Lumobiotics GmbH. The remaining authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Afonin, Koniev, Préau, Takamiya, Strizhak, Babii, Hrebonkin, Pivovarenko, Dathe, le Noble, Rastegar, Strähle, Ulrich and Komarov.)

Zebrafish is an attractive model to investigate regeneration of the nervous system. Despite major progress in our understanding of the underlying processes, the transcriptomic changes are largely unknown. We carried out a computational analysis of the transcriptome of the regenerating telencephalon integrating changes in the expression of mRNAs, their splice variants and investigated the putative role of regulatory RNAs in the modulation of these transcriptional changes. Profound changes in the expression of genes and their splice variants engaged in many distinct processes were observed. Differential transcription and splicing are important processes in response to injury of the telencephalon. As exemplified by the coordinated regulation of the cholesterol synthesizing enzymes and transporters, the genome responded to injury of the telencephalon in a multi-tiered manner with distinct and interwoven changes in expression of enzymes, transporters and their regulatory molecules. This coordinated genomic response involved a decrease of the mRNA of the key transcription factor SREBF2, induction of microRNAs ( miR-182 , miR-155 , miR-146 , miR-31 ) targeting cholesterol genes, shifts in abundance of splice variants as well as regulation of long non-coding RNAs. Cholesterol metabolism appears to be switched from synthesis to relocation of cholesterol. Based on our in silico analyses, this switch involves complementary and synergistic inputs by different regulatory principles. Our studies suggest that adaptation of cholesterol metabolism is a key process involved in regeneration of the injured zebrafish brain., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gourain, Armant, Lübke, Diotel, Rastegar and Strähle.)

The notochord defines the axial structure of all vertebrates during development. Notogenesis is a result of major cell reorganization in the mesoderm, the convergence and the extension of the axial cells. However, it is currently not fully understood how these processes act together in a coordinated way during notochord formation. The prechordal plate is an actively migrating cell population in the central mesoderm anterior to the trailing notochordal plate cells. We show that prechordal plate cells express Protocadherin 18a (Pcdh18a), a member of the cadherin superfamily. We find that Pcdh18a-mediated recycling of E-cadherin adhesion complexes transforms prechordal plate cells into a cohesive and fast migrating cell group. In turn, the prechordal plate cells subsequently instruct the trailing mesoderm. We simulated cell migration during early mesoderm formation using a lattice-based mathematical framework and predicted that the requirement for an anterior, local motile cell cluster could guide the intercalation and extension of the posterior, axial cells. Indeed, a grafting experiment validated the prediction and local Pcdh18a expression induced an ectopic prechordal plate-like cell group migrating towards the animal pole. Our findings indicate that the Pcdh18a is important for prechordal plate formation, which influences the trailing mesodermal cell sheet by orchestrating the morphogenesis of the notochord.

Kolmer-Agduhr (KA) cells are a subgroup of interneurons positioned adjacent to the neurocoele with cilia on the apical surface protruding into the central canal of the spinal cord. Although KA cells were identified almost a century ago, their development and functions are only beginning to be unfolded. Recent studies have revealed the characteristics of KA cells in greater detail, including their spatial distribution, the timing of their differentiation, and their specification via extrinsic signaling and a unique combination of transcription factors in zebrafish and mouse. Cell lineage-tracing experiments have demonstrated that two subsets of KA cells, named KA' and KA" cells, differentiate from motoneuronal progenitors and floor-plate precursors, respectively, in both zebrafish and mouse. Although KA' and KA" cells originate from different progenitors/precursors, they each share a common set of transcription factors. Intriguingly, the combination of transcription factors that promote the acquisition of KA' cell characteristics differs from those that promote a KA" cell identity. In addition, KA' and KA" cells exhibit separable neuronal targets and differential responses to bending of the spinal cord. In this review, we summarize what is currently known about the genetic programs defining the identities of KA' and KA" cell identities. We then discuss how these two subgroups of KA cells are genetically specified., (Copyright © 2020 Yang, Wang and Strähle.)

The glucose-sensing Mondo pathway regulates expression of metabolic genes in mammals. Here, we characterized its function in the zebrafish and revealed an unexpected role of this pathway in vertebrate embryonic development. We showed that knockdown of mondoa impaired the early morphogenetic movement of epiboly in zebrafish embryos and caused microtubule defects. Expression of genes in the terpenoid backbone and sterol biosynthesis pathways upstream of pregnenolone synthesis was coordinately downregulated in these embryos, including the most downregulated gene nsdhl . Loss of Nsdhl function likewise impaired epiboly, similar to MondoA loss of function. Both epiboly and microtubule defects were partially restored by pregnenolone treatment. Maternal-zygotic mutants of mondoa showed perturbed epiboly with low penetrance and compensatory changes in the expression of terpenoid/sterol/steroid metabolism genes. Collectively, our results show a novel role for MondoA in the regulation of early vertebrate development, connecting glucose, cholesterol and steroid hormone metabolism with early embryonic cell movements., Competing Interests: MW, AS, MT, JS, AK, JM, NK, US, GN, RM, TD No competing interests declared, BW BDW was an employee of Nestlé Health Sciences SA. CG CG was an employee of Nestlé Health Sciences SA. AP AP is an employee of Nestlé Health Sciences SA. FG FG was an employee of Nestlé Health Sciences SA. PG PG is an employee of Nestlé Health Sciences SA., (© 2020, Weger et al.)

In contrast to mammals, the adult zebrafish brain shows neurogenic activity in a multitude of niches present in almost all brain subdivisions. Irrespectively, constitutive neurogenesis in the adult zebrafish and mouse telencephalon share many similarities at the cellular and molecular level. However, upon injury during tissue repair, the situation is entirely different. In zebrafish, inflammation caused by traumatic brain injury or by induced neurodegeneration initiates specific and distinct neurogenic programs that, in combination with signaling pathways implicated in constitutive neurogenesis, quickly, and efficiently overcome the loss of neurons. In the mouse brain, injury-induced inflammation promotes gliosis leading to glial scar formation and inhibition of regeneration. A better understanding of the regenerative mechanisms occurring in the zebrafish brain could help to develop new therapies to combat the debilitating consequences of brain injury, stroke, and neurodegeneration. The aim of this review is to compare the properties of neural progenitors and the signaling pathways, which control adult neurogenesis and regeneration in the zebrafish and mammalian telencephalon., (Copyright © 2020 Diotel, Lübke, Strähle and Rastegar.)

The ubiquitin-proteasome system degrades ubiquitin-modified proteins to maintain protein homeostasis and to control signalling. Whole-genome sequencing of patients with severe deafness and early-onset cataracts as part of a neurological, sensorial and cutaneous novel syndrome identified a unique deep intronic homozygous variant in the PSMC3 gene, encoding the proteasome ATPase subunit Rpt5, which lead to the transcription of a cryptic exon. The proteasome content and activity in patient's fibroblasts was however unaffected. Nevertheless, patient's cells exhibited impaired protein homeostasis characterized by accumulation of ubiquitinated proteins suggesting severe proteotoxic stress. Indeed, the TCF11/Nrf1 transcriptional pathway allowing proteasome recovery after proteasome inhibition is permanently activated in the patient's fibroblasts. Upon chemical proteasome inhibition, this pathway was however impaired in patient's cells, which were unable to compensate for proteotoxic stress although a higher proteasome content and activity. Zebrafish modelling for knockout in PSMC3 remarkably reproduced the human phenotype with inner ear development anomalies as well as cataracts, suggesting that Rpt5 plays a major role in inner ear, lens and central nervous system development., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

In the telencephalon of adult zebrafish, the inhibitor of DNA binding 1 (id1) gene is expressed in radial glial cells (RGCs), behaving as neural stem cells (NSCs), during constitutive and regenerative neurogenesis. Id1 controls the balance between resting and proliferating states of RGCs by promoting quiescence. Here, we identified a phylogenetically conserved cis-regulatory module (CRM) mediating the specific expression of id1 in RGCs. Systematic deletion mapping and mutation of conserved transcription factor binding sites in stable transgenic zebrafish lines reveal that this CRM operates via conserved smad1/5 and 4 binding motifs under both homeostatic and regenerative conditions. Transcriptome analysis of injured and uninjured telencephala as well as pharmacological inhibition experiments identify a crucial role of bone morphogenetic protein (BMP) signaling for the function of the CRM. Our data highlight that BMP signals control id1 expression and thus NSC proliferation during constitutive and induced neurogenesis., (©2020 The Authors. Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press 2020.)

Cranial neural crest (NC) contributes to the developing vertebrate eye. By multidimensional, quantitative imaging, we traced the origin of the ocular NC cells to two distinct NC populations that differ in the maintenance of sox10 expression, Wnt signalling, origin, route, mode and destination of migration. The first NC population migrates to the proximal and the second NC cell group populates the distal (anterior) part of the eye. By analysing zebrafish pax6a/b compound mutants presenting anterior segment dysgenesis, we demonstrate that Pax6a/b guide the two NC populations to distinct proximodistal locations. We further provide evidence that the lens whose formation is pax6a/b-dependent and lens-derived TGFβ signals contribute to the building of the anterior segment. Taken together, our results reveal multiple roles of Pax6a/b in the control of NC cells during development of the anterior segment., Competing Interests: The authors have declared that no competing interests exist.

Promoting the regeneration or survival of retinal ganglion cells (RGCs) is one focus of regenerative medicine. Homeobox Barhl transcription factors might be instrumental in these processes. In mammals, only barhl2 is expressed in the retina and is required for both subtype identity acquisition of amacrine cells and for the survival of RGCs downstream of Atoh7, a transcription factor necessary for RGC genesis. The underlying mechanisms of this dual role of Barhl2 in mammals have remained elusive. Whole genome duplication in the teleost lineage generated the barhl1a and barhl2 paralogues. In the Zebrafish retina, Barhl2 functions as a determinant of subsets of amacrine cells lineally related to RGCs independently of Atoh7. In contrast, barhl1a expression depends on Atoh7 but its expression dynamics and function have not been studied. Here we describe for the first time a Barhl1a reporter line in vivo showing that barhl1a turns on exclusively in subsets of RGCs and their post-mitotic precursors. We also show transient expression of barhl1a:GFP in diencephalic neurons extending their axonal projections as part of the post-optic commissure, at the time of optic chiasm formation. This work sets the ground for future studies on RGC subtype identity, axonal projections and genetic specification of Barhl1a-positive RGCs and commissural neurons.

Minamata disease in Japan and the large-scale poisoning by methylmercury (MeHg) in Iraq caused wide public concerns about the risk emanating from mercury for human health. Nowadays, it is widely known that all forms of mercury induce toxic effects in mammals, and increasing evidence supports the concern that environmentally relevant levels of MeHg could impact normal biological functions in wildlife. The information of mechanism involved in mercurial toxicity is growing but knowledge gaps still exist between the adverse effects and mechanisms of action, especially at the molecular level. A body of data obtained from experimental studies on mechanisms of mercurial toxicity in vivo and in vitro points to that disruption of the antioxidant system may play an important role in the mercurial toxic effects. Moreover, the accumulating evidence indicates that signaling transduction, protein or/and enzyme activity, and gene regulation are involving in mediating toxic and adaptive response to mercury exposure. We conducted here a comprehensive review of mercurial toxic effects on wildlife and human, in particular synthesized key findings of molecular pathways involved in mercurial toxicity from the cells to human. We discuss the molecular evidence related mercurial toxicity to the adverse effects, with particular emphasis on the gene regulation. The further studies relying on Omic analysis connected to adverse effects and modes of action of mercury will aid in the evaluation and validation of causative relationship between health outcomes and gene expression., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Despite the common knowledge that the reticuloendothelial system is largely responsible for blood clearance of systemically administered nanoparticles, the sequestration mechanism remains a "black box". Using transgenic zebrafish embryos with cell type-specific fluorescent reporters and fluorescently labeled model nanoparticles (70 nm SiO 2 ), we here demonstrate simultaneous three-color in vivo imaging of intravenously injected nanoparticles, macrophages, and scavenger endothelial cells (SECs). The trafficking processes were further revealed at ultrastructural resolution by transmission electron microscopy. We also find, using a correlative light-electron microscopy approach, that macrophages rapidly sequester nanoparticles via membrane adhesion and endocytosis (including macropinocytosis) within minutes after injection. In contrast, SECs trap single nanoparticles via scavenger receptor-mediated endocytosis, resulting in gradual sequestration with a time scale of hours. Inhibition of the scavenger receptors prevented SECs from accumulating nanoparticles but enhanced uptake in macrophages, indicating the competitive nature of nanoparticle clearance in vivo . To directly quantify the relative contributions of the two cell types to overall nanoparticle sequestration, the differential sequestration kinetics was studied within the first 30 min post-injection. This revealed a much higher and increasing relative contribution of SECs, as they by far outnumber macrophages in zebrafish embryos, suggesting the importance of the macrophage:SECs ratio in a given tissue. Further characterizing macrophages on their efficiency in nanoparticle clearance, we show that inflammatory stimuli diminish the uptake of nanoparticles per cell. Our study demonstrates the strength of transgenic zebrafish embryos for intravital real-time and ultrastructural imaging of nanomaterials that may provide mechanistic insights into nanoparticle clearance in rodent models and humans.

This study evaluates the embryotoxicity of dithienylethene-modified peptides upon photoswitching, using 19 analogues based on the β-hairpin scaffold of the natural membranolytic peptide gramicidin S. We established an in vivo assay in two variations (with ex vivo and in situ photoisomerization), using larvae of the model organism Danio rerio, and determined the toxicities of the peptides in terms of 50% lethal doses (LD 50 ). This study allowed us to: (i) demonstrate the feasibility of evaluating peptide toxicity with D. rerio larvae at 3-4 days post fertilization, (ii) determine the phototherapeutic safety windows for all peptides, (iii) demonstrate photoswitching of the whole-body toxicity for the dithienylethene-modified peptides in vivo, (iv) re-analyze previous structure-toxicity relationship data, and (v) select promising candidates for potential clinical development., (Copyright © 2020, Afonin et al.; licensee Beilstein-Institut.)

Otospiralin (OTOSP) is a small protein of unknown function, expressed in fibrocytes of the inner ear and required for normal cochlear auditory function. Despite its conservation from fish to mammals, expression of otospiralin was only investigated in mammals. Here, we report for the first time the expression profile of OTOS orthologous genes in zebrafish (Danio rerio): otospiralin and si:ch73-23l24.1 (designated otospiralin-like). In situ hybridization analyses in zebrafish embryos showed a specific expression of otospiralin-like in notochord (from 14 to 48 hpf) and similar expression patterns for otospiralin and otospiralin-like in gut (from 72 to 120 hpf), swim bladder (from 96 to 120 hpf) and inner ear (at 120 hpf). Morpholino knockdown of otospiralin and otospiralin-like showed no strong change of the body structure of the embryos at 5 dpf and the inner ear was normally formed. Nevertheless, knockdown embryos showed a reduced number of kinocilia in the lateral crista, indicating that these genes play an important role in kinocilium formation. RT-qPCR revealed that otospiralin is highly expressed in adult zebrafish inner ear comparing to the others analyzed tissues as previously shown for mice. Interestingly, otospiralin-like was not detected in the inner ear which suggests that otospiralin have a more important function in hearing than otospiralin-like. Phylogenetic analysis of otospiralin proteins in vertebrates indicated the presence of two subgroups and supported the functional divergence observed in zebrafish for otospiralin and otospiralin-like genes. This study offers the first insight into the expression of otospiralin and otospiralin-like in zebrafish. Expression data point to an important role for otospiralin in zebrafish hearing and a specific role for otospiralin-like in notochord vacuolization.

Three promising antibacterial peptides were studied with regard to their ability to inhibit the growth and kill the cells of clinical strains of Staphylococcus aureus, Enterococcus faecalis and Enterococcus faecium. The multifunctional gramicidin S (GS) was the most potent, compared to the membranotropic temporin L (TL), being more effective than the innate-defence regulator IDR-1018 (IDR). These activities, compared across 16 strains as minimal bactericidal and minimal inhibitory concentrations (MIC), are independent of bacterial resistance pattern, phenotype variations and/or biofilm-forming potency. For S. aureus strains, complete killing is accomplished by all peptides at 5 × MIC. For E. faecalis strains, only GS exhibits a rapid bactericidal effect at 5 × MIC, while TL and IDR require higher concentrations. The biofilm-preventing activities of all peptides against the six strains with the largest biofilm biomass were compared. GS demonstrates the lowest minimal biofilm inhibiting concentrations, whereas TL and IDR are consistently less effective. In mature biofilms, only GS completely kills the cells of all studied strains. We compare the physicochemical properties, membranolytic activities, model pharmacokinetics and eukaryotic toxicities of the peptides and explain the bactericidal, antipersister and antibiofilm activities of GS by its elevated stability, pronounced cell-penetration ability and effective utilization of multiple modes of antibacterial action.

Thioredoxin is an evolutionarily conserved antioxidant protein that plays a crucial role for fundamental cellular processes and embryonic development. Growing evidence support that Thioredoxin influences cellular response to chemicals insults, particularly those accompanying oxidative stress. The mechanisms underlying the functions of Thioredoxin1 in the embryonic development under the environmental toxicant exposure remain, however, largely unexplored. We report here that thioredoxin1 becomes differentially expressed in zebrafish embryos after exposure to 9 out of 11 environmental chemicals. In situ gene expression analysis show that thioredoxin1 is expressed in neurons, olfactory epithelia, liver and swim bladder under normal conditions. After MeHg exposure, however, thioredoxin1 is ectopically induced in the hair cells of the lateral line and in epithelia cells of the pharynx. Knockdown of Thioredoxin1 induces hydrocephalus and increases cell apoptosis in the brain ventricular epithelia cells. In comparison with 5% malformation in embryos injected with control morpholino, MeHg induces more than 77% defects in Thioredoxin1 knockdown embryos. Our data suggest that there is an association between hydrocephalus and Thioredoxin1 malfunction in embryonic development, and provide valuable information to elucidate the protective role of Thioredoxin1 against chemicals disruption., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Adiponectin and its receptors (adipor) have been initially characterized for their role in lipid and glucose metabolism. More recently, adiponectin signaling was shown to display anti-inflammatory effects and to participate in brain homeostasis and neuroprotection. In this study, we investigated adipor gene expression and its regulation under inflammatory conditions in two complementary models: mouse and zebrafish. We demonstrate that adipor1a, adipor1b, and adipor2 are widely distributed throughout the brain of adult fish, in neurons and also in radial glia, behaving as neural stem cells. We also show that telencephalic injury results in a decrease in adipor gene expression, inhibited by an anti-inflammatory treatment (Dexamethasone). Interestingly, adiponectin injection after brain injury led to a consistent decrease (a) in the recruitment of microglial cells at the lesioned site and (b) in the proliferation of neural progenitors, arguing for a neuroprotective role of adiponectin. In a comparative approach, we investigate Adipor1 and Adipor2 gene distribution in the brain of mice and demonstrated their expression in regions shared with fish including neurogenic regions. We also document Adipor gene expression in mice after middle cerebral artery occlusion and lipopolysaccharide injection. In contrast to zebrafish, these inflammatory stimuli do no impact cerebral adiponectin receptor gene expression in mouse. This work provides new insights regarding adipor expression in the brain of fish, and demonstrates evolutionary conserved distribution of adipor with mouse. This is the first report of adipor expression in adult neural stem cells of fish, suggesting a potential role of adiponectin signaling during vertebrate neurogenesis. It also suggests a potential contribution of inflammation in the regulation of adipor in fish., (© 2019 Wiley Periodicals, Inc.)

We report the synthesis of poly(2-ethyl-2-oxazoline)-based (PEtOx) nanoobjects by polymerization-induced self-assembly (PISA). First, well-defined PEtOx macromolecular chain transfer agents were synthesized by cationic ring-opening polymerization and click chemistry. The photoinitiated PISA of 2-hydroxypropyl methacrylate mediated by these PEtOx produced nanoobjects spanning the full range of core-shell morphologies. The nanoparticles exhibited high biocompatibility and stealth properties in vitro or in vivo, as well as thermoresponsive behavior.

Specification of neurons in the spinal cord relies on extrinsic and intrinsic signals, which in turn are interpreted by expression of transcription factors. V2 interneurons develop from the ventral aspects of the spinal cord. We report here a novel neuronal V2 subtype, named V2s, in zebrafish embryos. Formation of these neurons depends on the transcription factors sox1a and sox1b. They develop from common gata2a - and gata3 -dependent precursors co-expressing markers of V2b and V2s interneurons. Chemical blockage of Notch signalling causes a decrease in V2s and an increase in V2b cells. Our results are consistent with the existence of at least two types of precursor arranged in a hierarchical manner in the V2 domain. V2s neurons grow long ipsilateral descending axonal projections with a short branch at the ventral midline. They acquire a glycinergic neurotransmitter type during the second day of development. Unilateral ablation of V2s interneurons causes a delay in touch-provoked escape behaviour, suggesting that V2s interneurons are involved in fast motor responses., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Silica nanoparticles are widely used platform materials for the immobilization of proteins to realize applications in biomedicine and biotechnology. We here report on the use of a highly delicate protein for the systematic evaluation of routes for the surface modification of multifunctional silica nanoparticles. To investigate how surface immobilization methods affect the functionality of surface-bound proteins, we constructed a novel fusion protein, dubbed FlipHOB, that combines the glucose sensor protein FLIP with a variant of the commercially-available self-ligating Halo-tag. As indicated by the spectroscopic properties and sensing capabilities of FlipHOB, the oriented immobilization of this protein through its HOB tag domain or DNA-directed immobilization were superior over the non-directional statistical immobilization via glutardialdehyde-mediated cross-coupling. Immobilization through double-stranded DNA bridges also allows for the triggered disassembly of FlipHOB nanosensors and the controlled recovery of the sensor protein. We demonstrate that the nanosensors are functional in in vitro settings and can be used for imaging in vivo. We believe that our results show generic strategies and provide essential guidelines for the development of protein-based nanoparticle sensors for applications in the life sciences., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In zebrafish, the gene choline acetyltransferase a (chata) encodes one of the two ChAT orthologs responsible for the synthesis of acetylcholine. Acetylcholine (ACh) is essential for neuromuscular transmission and its impaired synthesis by ChAT can lead to neuromuscular junction disorders such as congenital myasthenic syndromes in humans. We have identified a novel mutation in the chata gene of zebrafish, chatatk64, in a collection of uncharacterised ENU-induced mutants. This mutant carries a missense mutation in the codon of a highly conserved serine changing it to an arginine (S102R). This serine is conserved among ChATs from zebrafish, rat, mice and chicken to humans. It resides within the catalytic domain and in the vicinity of the active site of the enzyme. However, it has not been reported so far to be required for enzymatic activity. Modelling of the S102R variant change in the ChAT protein crystal structure suggests that the change affects protein structure and has a direct impact on the catalytic domain of the protein which abolishes embryo motility almost completely., Competing Interests: The authors have declared that no competing interests exist.

The knowledge on environmentally relevant chemicals that may interfere with thyroid signaling is scarce. Here, we present a method for the screening of goitrogens, compounds that disrupt the thyroid gland function, based on the automatic orientation of zebrafish in a glass capillary and a subsequent imaging of reporter gene fluorescence in the thyroid gland of embryos of the transgenic zebrafish line tg(tg:mCherry). The tg(tg:mCherry) reporter gene indicates a compensatory upregulation of thyroglobulin, the thyroid hormone precursor, in response to inhibition of thyroid hormone synthesis. Fish embryos were exposed to a negative control compound (3,4-dichloroaniline), or a concentration series of known goitrogenic compounds (resorcinol, methimazole, potassium perchlorate, 6-propyl-2-thiouracil, ethylenethiourea, phloroglucinol, pyrazole) with maximum exposure concentration selected based on mortality and/or solubility. Exposure to 3,4-dichloroaniline decreased the fluorescence signal. All goitrogenic compounds exhibited clear concentration-dependent inductions of reporter fluorescence 1.4 to 2.6 fold above control levels. Concentration-response modelling was used to calculate goitrogenic potencies based on EC50 values. The new automated method offers an efficient screening approach for goitrogenic activity., Competing Interests: The authors have declared that no competing interests exist.