Search

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., The agrochemical mandipropamid (Mandi), which induces dimerization of a variant of the abscisic acid receptor, has been developed as a new chemical inducer of proximity for cellular and organismal applications.

SummaryPrimary 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 new 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 isolated OSs show that Bbs1 is required for BBSome-entry into OSs 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 new role for Bbs1/BBSome in OS lipid homeostasis and suggest a new pathomechanism underlying retinal degeneration in BBS.

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.

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.

The mechanisms of the management of the neural stem cell (NSC) pool underlying the regenerative capacity of the adult zebrafish brain are not understood. We show that Bone Morphogenetic Proteins (BMPs) which are exclusively expressed by neurons in the adult telencephalon and the helix-loop-helix (HLH) transcription co-regulator, Inhibitor of differentiation 1 (Id1), control quiescence of NSCs. Upon injury, lack ofid1function leads to an initial over-proliferation and subsequent loss of NSCs and the regenerative capacity. BMP/Id1 signaling up-regulates the transcription factorher4.1which is also a target of Notch signaling mediating short-range control of NSC quiescence. Hence, the two signaling systems converge onto Her4.1. Our data show that neurons feedback on NSC proliferation. BMP1/Id1 signaling appears as the predominant safeguard of the NSC pool under regenerative conditions while Notch signaling is sufficient to maintain NSCs under homeostatic baseline neurogenesis in the uninjured animal.

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.

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.

International audience; 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, injuryinduced 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.

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.

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 (SBMs) 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.

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 (LD50). 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.

The increasing importance of zebrafish as a biomedical model organism is reflected by the steadily growing number of publications and laboratories working with this species. Regulatory recommendations for euthanasia as issued in Directive 2010/63/EU are, however, based on experience with fish species used for food production and do not take the small size and specific physiology of zebrafish into account. Consequently, the currently recommended methods of euthanasia in the Directive 2010/63/EU are either not applicable or may interfere with research goals. An international workshop was held in Karlsruhe, Germany, March 9, 2017, to discuss and propose alternative methods for euthanasia of zebrafish. The aim was to identify methods that adequately address the physiology of zebrafish and its use as a biomedical research model, follow the principles of the 3Rs (Replacement, Reduction, and Refinement) in animal experimentation and consider animal welfare during anesthesia and euthanasia. The results of the workshop are summarized here in the form of a white paper.

Biomolecule decoration of nanoparticles provides a corona that modulates how the nanoparticles interact with biological milieus. The corona composition has proved to reflect the differences in the repertoire of proteins to which the nanoparticles are exposed, and as a result the same nanoparticles can acquire a differential biological identity. Here we examined whether a unique biological identity acquired from sex-specific protein repertoires could alter the degree of nanoparticle uptake by cognate immune cells. We chose zebrafish as a model species of which blood plasma is sexually contrasted by the unique presence/absence of the egg yolk precursor protein vitellogenin. Sex-specific protein coronas were thus formed around 70 nm SiO2 nanoparticles using female/male blood plasma from zebrafish or fetal bovine serum as a non-native reference. In contrast to protein coronas formed of male blood plasma, a “female” biological identity of the nanoparticles was represented by prevailing contribution of vitellogenins to the corona proteome. We then exposed zebrafish blood cells to the three types of pre-formed nanoparticle–protein complexes and compared nanoparticle uptake using flow cytometry. Lymphoid and myeloid populations of the blood cells preferentially accumulated the nanoparticles with a female biological identity, irrespective of the sex of the fish from which the cells were obtained. The concept of repertoire differences in the corona proteome therefore deserves further attention, as various factors such as sex-specific biological conditions exemplified in this study could alter the nanoparticle–cell interactions.

Whole-genome sequencing was performed on patients with severe deafness and early-onset cataracts as part of a neurological, sensorial and cutaneous novel syndrome. A unique deep intronic homozygous variant in thePSMC3gene (c.1127+337A>G, p.Ser376Argfs15*), encoding the 26S proteasome ATPase ring subunit 5 (Rpt5) was identified leading to the transcription of a cryptic exon. Patient’s fibroblasts 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 is however impaired. These cells were unable to compensate for proteotoxic stress although a higher proteasome content. Two different zebrafish studies led to inner ear development anomalies as well as cataracts. PSMC3 proteasome subunit dysfunction leads to various neurological manifestations, early onset cataracts and deafness and suggest that Rpt5 plays a major role in inner ear, lens and central nervous system development.

Polydactyly is one of the most frequent inherited defects of the limbs characterized by supernumerary digits and high-genetic heterogeneity. Among the many genes involved, either in isolated or syndromic forms, eight have been implicated in postaxial polydactyly (PAP). Among those, IQCE has been recently identified in a single consanguineous family. Using whole-exome sequencing in patients with uncharacterized ciliopathies, including PAP, we identified three families with biallelic pathogenic variations in IQCE. Interestingly, the c.895_904del (p.Val301Serfs*8) was found in all families without sharing a common haplotype, suggesting a recurrent mechanism. Moreover, in two families, the systemic phenotype could be explained by additional pathogenic variants in known genes (TULP1, ATP6V1B1). RNA expression analysis on patients' fibroblasts confirms that the dysfunction of IQCE leads to the dysregulation of genes associated with the hedgehog-signaling pathway, and zebrafish experiments demonstrate a full spectrum of phenotypes linked to defective cilia: Body curvature, kidney cysts, left-right asymmetry, misdirected cilia in the pronephric duct, and retinal defects. In conclusion, we identified three additional families confirming IQCE as a nonsyndromic PAP gene. Our data emphasize the importance of taking into account the complete set of variations of each individual, as each clinical presentation could finally be explained by multiple genes.

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.

International audience; 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.

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/gata3 dependent precursors co-expressing markers of V2b and V2s interneurons. Chemical blockage of Notch signaling causes a decrease of V2s and an increase of V2b cells. Our results are consistent with the existence of at least two types of precursors 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 behavior suggesting that V2s interneurons are involved in fast motor responses.

The European Zebrafish Principal Investigator Meeting (EZPM) is an ideal forum for group leaders using this fantastic animal model not only to discuss science but also to strengthen their interactions, to push forward technological advances, and to define guidelines for the use of this fish in research. The city of Lisbon (Portugal) was voted by the European group leaders to be the setting for the 4th EZPM, and the organizing committee, composed by Leonor Saude (iMM Lisboa, PT), Susana Lopes (CEDOC, PT), Michael Orger (Champalimaud Foundation, PT), Rui Oliveira (ISPA, PT), and Antonio Jacinto (CEDOC, PT), was very enthusiastic to organize a productive event. The 4th EZPM took place from March 15 to 19 at PavilhAo do Conhecimento, a Science Museum and Educational Center winner of The Great Prize FAD of Arquitecture 1999 and The Society for Environmental Graphic Design Award 2011. Over 5 days, 135 group leaders (89 men and 46 women) coming from 19 different European countries and also from the United States, Turkey, Israel, Chile, and Singapore presented and discussed their recent research achievements. In addition to the scientific oral and poster presentations, the group leaders gathered in very lively community sessions on morphants versus mutants (chaired by Didier Stainier, Max Planck Institute for Heart and Lung Research, DE), funding issues (chaired by Uwe Strahle, KIT-ITG, DE), and gender equality (chaired by Corinne Houart, KCL, United Kingdom). One of the highlights of the 4th EZPM was the guided visit to Oceanario de Lisboa, an international award-winning place that celebrates life with a stunning display of living aquatic creatures.

Summary: Gene therapeutic approaches to cure genetic diseases require tools to express the rescuing gene exclusively within the affected tissues. Viruses are often chosen as gene transfer vehicles but they have limited capacity for genetic information to be carried and transduced. In addition, to avoid off‐target effects the therapeutic gene should be driven by a tissue‐specific promoter in order to ensure expression in the target organs, tissues, or cell populations. The larger the promoter, the less space will be left for the respective gene. Thus, there is a need for small but tissue‐specific promoters. Here, we describe a compact unc45b promoter fragment of 195 bp that retains the ability to drive gene expression exclusively in skeletal and cardiac muscle in zebrafish and mouse. Remarkably, the described unc45b promoter fragment not only drives muscle‐specific expression but presents heat‐shock inducibility, allowing a temporal and spatial quantity control of (trans)gene expression. Here, we demonstrate that the transgenic expression of the smyd1b gene driven by the unc45b promoter fragment is able to rescue the embryonically lethal heart and skeletal muscle defects in smyd1b‐deficient flatline mutant zebrafish. Our findings demonstrate that the described muscle‐specific unc45b promoter fragment might be a valuable tool for the development of genetic therapies in patients suffering from myopathies. genesis 54:431–438, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.

In our previous work, we established an in vitro variant of the currently developed in vivo PIG-A assay as promising mutagenicity test system. We applied the human B-lymphoblastoid cell line TK6 for the in vitro assay development, which is based on the cellular glycosylphosphatidylinositol (GPI) status. At least 22 genes are involved in GPI biosynthesis, leading to the complex situation that, in principle, multiple genes could induce a GPI-deficient phenotype by acquiring inactivating mutations. However, only the PIG-A gene is located on the X-chromosome, rendering PIG-A more sensitive compared to autosomal linked, GPI-relevant genes. In this work, we investigated the GPI-related genotype-to-phenotype relationship in TK6 cells. By a next-generation sequencing approach, we identified a heterozygous chromosomal deletion on chromosome 17, where the PIG-L gene is located. In the analyzed TK6 cell clones, the GPI-deficient phenotype was induced either by mutations in PIG-A, by the complete absence of PIG-A mRNA, or by deletions in the remaining functional PIG-L gene, causing loss of heterozygosity. The identified PIG-L heterozygosity could also be responsible for the increased sensitivity toward mutagenic ethyl methanesulfonate or UV-C treatments of p53-proficient TK6 compared to the TK6-related, but p53-deficient WI-L2-NS cell line. Moreover, the WI-L2-NS cell line was found to exhibit a much lower number of GPI-deficient mutant cells in the purchased cell batch, and WI-L2-NS exerted a lower spontaneous rate of GPI deficiency compared to TK6 cells.

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.

In the originally published article, the first names and family names of the authors were interchanged, hence not correct. The correct presentation of names is presented above.

General anesthetics are small molecules that interact with and effect the function of many different proteins to promote loss of consciousness, amnesia, and sometimes, analgesia. Owing to the complexity of this state transition and the transient nature of these drug/protein interactions, anesthetics can be difficult to study. The zebrafish is an emerging model for the discovery of both new genes required for the response to and side effects of anesthesia. Here we discuss the tools available to manipulate the zebrafish genome, including both genetic screens and genome engineering approaches. Additionally, there are various robust behavior assays available to study anesthetic and other drug responses. These assays are available for single-gene study or high throughput for genetic or drug discovery. Finally, we present a case study of using propofol as an anesthetic in the zebrafish. These techniques and protocols make the zebrafish a powerful model to study anesthetic mechanisms and drug discovery.

Transcriptomics is often used to investigate changes in an organism's genetic response to environmental contamination. Data noise can mask the effects of contaminants making it difficult to detect responding genes. Because the number of genes which are found differentially expressed in transcriptome data is often very large, algorithms are needed to reduce the number down to a few robust discriminative genes. We present an algorithm for aggregated analysis of transcriptome data which uses multiple fold-change thresholds (threshold screening) and p values from Bayesian generalized linear model in order to assess the robustness of a gene as a potential indicator for the treatments tested. The algorithm provides a robustness indicator (ROBI) as well as a significance profile, which can be used to assess the statistical significance of a given gene for different fold-change thresholds. Using ROBI, eight discriminative genes were identified from an exemplary dataset (Danio rerio FET treated with chlorpyrifos, methylmercury, and PCB) which could be potential indicators for a given substance. Significance profiles uncovered genetic effects and revealed appropriate fold-change thresholds for single genes or gene clusters. Fold-change threshold screening is a powerful tool for dimensionality reduction and feature selection in transcriptome data, as it effectively reduces the number of detected genes suitable for environmental monitoring. In addition, it is able to unmask patterns in altered genetic expression hidden by data noise and reduces the chance of type II errors, e.g., in environmental screening.

Protein trafficking in and out of the nucleus represents a key step in controlling cell fate and function. Here we report the development of a red light-inducible and far-red light-reversible synthetic system for controlling nuclear localization of proteins in mammalian cells and zebrafish. First, we synthetically reconstructed and validated the red light-dependent Arabidopsis phytochrome B nuclear import mediated by phytochrome-interacting factor 3 in a nonplant environment and support current hypotheses on the import mechanism in planta. On the basis of this principle we next regulated nuclear import and activity of target proteins by the spatiotemporal projection of light patterns. A synthetic transcription factor was translocated into the nucleus of mammalian cells and zebrafish to drive transgene expression. These data demonstrate the first in vivo application of a plant phytochrome-based optogenetic tool in vertebrates and expand the repertoire of available light-regulated molecular devices.

The teleost brain has the remarkable ability to generate new neurons and to repair injuries during adult life stages. Maintaining life-long neurogenesis requires careful management of neural stem cell pools. In a genome-wide expression screen for transcription regulators, the id1 gene, encoding a negative regulator of E-proteins, was found to be upregulated in response to injury. id1 expression was mapped to quiescent type I neural stem cells in the adult telencephalic stem cell niche. Gain and loss of id1 function in vivo demonstrated that Id1 promotes stem cell quiescence. The increased id1 expression observed in neural stem cells in response to injury appeared independent of inflammatory signals, suggesting multiple antagonistic pathways in the regulation of reactive neurogenesis. Together, we propose that Id1 acts to maintain the neural stem cell pool by counteracting neurogenesis-promoting signals. Stem Cells 2015;33:892–903

The zebrafish has become a model to study adult vertebrate neurogenesis. In particular, the adult telencephalon has been an intensely studied structure in the zebrafish brain. Differential expression of transcriptional regulators (TRs) is a key feature of development and tissue homeostasis. Here we report an expression map of 1,202 TR genes in the telencephalon of adult zebrafish. Our results are summarized in a database with search and clustering functions to identify genes expressed in particular regions of the telencephalon. We classified 562 genes into 13 distinct patterns, including genes expressed in the proliferative zone. The remaining 640 genes displayed unique and complex patterns of expression and could thus not be grouped into distinct classes. The neurogenic ventricular regions express overlapping but distinct sets of TR genes, suggesting regional differences in the neurogenic niches in the telencephalon. In summary, the small telencephalon of the zebrafish shows a remarkable complexity in TR gene expression. The adult zebrafish telencephalon has become a model to study neurogenesis. We established the expression pattern of more than 1200 transcription regulators (TR) in the adult telencephalon. The neurogenic regions express overlapping but distinct sets of TR genes suggesting regional differences in the neurogenic potential. J. Comp. Neurol. 523:1202–1221, 2015. © 2015 Wiley Periodicals, Inc.

Sarcomeric protein turnover needs to be tightly balanced to assure proper assembly and renewal of sarcomeric units within muscle tissues. The mechanisms regulating these fundamental processes are only poorly understood, but of great clinical importance since many cardiac and skeletal muscle diseases are associated with defective sarcomeric organization. The SET- and MYND domain containing protein 1b (Smyd1b) is known to play a crucial role in myofibrillogenesis by functionally interacting with the myosin chaperones Unc45b and Hsp90α1. In zebrafish, Smyd1b, Unc45b and Hsp90α1 are part of the misfolded myosin response (MMR), a regulatory transcriptional response that is activated by disturbed myosin homeostasis. Genome duplication in zebrafish led to a second smyd1 gene, termed smyd1a. Morpholino- and CRISPR/Cas9-mediated knockdown of smyd1a led to significant perturbations in sarcomere structure resulting in decreased cardiac as well as skeletal muscle function. Similar to Smyd1b, we found Smyd1a to localize to the sarcomeric M-band in skeletal and cardiac muscles. Overexpression of smyd1a efficiently compensated for the loss of Smyd1b in flatline (fla) mutant zebrafish embryos, rescued the myopathic phenotype and suppressed the MMR in Smyd1b-deficient embryos, suggesting overlapping functions of both Smyd1 paralogs. Interestingly, Smyd1a is not transcriptionally activated in Smyd1b-deficient fla mutants, demonstrating lack of genetic compensation despite the functional redundancy of both zebrafish Smyd1 paralogs.

A bottleneck in CRISPR/Cas9 genome editing is variable efficiencies of in silico-designed gRNAs. We evaluated the sensitivity of the TIDE method (Tracking of Indels by DEcomposition) introduced by Brinkman et al. in 2014 for assessing the cutting efficiencies of gRNAs in zebrafish. We show that this simple method, which involves bulk polymerase chain reaction amplification and Sanger sequencing, is highly effective in tracking well-performing gRNAs in pools of genomic DNA derived from injected embryos. The method is equally effective for tracing INDELs in heterozygotes.

Motivation: To reliably assess the effects of unknown chemicals on the development of fluorescently labeled sensory-, moto- and interneuron populations in the spinal cord of zebrafish, automated data analysis is essential. Results: For the evaluation of a high-throughput screen of a large chemical library, we developed a new method for the automated extraction of quantitative information from green fluorescent protein (eGFP) and red fluorescent protein (RFP) labeled spinal cord neurons in double-transgenic zebrafish embryos. The methodology comprises region of interest detection, intensity profiling with reference comparison and neuron distribution histograms. All methods were validated on a manually evaluated pilot study using a Notch inhibitor dose-response experiment. The automated evaluation showed superior performance to manual investigation regarding time consumption, information detail and reproducibility. Availability and implementation: Being part of GNU General Public Licence (GNU-GPL) licensed open-source MATLAB toolbox Gait-CAD, an implementation of the presented methods is publicly available for download at http://sourceforge.net/projects/zebrafishimage/. Contact: johannes.stegmaier@kit.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Methyl mercury (MeHg) is a neurotoxicant with adverse effects on the development of the nervous system from fish to man. Despite a detailed understanding of the molecular mechanisms by which MeHg affects cellular homeostasis, it is still not clear how MeHg causes developmental neurotoxicity. We performed here a genome-wide transcriptional analysis of MeHg-exposed zebrafish embryos and combined this with a whole-mount in situ expression analysis of 88 MeHg-affected genes. The majority of the analyzed genes showed tissue- and region-restricted responses in various organs and tissues. The genes were linked to gene ontology terms like oxidative stress, transport and cell protection. Areas even within the central nervous system (CNS) are affected differently resulting in distinct cellular stress responses. Our study revealed an unexpected heterogeneity in gene responses to MeHg exposure in different tissues and neuronal subregions, even though the known molecular action of MeHg would predict a similar burden of exposed cells. The overall structure of the developing brain of MeHg-exposed embryos appeared normal, suggesting that the mechanism leading to differentiation of the CNS is not overtly affected by exposure to MeHg. We propose that MeHg disturbs the function of the CNS by disturbing the cellular homeostasis. As these cellular stress responses comprise genes that are also involved in normal neuronal activity and learning, MeHg may affect the developing CNS in a subtle manner that manifests itself in behavioral deficits.

The use of animals in scientific research in Europe is governed by the DIRECTIVE 2010/63/EU of 22 September 2010. This framework is built on the 3R principle with the aim to reduce, refine and replace animal experiments to the indispensable minimum and to conduct them humanely [1]. Careful experimental planning and statistical evaluation can reduce the number of animals necessary for an experiment. Refinement can, for example, include appropriate analgesia and anaesthesia, the improvement of assay procedures and non‐invasive methods. The third and most rigorous strategic line of the 3R principle is the replacement of animals altogether by in vitro methods including cell, organ and embryo culture or by in silico simulation. Since its adoption in 2010, DIRECTIVE 2010/63/EU has been implemented by national legal guidelines such as the Animal Protection Act (TierSchG—Tierschutzgesetz) in Germany. The administrative burden for scientists has increased along with the new guidelines, prompting researchers to avoid this additional workload by employing of alternative research models. Big hopes rest on artificial stem cell‐derived in vitro organ systems. However, significant technological advances are still required to faithfully reproduce organ function in vitro . In addition, many studies involving interactions at a system level or complex behavioural outcomes require the intact animal model. The zebrafish embryo is increasingly used as an alternative model system. Many aspects of biology, including disease …

Ciliopathies are a group of diseases that affect kidney and retina among other organs. Here, we identify a missense mutation in PIK3R4 (phosphoinositide 3-kinase regulatory subunit 4, named VPS15) in a family with a ciliopathy phenotype. Besides being required for trafficking and autophagy, we show that VPS15 regulates primary cilium length in human fibroblasts, as well as ciliary processes in zebrafish. Furthermore, we demonstrate its interaction with the golgin GM130 and its localization to the Golgi. The VPS15-R998Q patient mutation impairs Golgi trafficking functions in humanized yeast cells. Moreover, in VPS15-R998Q patient fibroblasts, the intraflagellar transport protein IFT20 is not localized to vesicles trafficking to the cilium but is restricted to the Golgi. Our findings suggest that at the Golgi, VPS15 and GM130 form a protein complex devoid of VPS34 to ensure the IFT20-dependent sorting and transport of membrane proteins from the cis-Golgi to the primary cilium., VPS15 is known as a VPS34-associated protein that functions in intracellular trafficking and autophagy. Here the authors identify a role for VPS15 in ciliopathy and ciliary phenotypes, and show that it interacts with GM130 and functions in IFT20-dependent cis-Golgi to cilium trafficking.

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.

Pufferfish such as fugu and tetraodon carry the smallest genomes among all vertebrates and are ideal for studying genome evolution. However, comparative genomics using these species is hindered by the poor annotation of their genomes. We performed RNA sequencing during key stages of maternal to zygotic transition of Tetraodon nigroviridis and report its first developmental transcriptome. We assembled 61,033 transcripts (23,837 loci) representing 80% of the annotated gene models and 3816 novel coding transcripts from 2667 loci. We demonstrate the similarities of gene expression profiles between pufferfish and zebrafish during maternal to zygotic transition and annotated 1120 long noncoding RNAs (lncRNAs) many of which differentially expressed during development. The promoters for 60% of the assembled transcripts result validated by CAGE-seq. Despite the extreme compaction of the tetraodon genome and the dramatic loss of transposons, the length of lncRNA exons remain comparable to that of other vertebrates and a small set of lncRNAs appears enriched for transposable elements suggesting a selective pressure acting on lncRNAs length and composition. Finally, a set of lncRNAs are microsyntenic between teleost and vertebrates, which indicates potential regulatory interactions between lncRNAs and their flanking coding genes. Our work provides a fundamental molecular resource for vertebrate comparative genomics and embryogenesis studies.

The zebrafish (Danio rerio) is a well-established vertebrate model organism. Its embryos are used extensively in biology and medicine to perform chemical screens to identify drug candidates or to evaluate teratogenicity and embryotoxicity of substances. Behavioral readouts are increasingly used to assess the effects of compounds on the nervous system. Early stage zebrafish show characteristic behavioral features at stages between 30 and 42 hours post fertilization (hpf) when exposed to a short and bright light flash. This so-called Photomotor Response (PMR) is a reaction of the nervous system of the fish and can be used as a marker in screenings for neuroactive chemicals. To probe a broad and diverse chemical space, many different substances have to be tested and repeated observations are necessary to warrant statistical significance of the results. Although PMR-based chemical screens must use a large number of specimens, there is no sophisticated, automated high-throughput platform available which ensures minimal human intervention. Here we report a PMR platform that was developed by combining an improved automatic sample handling with a remotely controllable microscope setup and an image analysis pipeline. Using infrared illumination during automatic sample preparation, we were able to eliminate excess amounts of visible light that could potentially alter the response results. A remotely controlled microscope setup allows us to screen entire 96-well microtiter plates without human presence that could disturb the embryos. The development of custom video analysis software, including single egg detection, enables us to detect variance among treated specimens and extract easy to interpret numerical values representing the PMR motion. By testing several neuroactive compounds we validated the workflow that can be used to analyze more than one thousand zebrafish eggs on a single 96-well plate.

We have established a European Zebrafish Resource Center (EZRC) at the KIT. This center not only maintains and distributes a large number of existing mutant and transgenic zebrafish lines but also gives zebrafish researchers access to screening services and technologies such as imaging and high-throughput sequencing, provided by the Institute of Toxicology and Genetics (ITG). The EZRC maintains and distributes the stock collection of the Nüsslein-Volhard laboratory, comprising over 2000 publicly released mutations, as frozen sperm samples. Within the framework of the ZF-HEALTH EU project, the EZRC distributes over 10,000 knockout mutations from the Sanger Institute (United Kingdom), as well as over 100 mutant and transgenic lines from other sources. In this article, we detail the measures we have taken to ensure the health of our fish, including hygiene, quarantine, and veterinary inspections.

Despite enormous progress to map cis-regulatory modules (CRMs), like enhancers and promoters in genomes, elucidation of the regulatory landscape of the developing embryo remains a challenge. The zebrafish embryo with its experimental virtues has a great potential to contribute to this endeavor. However, so far progress remained behind expectation. We discuss here available methods and their limitations and how the zebrafish embryo could contribute in the future to unravel the wiring of the vertebrate genome.

The zebrafish embryo has repeatedly proved to be a useful model for the analysis of effects by environmental toxicants. This proof-of-concept study was performed to investigate if an approach combining mechanism-specific bioassays with microarray techniques can obtain more in-depth insights into the ecotoxicity of complex pollutant mixtures as present, e.g., in sediment extracts. For this end, altered gene expression was compared to data from established bioassays as well as to results from chemical analysis. Mechanism-specific biotests indicated a defined hazard potential of the sediment extracts, and microarray analysis revealed several classes of significantly regulated genes which could be related to the hazard potential. Results indicate that potential classes of contaminants can be assigned to sediment extracts by both classical biomarker genes and corresponding expression profile analyses of known substances. However, it is difficult to distinguish between specific responses and more universal detoxification of the organism.

Worldwide, the zebrafish has become a popular model for biomedical research and (eco)toxicology. Particularly the use of embryos is receiving increasing attention, since they are considered as replacement method for animal experiments. Zebrafish embryos allow the analysis of multiple endpoints ranging from acute and developmental toxicity determination to complex functional genetic and physiological analysis. Particularly the more complex endpoints require the use of post-hatched eleutheroembryo stages. According to the new EU Directive 2010/63/EU on the protection of animals used for scientific purposes, the earliest life-stages of animals are not defined as protected and, therefore, do not fall into the regulatory frameworks dealing with animal experimentation. Independent feeding is considered as the stage from which free-living larvae are subject to regulations for animal experimentation. However, despite this seemingly clear definition, large variations exist in the interpretation of this criterion by national and regional authorities. Since some assays require the use of post-hatched stages up to 120 h post fertilization, the literature and available data are reviewed in order to evaluate if this stage could still be considered as non-protected according to the regulatory criterion of independent feeding. Based on our analysis and by including criteria such as yolk consumption, feeding and swimming behavior, we conclude that zebrafish larvae can indeed be regarded as independently feeding from 120 h after fertilization. Experiments with zebrafish should thus be subject to regulations for animal experiments from 120 h after fertilization onwards.

Dysferlin (Dysf) has been implicated as a key protein for membrane repair in muscle cells, although its precise biological function is still unknown. Dysf is a transmembrane protein, and its rapid recruitment of Dysf to membrane lesions has been observed; direct observation of Dysf transport has so far remained elusive [1]. Moreover, interaction of Dysf with various other proteins has been investigated for many years to understand its role in cell membrane repair; however, studies addressing the role of phospholipids, the main components of the plasma membrane, are still scarce.

Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on a severe developmental dental defect that results in a dentin dysplasia phenotype with major microdontia, oligodontia, and shape abnormalities in a highly consanguineous family. Homozygosity mapping revealed a unique zone on 6q27-ter. The two affected children were found to carry a homozygous mutation in SMOC2. Knockdown of smoc2 in zebrafish showed pharyngeal teeth that had abnormalities reminiscent of the human phenotype. Moreover, smoc2 depletion in zebrafish affected the expression of three major odontogenesis genes: dlx2, bmp2, and pitx2.

In contrast to mammals, the brain of the adult zebrafish has a remarkable ability to regenerate. In mammals, injuries induce proliferation of astrocytes and oligodendrocyte progenitors contributing to the formation of a glial scar. We analyzed the proliferation of glial cells and microglia in response to stab injury in the adult zebrafish telencephalon: Radial glial markers were up-regulated at the ventricle and co-expressed the proliferation nuclear antigen (PCNA). Microglia and oligodendrocyte progenitors accumulated transiently at the site of lesion. However, we could not find evidence of permanent scar formation. Parenchymal proliferation was almost negligible in comparison to the increase in proliferation at the ventricular zone. This suggests that most of the cellular material for regeneration is derived from regions of constitutive neurogenesis. Remarkably, the proliferative response is almost completely restricted to the lesioned hemisphere indicating that signals inducing regeneration remain mainly confined within the lesioned half of the telencephalon.

Zusammenfassung Die vorliegende Arbeit stellt eine neue bildbasierte Methode zur automatischen Klassifikation der Spontanbewegung und des Herzschlags in toxikologisch behandelten Zebrabärblingslarven vor, die sich für Hochdurchsatz-Verfahren in der Toxikologie eignet. Dabei werden neue Akquisestrategien vorgeschlagen, Merkmale aus Bildzeitreihen extrahiert und Klassifikatoren entworfen. Diese werden iterativ kombiniert und anhand von drei Substanzen prototypisch experimentell erprobt.

Assembly, maintenance and renewal of sarcomeres require highly organized and balanced folding, transport, modification and degradation of sarcomeric proteins. However, the molecules that mediate these processes are largely unknown. Here, we isolated the zebrafish mutant flatline (fla), which shows disturbed sarcomere assembly exclusively in heart and fast-twitch skeletal muscle. By positional cloning we identified a nonsense mutation within the SET- and MYND-domain-containing protein 1 gene (smyd1) to be responsible for the fla phenotype. We found SMYD1 expression to be restricted to the heart and fast-twitch skeletal muscle cells. Within these cell types, SMYD1 localizes to both the sarcomeric M-line, where it physically associates with myosin, and the nucleus, where it supposedly represses transcription through its SET and MYND domains. However, although we found transcript levels of thick filament chaperones, such as Hsp90a1 and UNC-45b, to be severely upregulated in fla, its histone methyltransferase activity – mainly responsible for the nuclear function of SMYD1 – is dispensable for sarcomerogenesis. Accordingly, sarcomere assembly in fla mutant embryos can be reconstituted by ectopically expressing histone methyltransferase-deficient SMYD1. By contrast, ectopic expression of myosin-binding-deficient SMYD1 does not rescue fla mutants, implicating an essential role for the SMYD1–myosin interaction in cardiac and fast-twitch skeletal muscle thick filament assembly.

In contrast to mammals where the formation of new neurons in the adult is very limited, teleost fish like the zebrafish form new neurons abundantly and have a high regenerative potential to repair lesions of the central nervous system. These news neurons will integrate into the existing neuronal networks. The challenge will be to unravel the molecular mechanisms of nervous tissue regeneration in zebrafish in the hope to employ this knowledge for development of new therapies for human patients.

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.