Pax6 organizes the anterior eye segment by guiding two distinct neural crest waves.

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 TGFß 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.

Genetically encoded thiol redox-sensors in the zebrafish model: lessons for embryonic development and regeneration.

Important roles for reactive oxygen species (ROS) and redox signaling in embryonic development and regenerative processes are increasingly recognized. However, it is difficult to obtain information on spatiotemporal dynamics of ROS production and signaling in vivo. The zebrafish is an excellent model for in vivo bioimaging and possesses a remarkable regenerative capacity upon tissue injury. Here, we review data obtained in this model system with genetically encoded redox-sensors targeting H2O2 and glutathione redox potential. We describe how such observations have prompted insight into regulation and downstream effects of redox alterations during tissue differentiation, morphogenesis and regeneration. We also discuss the properties of the different sensors and their consequences for the interpretation of in vivo imaging results. Finally, we highlight open questions and additional research fields that may benefit from further application of such sensor systems in zebrafish models of development, regeneration and disease.

Inductively coupled magic angle spinning microresonators benchmarked for high-resolution single embryo metabolomic profiling.

The magic angle coil spinning (MACS) technique has been introduced as a very promising extension for solid state NMR detection, demonstrating sensitivity enhancements by a factor of 14 from the very first time it has been reported. The main beneficiary of this technique is the scientific community dealing with mass- and volume-limited, rare, or expensive samples. However, more than a decade after the first report on MACS, there is a very limited number of groups who have continued to develop the technique, let alone it being widely adopted by practitioners. This might be due to several drawbacks associated with the MACS technology until now, including spectral linewidth, heating due to eddy currents, and imprecise manufacturing. Here, we report a device overcoming all these remaining issues, therefore achieving: (1) spectral resolution of approx 0.01 ppm and normalized limit of detection of approx. 13 nmol s0.5 calculated using the anomeric proton of sucrose at 3 kHz MAS frequency; (2) limited temperature increase inside the MACS insert of only 5 °C at 5 kHz MAS frequency in an 11.74 T magnetic field, rendering MACS suitable to study live biological samples. The wafer-scale fabrication process yields MACS inserts with reproducible properties, readily available to be used on a large scale in bio-chemistry labs. To illustrate the potential of these devices for metabolomic studies, we further report on: (3) ultra-fine 1H-1H and 13C-13C J-couplings resolved within 10 min for a 340 mM uniformly 13C-labeled glucose sample; and (4) single zebrafish embryo measurements through 1H-1H COSY within 4.5 h, opening the gate for the single embryo NMR studies.

Extensive Regulation of Diurnal Transcription and Metabolism by Glucocorticoids.

Altered daily patterns of hormone action are suspected to contribute to metabolic disease. It is poorly understood how the adrenal glucocorticoid hormones contribute to the coordination of daily global patterns of transcription and metabolism. Here, we examined diurnal metabolite and transcriptome patterns in a zebrafish glucocorticoid deficiency model by RNA-Seq, NMR spectroscopy and liquid chromatography-based methods. We observed dysregulation of metabolic pathways including glutaminolysis, the citrate and urea cycles and glyoxylate detoxification. Constant, non-rhythmic glucocorticoid treatment rescued many of these changes, with some notable exceptions among the amino acid related pathways. Surprisingly, the non-rhythmic glucocorticoid treatment rescued almost half of the entire dysregulated diurnal transcriptome patterns. A combination of E-box and glucocorticoid response elements is enriched in the rescued genes. This simple enhancer element combination is sufficient to drive rhythmic circadian reporter gene expression under non-rhythmic glucocorticoid exposure, revealing a permissive function for the hormones in glucocorticoid-dependent circadian transcription. Our work highlights metabolic pathways potentially contributing to morbidity in patients with glucocorticoid deficiency, even under glucocorticoid replacement therapy. Moreover, we provide mechanistic insight into the interaction between the circadian clock and glucocorticoids in the transcriptional regulation of metabolism.

Stem cells and the circadian clock.

The circadian timing system is a complex biological network of interacting circadian clocks that regulates 24h rhythms of behavioral and physiological processes. One intriguing observation is that stem cell homeostasis is subject to circadian clock regulation. Rhythmic oscillations have been observed in a variety of embryonic and adult stem cell dependent processes, such as hematopoietic progenitor cell migration, the hair follicle cycle, bone remodeling, regenerative myogenesis and neurogenesis. This review aims to discuss the nature of the circadian clock in embryonic stem cells and how it changes during differentiation. Furthermore, it will examine how the circadian clock contributes to adult stem cell function in different tissues of the body with an emphasis on the brain and adult neurogenesis.

Glycolysis supports embryonic muscle growth by promoting myoblast fusion.

Muscles ensure locomotion behavior of invertebrate and vertebrate organisms. They are highly specialized and form using conserved developmental programs. To identify new players in muscle development we screened Drosophila and zebrafish gene expression databases for orthologous genes expressed in embryonic muscles. We selected more than 100 candidates. Among them is the glycolysis gene Pglym78/pgam2, the attenuated expression of which results in the formation of thinner muscles in Drosophila embryos. This phenotype is also observed in fast muscle fibers of pgam2 zebrafish morphants, suggesting affected myoblast fusion. Indeed, a detailed analysis of developing muscles in Pglym78 RNAi embryos reveals loss of fusion-associated actin foci and an inefficient Notch decay in fusion competent myoblasts, both known to be required for fusion. In addition to Pglym78, our screen identifies six other genes involved in glycolysis or in pyruvate metabolism (Pfk, Tpi, Gapdh, Pgk, Pyk, and Impl3). They are synchronously activated in embryonic muscles and attenuation of their expression leads to similar muscle phenotypes, which are characterized by fibers with reduced size and the presence of unfused myoblasts. Our data also show that the cell size triggering insulin pathway positively regulates glycolysis in developing muscles and that blocking the insulin or target of rapamycin pathways phenocopies the loss of function phenotypes of glycolytic genes, leading to myoblast fusion arrest and reduced muscle size. Collectively, these data suggest that setting metabolism to glycolysis-stimulated biomass production is part of a core myogenic program that operates in both invertebrate and vertebrate embryos and promotes formation of syncytial muscles.

Real-time in vivo monitoring of circadian E-box enhancer activity: a robust and sensitive zebrafish reporter line for developmental, chemical and neural biology of the circadian clock.

The circadian clock co-ordinates physiology and behavior with the day/night cycle. It consists of a transcriptional-translational feedback loop that generates self-sustained oscillations in transcriptional activity with a roughly 24h period via E-box enhancer elements. Numerous in vivo aspects of core clock feedback loop function are still incompletely understood, including its maturation during development, tissue-specific activity and perturbation in disease states. Zebrafish are promising models for biomedical research due to their high regenerative capacity and suitability for in vivo drug screens, and transgenic zebrafish lines are valuable tools to study transcriptional activity in vivo during development. To monitor the activity of the core clock feedback loop in vivo, we created a transgenic zebrafish line expressing a luciferase reporter gene under the regulation of a minimal promoter and four E-boxes. This Tg(4xE-box:Luc) line shows robust oscillating reporter gene expression both under light-dark cycles and upon release into constant darkness. Luciferase activity starts to oscillate during the first days of development, indicating that the core clock loop is already functional at an early stage. To test whether the Tg(4xE-box:Luc) line could be used in drug screens aimed at identifying compounds that target the circadian clock in vivo, we examined drug effects on circadian period. We were readily able to detect period changes as low as 0.7h upon treatment with the period-lengthening drugs lithium chloride and longdaysin in an assay set-up suitable for large-scale screens. Reporter gene mRNA expression is also detected in the adult brain and reveals differential clock activity across the brain, overlapping with endogenous clock gene expression. Notably, core clock activity is strongly correlated with brain regions where neurogenesis takes place and can be detected in several types of neural progenitors. Our results demonstrate that the Tg(4xE-box:Luc) line is an excellent tool for studying the regulation of the circadian clock and its maturation in vivo and in real time. Furthermore, it is highly suitable for in vivo screens targeting the core clock mechanism that take into account the complexity of an intact organism. Finally, it allows mapping of clock activity in the brain of a vertebrate model organism with prominent adult neurogenesis and high regeneration capacity.

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System.

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.

Glucocorticoids play a key role in circadian cell cycle rhythms.

Clock output pathways play a pivotal role by relaying timing information from the circadian clock to a diversity of physiological systems. Both cell-autonomous and systemic mechanisms have been implicated as clock outputs; however, the relative importance and interplay between these mechanisms are poorly understood. The cell cycle represents a highly conserved regulatory target of the circadian timing system. Previously, we have demonstrated that in zebrafish, the circadian clock has the capacity to generate daily rhythms of S phase by a cell-autonomous mechanism in vitro. Here, by studying a panel of zebrafish mutants, we reveal that the pituitary-adrenal axis also plays an essential role in establishing these rhythms in the whole animal. Mutants with a reduction or a complete absence of corticotrope pituitary cells show attenuated cell-proliferation rhythms, whereas expression of circadian clock genes is not affected. We show that the corticotrope deficiency is associated with reduced cortisol levels, implicating glucocorticoids as a component of a systemic signaling pathway required for circadian cell cycle rhythmicity. Strikingly, high-amplitude rhythms can be rescued by exposing mutant larvae to a tonic concentration of a glucocorticoid agonist. Our work suggests that cell-autonomous clock mechanisms are not sufficient to establish circadian cell cycle rhythms at the whole-animal level. Instead, they act in concert with a systemic signaling environment of which glucocorticoids are an essential part.

MondoA regulates gene expression in cholesterol biosynthesis-associated pathways required for zebrafish epiboly.

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.

In most animals, a protein called MondoA closely monitors the amount of glucose in the body, as this type of sugar is the fuel required for many life processes. Glucose levels also act as a proxy for the availability of other important nutrients. Once MondoA has detected glucose molecules, it turns genetic programmes on and off depending on the needs of the cell. So far, these mechanisms have mainly been studied in adult cells. However, recent studies have shown that proteins that monitor nutrient availability, and their associated pathways, can control early development. MondoA had not been studied in this context before, so Weger et al. decided to investigate its role in embryonic development. The experiments used embryos from zebrafish, a small freshwater fish whose early development is easily monitored and manipulated in the laboratory. Inhibiting production of the MondoA protein in zebrafish embryos prevented them from maturing any further, stopping their development at an early key stage. This block was caused by defects in microtubules, the tubular molecules that act like a microscopic skeleton to provide structural support for cells and guide transport of cell components. In addition, the pathway involved in the production of cholesterol and cholesterol-based hormones was far less active in embryos lacking MondoA. Treating MondoA-deficient embryos with one of these hormones corrected the microtubule defects and let the embryos progress to more advanced stages of development. These results reveal that, during development, the glucose sensor MondoA also controls pathways involved in the creation of cholesterol and associated hormones. These new insights into the metabolic regulation of development could help to understand certain human conditions; for example, certain patients with defective cholesterol pathway genes also show developmental perturbations. In addition, the work highlights a biological link between cholesterol production and cellular responses to glucose, which Weger et al. hope could one day help to identify new cholesterol-lowering drugs.

The words of the regulatory code are arranged in a variable manner in highly conserved enhancers.

The cis-regulatory regions of many developmental regulators and transcription factors are believed to be highly conserved in the genomes of vertebrate species, suggesting specific regulatory mechanisms for these gene classes. We functionally characterized five notochord enhancers, whose sequence is highly conserved, and systematically mutated two of them. Two subregions were identified to be essential for expression in the notochord of the zebrafish embryo. Synthetic enhancers containing the two essential regions in front of a TATA-box drive expression in the notochord while concatemerization of the subregions alone is not sufficient, indicating that the combination of the two sequence elements is required for notochord expression. Both regions are present in the five functionally characterized notochord enhancers. However, the position, the distance and relative orientation of the two sequence motifs can vary substantially within the enhancer sequences. This suggests that the regulatory grammar itself does not dictate the high evolutionary conservation between these orthologous cis-regulatory sequences. Rather, it represents a less well-conserved layer of sequence organization within these sequences.

Nano-Sampling and Reporter Tools to Study Metabolic Regulation in Zebrafish.

In the past years, evidence has emerged that hallmarks of human metabolic disorders can be recapitulated in zebrafish using genetic, pharmacological or dietary interventions. An advantage of modeling metabolic diseases in zebrafish compared to other "lower organisms" is the presence of a vertebrate body plan providing the possibility to study the tissue-intrinsic processes preceding the loss of metabolic homeostasis. While the small size of zebrafish is advantageous in many aspects, it also has shortcomings such as the difficulty to obtain sufficient amounts for biochemical analyses in response to metabolic challenges. A workshop at the European Zebrafish Principal Investigator meeting in Trento, Italy, was dedicated to discuss the advantages and disadvantages of zebrafish to study metabolic disorders. This perspective article by the participants highlights strategies to achieve improved tissue-resolution for read-outs using "nano-sampling" approaches for metabolomics as well as live imaging of zebrafish expressing fluorescent reporter tools that inform on cellular or subcellular metabolic processes. We provide several examples, including the use of reporter tools to study the heterogeneity of pancreatic beta-cells within their tissue environment. While limitations exist, we believe that with the advent of new technologies and more labs developing methods that can be applied to minimal amounts of tissue or single cells, zebrafish will further increase their utility to study energy metabolism.

Oriented immobilization of a delicate glucose-sensing protein on silica nanoparticles.

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.

Enhancer sequence conservation between vertebrates is favoured in developmental regulator genes.

Sequence conservation has been used to find genes and to pinpoint functional non-coding sequences such as transcriptional regulatory elements. In this article, we analysed the conservation of 104 experimentally validated murine enhancer sequences between the mouse and zebrafish genomes. Surprisingly, only 10.5% of the mouse enhancers have homologues in zebrafish. All of the genes with conserved cis-elements have regulatory functions during embryonic development, perhaps reflecting substantial structural constraints on the integration of spatio-temporal signalling cues during the formation of the vertebrate body.

Temperature regulates transcription in the zebrafish circadian clock.

It has been well-documented that temperature influences key aspects of the circadian clock. Temperature cycles entrain the clock, while the period length of the circadian cycle is adjusted so that it remains relatively constant over a wide range of temperatures (temperature compensation). In vertebrates, the molecular basis of these properties is poorly understood. Here, using the zebrafish as an ectothermic model, we demonstrate first that in the absence of light, exposure of embryos and primary cell lines to temperature cycles entrains circadian rhythms of clock gene expression. Temperature steps drive changes in the basal expression of certain clock genes in a gene-specific manner, a mechanism potentially contributing to entrainment. In the case of the per4 gene, while E-box promoter elements mediate circadian clock regulation, they do not direct the temperature-driven changes in transcription. Second, by studying E-box-regulated transcription as a reporter of the core clock mechanism, we reveal that the zebrafish clock is temperature-compensated. In addition, temperature strongly influences the amplitude of circadian transcriptional rhythms during and following entrainment by light-dark cycles, a property that could confer temperature compensation. Finally, we show temperature-dependent changes in the expression levels, phosphorylation, and function of the clock protein, CLK. This suggests a mechanism that could account for changes in the amplitude of the E-box-directed rhythm. Together, our results imply that several key transcriptional regulatory elements at the core of the zebrafish clock respond to temperature.

Fully Automated Pipetting Sorting System for Different Morphological Phenotypes of Zebrafish Embryos.

Systems biology methods, such as transcriptomics and metabolomics, require large numbers of small model organisms, such as zebrafish embryos. Manual separation of mutant embryos from wild-type embryos is a tedious and time-consuming task that is prone to errors, especially if there are variable phenotypes of a mutant. Here we describe a zebrafish embryo sorting system with two cameras and image processing based on template-matching algorithms. In order to evaluate the system, zebrafish rx3 mutants that lack eyes due to a patterning defect in brain development were separated from their wild-type siblings. These mutants show glucocorticoid deficiency due to pituitary defects and serve as a model for human secondary adrenal insufficiencies. We show that the variable phenotypes of the mutant embryos can be safely distinguished from phenotypic wild-type zebrafish embryos and sorted from one petri dish into another petri dish or into a 96-well microtiter plate. On average, classification of a zebrafish embryo takes approximately 1 s, with a sensitivity and specificity of 87% to 95%, respectively. Other morphological phenotypes may be classified and sorted using similar techniques.

Glucocorticoid deficiency causes transcriptional and post-transcriptional reprogramming of glutamine metabolism.

Background: Deficient glucocorticoid biosynthesis leading to adrenal insufficiency is life-threatening and is associated with significant co-morbidities. The affected pathways underlying the pathophysiology of co-morbidities due to glucocorticoid deficiency remain poorly understood and require further investigation. Methods: To explore the pathophysiological processes related to glucocorticoid deficiency, we have performed global transcriptional, post-transcriptional and metabolic profiling of a cortisol-deficient zebrafish mutant with a disrupted ferredoxin (fdx1b) system. Findings: fdx1b−/− mutants show pervasive reprogramming of metabolism, in particular of glutamine-dependent pathways such as glutathione metabolism, and exhibit changes of oxidative stress markers. The glucocorticoid-dependent post-transcriptional regulation of key enzymes involved in de novo purine synthesis was also affected in this mutant. Moreover, fdx1b−/− mutants exhibit crucial features of primary adrenal insufficiency, and mirror metabolic changes detected in primary adrenal insufficiency patients. Interpretation: Our study provides a detailed map of metabolic changes induced by glucocorticoid deficiency as a consequence of a disrupted ferredoxin system in an animal model of adrenal insufficiency. This improved pathophysiological understanding of global glucocorticoid deficiency informs on more targeted translational studies in humans suffering from conditions associated with glucocorticoid deficiency. Fund: Marie Curie Intra-European Fellowships for Career Development, HGF-programme BIFTM, Deutsche Forschungsgemeinschaft, BBSRC.

Absence of 11-keto reduction of cortisone and 11-ketotestosterone in the model organism zebrafish.

Zebrafish are widely used as model organism. Their suitability for endocrine studies, drug screening and toxicity assessements depends on the extent of conservation of specific genes and biochemical pathways between zebrafish and human. Glucocorticoids consist of inactive 11-keto (cortisone and 11-dehydrocorticosterone) and active 11ß-hydroxyl forms (cortisol and corticosterone). In mammals, two 11ß-hydroxysteroid dehydrogenases (11ß-HSD1 and 11ß-HSD2) interconvert active and inactive glucocorticoids, allowing tissue-specific regulation of glucocorticoid action. Furthermore, 11ß-HSDs are involved in the metabolism of 11-oxy androgens. As zebrafish and other teleost fish lack a direct homologue of 11ß-HSD1, we investigated whether they can reduce 11-ketosteroids. We compared glucocorticoid and androgen metabolism between human and zebrafish using recombinant enzymes, microsomal preparations and zebrafish larvae. Our results provide strong evidence for the absence of 11-ketosteroid reduction in zebrafish. Neither human 11ß-HSD3 nor the two zebrafish 11ß-HSD3 homologues, previously hypothesized to reduce 11-ketosteroids, converted cortisone and 11-ketotestosterone (11KT) to their 11ß-hydroxyl forms. Furthermore, zebrafish microsomes were unable to reduce 11-ketosteroids, and exposure of larvae to cortisone or the synthetic analogue prednisone did not affect glucocorticoid-dependent gene expression. Additionally, a dual-role of 11ß-HSD2 by inactivating glucocorticoids and generating the main fish androgen 11KT was supported. Thus, due to the lack of 11-ketosteroid reduction, zebrafish and other teleost fish exhibit a limited tissue-specific regulation of glucocorticoid action, and their androgen production pathway is characterized by sustained 11KT production. These findings are of particular significance when using zebrafish as a model to study endocrine functions, stress responses and effects of pharmaceuticals.

Automated phenotype pattern recognition of zebrafish for high-throughput screening.

Over the last years, the zebrafish (Danio rerio) has become a key model organism in genetic and chemical screenings. A growing number of experiments and an expanding interest in zebrafish research makes it increasingly essential to automatize the distribution of embryos and larvae into standard microtiter plates or other sample holders for screening, often according to phenotypical features. Until now, such sorting processes have been carried out by manually handling the larvae and manual feature detection. Here, a prototype platform for image acquisition together with a classification software is presented. Zebrafish embryos and larvae and their features such as pigmentation are detected automatically from the image. Zebrafish of 4 different phenotypes can be classified through pattern recognition at 72 h post fertilization (hpf), allowing the software to classify an embryo into 2 distinct phenotypic classes: wild-type versus variant. The zebrafish phenotypes are classified with an accuracy of 79-99% without any user interaction. A description of the prototype platform and of the algorithms for image processing and pattern recognition is presented.