Analytical method for determination of nitric oxide in zebrafish larvae: toxicological and pharmacological applications

Zebrafish are currently used at various stages of the drug discovery process and can be a useful and cost-effective alternative to some mammalian models. Nitric oxide (NO) plays an important role in physiology of zebrafish. The availability of appropriate analytical techniques to quantify the NO is crucial for studying its role in physiological and pathological conditions. This work aimed at establishing a high-performance liquid chromatography method for determination of NO levels in zebrafish larvae. Attempts were also made to assess the normal levels of NO at the first days postfertilization and the possible changes under pathological conditions. The method validation was quantitatively evaluated in terms of sensitivity, specificity, precision, accuracy, linearity, and recovery. NO levels from zebrafish larvae at the first days postfertilization and larvae challenged to N(G)-nitro-L-arginine methyl ester, sodium nitroprusside, Escherichia coli lipopolysaccharide, and copper sulfate were analyzed. The samples were derivatized with 2,3-diaminonaphthalene, and fluorescence detection was used for the indirect determination of NO. The method showed a good performance for all validation parameters evaluated and was efficient to monitor changes in NO concentration under physiological and pathophysiological conditions. This method might represent a powerful tool to be applied in NO studies with zebrafish larvae.     

Solving an enigma: arterial pole development in the zebrafish heart

It is a widely held belief that the arterial pole of the zebrafish heart is unusual among models of comparative cardiogenesis. This is based, in part, on the report that the bulbus arteriosus undergoes a striated-to-smooth muscle phenotypic transition during development. An implication of this is that the zebrafish, a model almost ubiquitously accepted in other fields of comparative biology, may be poorly suited to the study of conotruncal abnormalities in human disease. However, while the use of atrioventricular-specific molecular markers has allowed extensive characterization of the development of the atrium and ventricle, the lack of any bulbus-specific markers has meant that this region of the zebrafish heart is poorly characterized and quite possibly misunderstood. We have discovered that the fluorescent nitric oxide indicator 4,5-diaminofluorescein diacetate (DAF-2DA) specifically labels the bulbus arteriosus throughout development from approximately 48 h post-fertilization. Therefore, using DAF-2DA and an immunohistochemical approach, we attempted to further characterize the development of the bulbus. We have concluded that no such phenotypic transition occurs, that contrary to current thinking, aspects of zebrafish arterial pole development are evolutionarily conserved, and that the bulbus should not be considered a chamber, being more akin to the arterial trunk(s) of higher vertebrates.     

Proteomic analysis of zebrafish caudal fin regeneration

The epimorphic regeneration of zebrafish caudal fin is rapid and complete. We have analyzed the biomechanism of zebrafish caudal fin regeneration at various time points based on differential proteomics approaches. The spectrum of proteome changes caused by regeneration were analyzed among controls (0 h) and 1, 12, 24, 48, and 72 h postamputation involving quantitative differential proteomics analysis based on two-dimensional gel electrophoresis matrix-assisted laser desorption/ionization and differential in-gel electrophoresis Orbitrap analysis. A total of 96 proteins were found differentially regulated between the control nonregenerating and regenerating tissues of different time points for having at least 1.5-fold changes. 90 proteins were identified as differentially regulated for regeneration based on differential in-gel electrophoresis analysis between the control and regenerating tissues. 35 proteins were characterized for its expression in all of the five regenerating time points against the control samples. The proteins identified and associated with regeneration were found to be directly allied with various molecular, biological, and cellular functions. Based on network pathway analysis, the identified proteome data set for regeneration was majorly associated in maintaining cellular structure and architecture. Also the proteins were found associated for the cytoskeleton remodeling pathway and cellular immune defense mechanism. The major proteins that were found differentially regulated during zebrafish caudal fin regeneration includes keratin and its 10 isoforms, cofilin 2, annexin a1, skeletal α1 actin, and structural proteins. Annexin A1 was found to be exclusively undergoing phosphorylation during regeneration. The obtained differential proteome and the direct association of the various proteins might lead to a new understanding of the regeneration mechanism.     

Differential expression of two tropoelastin genes in zebrafish.

Elastin is the extracellular matrix protein responsible for properties of extensibility and elastic recoil in large blood vessels, lung and skin of most vertebrates. Elastin is synthesized as a monomer, tropoelastin, but is rapidly transformed into its final polymeric form in the extracellular matrix. Until recently information on sequence and developmental expression of tropoelastins was limited to mammalian and avian species. We have recently identified and characterized two expressed tropoelastin genes in zebrafish. This was the first example of a species with multiple tropoelastin genes, raising the possibility of differential expression and function of these tropoelastins in elastic tissues of the zebrafish. Here we have investigated the temporal expression and tissue distribution of the two tropoelastin genes in developing and adult zebrafish. Expression was detected early in skeletal cartilage structures of the head, in the developing outflow tract of the heart, including the bulbus arteriosus and the ventral aorta, and in the wall of the swim bladder. While the temporal pattern of expression was similar for both genes, the upregulation of eln2 was much stronger than that of eln1. In general, both genes were expressed and their gene products deposited in most of the elastic tissues examined, with the notable exception of the bulbus arteriosus in which eln2 expression and its gene product was predominant. This finding may represent a sub-specialization of eln2 to provide the unique architecture of elastin and the specific mechanical properties required by this organ.     

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.     

Venular endothelium-derived NO can affect paired arteriole: a computational model

Venular endothelial cells can release nitric oxide (NO) in response to intraluminal flow both in isolated venules and in vivo. Experimental studies suggest that venular endothelium-released NO causes dilation of the adjacent paired arteriole. In the vascular wall, NO stimulates its target hemoprotein, soluble guanylate cyclase (sGC), which relaxes smooth muscle cells. In this study, a computational model of NO transport for an arteriole and venule pair was developed to determine the importance of the venular endothelium-released NO and its transport to the adjacent arteriole in the tissue. The model predicts that the tissue NO levels are affected within a wide range of parameters, including NO-red blood cell reaction rate and NO production rate in the arteriole and venule. The results predict that changes in the venular NO production affected not only venular endothelial and smooth muscle NO concentration but also endothelial and smooth muscle NO concentration in the adjacent arteriole. This suggests that the anatomy of microvascular tissue can permit the transport of NO from arteriolar to venular side, and vice versa, and may provide a mechanism for dilation of proximal arterioles by venules. These results will have significant implications for our understanding of tissue NO levels in both physiological and pathophysiological conditions.     

The regenerative capacity of the zebrafish caudal fin is not affected by repeated amputations

Background

The zebrafish has the capacity to regenerate many tissues and organs. The caudal fin is one of the most convenient tissues to approach experimentally due to its accessibility, simple structure and fast regeneration. In this work we investigate how the regenerative capacity is affected by recurrent fin amputations and by experimental manipulations that block regeneration.

Methodology/Principal Findings

We show that consecutive repeated amputations of zebrafish caudal fin do not reduce its regeneration capacity and do not compromise any of the successive regeneration steps: wound healing, blastema formation and regenerative outgrowth. Interfering with Wnt/ß-catenin signalling using heat-shock-mediated overexpression of Dickkopf1 completely blocks fin regeneration. Notably, if these fins were re-amputated at the non-inhibitory temperature, the regenerated caudal fin reached the original length, even after several rounds of consecutive Wnt/ß-catenin signalling inhibition and re-amputation.

Conclusions/Significance

We show that the caudal fin has an almost unlimited capacity to regenerate. Even after inhibition of regeneration caused by the loss of Wnt/ß-catenin signalling, a new amputation resets the regeneration capacity within the caudal fin, suggesting that blastema formation does not depend on a pool of stem/progenitor cells that require Wnt/ß-catenin signalling for their survival.     

Two different transgenes to study gene silencing and re-expression during zebrafish caudal fin and retinal regeneration

We used the 500-bp Xenopus ef1-alpha promoter and the 2-kb zebrafish histone 2A.F/Z promoter to generate several independent transgenic zebrafish lines expressing EGFP. While both promoters drive ubiquitous EGFP expression in early zebrafish development, they are systematically silenced in several adult tissues, including the retina and caudal fin. However, EGFP expression is temporarily renewed in the adult during either caudal fin or retinal regeneration. In the Tg(H2A.F/Z:EGFP)nt line, EGFP is moderately expressed in both the wound epithelium and blastema of the regenerating caudal fin. In the Tg(ef1-alpha:EGFP)nt line, EGFP expression is reinitiated and restricted to the blastema of the regenerating caudal fin and colabels with BrdU, PCNA, and msxc-positive cells. Thus, these two ubiquitous promoters drive EGFP transgene expression in different cell populations during caudal fin regeneration. We further analyzed the ability of the ef1-alpha:EGFP transgene to label nonterminally differentiated cells during adult tissue regeneration. First, we demonstrated that the transgene is highly methylated in adult zebrafish caudal fin tissue, but not during fin regeneration, implicating methylation as a potential means of transgene silencing in this line. Next, we determined that the ef1-alpha:EGFP transgene is also re-expressed during adult retinal regeneration. Specifically, the ef1-alpha:EGFP transgene colabels with PCNA in the Müller glia, a specialized cell that is the source of neuronal progenitors during zebrafish retinal regeneration. Thus, we concluded that Tg(ef1-alpha:EGFP)nt line visually marks nonterminally differentiated cells in multiple adult regeneration environments and may prove to be a useful marker in tissue regeneration studies in zebrafish.     

Vestiges,rudiments and fusion events: the zebrafish caudal fin endoskeleton in an evo‐devo perspective

The vertebral column results from a controlled segmentation process associated with two main structures, the notochord and the somites. Pathological fusion of vertebral bodies can result from impaired segmentation during embryonic development or occur postnatally. Here, we explore the process of formation and subsequent fusion of the caudalmost vertebral bodies in zebrafish, where fusion is a normal process, mechanically required to support the caudal fin. To reveal whether the product of fusion is on an evolutionary or a developmental scale, we analyze the mode of formation of vertebral bodies, identify transitory rudiments, and characterize vestiges that indicate previous fusion events. Based on a series of closely spaced ontogenetic stages of cleared and stained zebrafish, parasagittal sections, and detection methods for elastin and mineral, we conclude that the formation of the urostyle involves four fusion events. Although fusion of preural 1 (PU1⁺) with ural 1 (U1) and fusion within ural 2 (U2⁺) are no longer traceable during centrum formation (phylogenetic fusion), fusion between the compound centrum [PU1⁺+U1] and U2⁺ (ontogenetic fusion) occurs after individualization of the centra. This slow process is the last fusion and perhaps the latest fusion during the evolution of the zebrafish caudal fin endoskeleton. Newly described characters, such as a mineralized subdivision within U2⁺, together with the reinterpretation of known features in an evolutionary–developmental context, strongly suggest that the zebrafish caudal fin endoskeleton is made from more fused vertebral bodies than previously assumed. In addition, these fusion events occur at different developmental levels depending on their evolutionary status, allowing the dissection of fusion processes that have taken place over different evolutionary times.     

Functional morphology of the bulbus arteriosus of rainbow trout (Salmo gairdneri Richardson)

The rate of expansion in volume of the bulbus arteriosus with increase in pressure is measured. From this it is calculated the elastic rebound of the bulbus can account for approximately 25 % of blood flow in the ventral aorta; this proportion decreases as cardiac output increases. The structure of the wall of the bulbus is shown to consist of a compact outer layer with a series of separate longitudinal elements on the inner surface. These elements are connected to the compact layer by numerous radial fibres. This structure equalizes strain in all the structural elements of the wall during large changes in volume. Evidence is discussed which shows that the bulbus arteriosus in teleosts is morphologically and biochemically distinct from the ventral aorta. The bulbus is probably of cardiac origin rather than an expansion of the posterior end of the aorta as generally supposed.     

Culture of cells from zebrafish (Brachydanio rerio) embryo and adult tissues

The zebrafish is a popular model for studies of vertebrate development and toxicology. However, in vitro approaches with this organism have not been fully exploited because cell culture systems have been unavailable. We developed methods for the culture of cells from blastula-stage diploid and haploid zebrafish embryos, as well as cells from the caudal and pelvic fin, gill, liver, and viscera of adult fish. The haploid embryo-derived cells differentiated in culture to a pigmented phenotype and expressed, upon exposure to 2,3,7,8-tetrachlorodibenzo p-dioxin, a protein that was immunologically and functionally similar to rainbow trout cytochrome P450IA1 Zebrafish cultures were grown in a complex basal nutrient medium supplemented with insulin, trout embryo extract, and low concentrations of trout and fetal bovine serum; they could not be maintained in conventional culture medium containing a high concentration of mammalian serum. Using calcium phosphate-mediated transfection, a plasmid constructed for use in mammalian cells was introduced into zebrafish embryo cell cultures and expressed in a stable manner. These results indicated that the transfection procedures utilized in mammalian systems can also be applied to zebrafish cell cultures, providing a means for in vitro alteration of the genotype and phenotype of the cells.[/ p]Abbreviations TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin - EROD, 7-ethyoxyresorufin - HDPDS, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - EDTA, ethylanediaminetetraacetic acid - FBS, fetal bovine serum - LDF, limit dilution factor - DMSO, dimethyl sulfoxide - ES, embryonal stem - PAH, polycylic aromatic hydrocarbons - ZG, zebrafish gill - ZBF, zebrafish pelvic fin - ZV, Zebrafish viscera - ZCF, zebrafish caudal fin - ZEM, diploid blastula-derived     

Differentiation of the cardiac outflow tract components in alevins of the sturgeon Acipenser naccarii (Osteichthyes, Acipenseriformes): implications for heart evolution

Previous work showed that in the adult sturgeon an intrapericardial, nonmyocardial segment is interposed between the conus arteriosus of the heart and the ventral aorta. The present report illustrates the ontogeny of this intermediate segment in Acipenser naccarii. The sample studied consisted of 178 alevins between 1 and 24 days posthatching. They were examined using light and electron microscopy. Our observations indicate that the entire cardiac outflow tract displays a myocardial character during early development. Between the fourth and sixth days posthatching, the distal portion of the cardiac outflow tract undergoes a phenotypical transition, from a myocardial to a smooth muscle-like phenotype. The length of this region with regard to the whole outflow tract increases only moderately during subsequent developmental stages, becoming more and more cellularized. The cells soon organize into a pattern that resembles that of the arterial wall. Elastin appears at this site by the seventh day posthatching. Therefore, two distinct components, proximal and distal, can be recognized from the fourth day posthatching in the cardiac outflow tract of A. naccarii. The proximal component is the conus arteriosus, characterized by its myocardial nature and the presence of endocardial cushions. The distal component transforms into the intrapericardial, nonmyocardial segment mentioned above, which is unequivocally of cardiac origin. We propose to designate this segment the "bulbus arteriosus" because it is morphogenetically equivalent to the bulbus arteriosus of teleosts. The present findings, together with data from the literature, point to the possibility that cells from the cardiac neural crest are involved in the phenotypical transition that takes place at the distal portion of the cardiac outflow tract, resulting in the appearance of the bulbus arteriosus. Moreover, they suggest that the cardiac outflow tract came to be formed by a bulbus arteriosus and a conus arteriosus from an early period of the vertebrate evolutionary story. Finally, we hypothesize that the embryonic truncus of birds and mammals is homologous to the bulbus arteriosus of fish.     

Development of a Whole-organism Model to Screen New Compounds for Sun Protection

We used zebrafish as a whole-organism model to screen new compounds for sun protection activity. First of all, we designed a series of UVB exposure experiments and recorded the phenotypic changes of zebrafish embryos. Results showed that 100 mJ/cm² of UVB given six times separated by 30 min intervals is the best condition. Fin malformation (reduced and/or absent fin) phenotypes are the most evident consequences after exposure to UVB. Each fin was affected by UVB, including pelvic, ventral, caudal, and dorsal fin, but pelvic fin seemed to be the most sensitive target after UVB exposure. We furthermore carried out “prevention” and “treatment” experiments using green tea extract and/or (−)-epigallocatechin (EGCG) to test this whole-organism model by observing the morphological changes of all fins (especially pelvic fin) after UVB exposure. Effects of UVB, green tea extract and EGCG on fin development were assessed using the Kaplan–Meier analysis, log-rank test and Cox proportional hazards regression. Results showed that a zebrafish pelvic fin in the UVB + green tea (treatment) group is 5.51 (range from 2.39 to 14.90) times, one in the UVB + green tea (prevention) group is 7.04 (range from 3.11 to 18.92) times, and one in the 25 ppm of EGCG (prevention) group is 22.19 (range from 9.40 to 61.50) times more likely to return to normal fin than one in the UVB only group. On the basis of these observations, we believe this model is effective for screening the higher stability and lower toxicity of new compounds, such as small chemicals which are derivative from EGCG or other dietary agents for sun protection. Yun-Hsin Wang and Chi-Chung Wen contributed equally.     

pyewacket, a new zebrafish fin pigment pattern mutant

Many mutants that disrupt zebrafish embryonic pigment pattern have been isolated, and subsequent cloning of the mutated genes causing these phenotypes has contributed to our understanding of pigment cell development. However, few mutants have been identified that specifically affect development of the adult pigment pattern. Through a mutant screen for adult pigment pattern phenotypes, we identified pyewacket (pye), a novel zebrafish mutant in which development of the adult caudal fin pigment pattern is aberrant. Specifically, pye mutants have fin melanocyte pigment pattern defects and fewer xanthophores than wild-type fins. We mapped pye to an interval where a single gene, the zebrafish ortholog of the human gene DHRSX, is present. pye will be an informative mutant for understanding how xanthophores and melanocytes interact to form the pigment pattern of the adult zebrafish fin.     

Morphogen-based simulation model of ray growth and joint patterning during fin development and regeneration

The fact that some organisms are able to regenerate organs of the correct shape and size following amputation is particularly fascinating, but the mechanism by which this occurs remains poorly understood. The zebrafish (Danio rerio) caudal fin has emerged as a model system for the study of bone development and regeneration. The fin comprises 16 to 18 bony rays, each containing multiple joints along its proximodistal axis that give rise to segments. Experimental observations on fin ray growth, regeneration and joint formation have been described, but no unified theory has yet been put forward to explain how growth and joint patterns are controlled. We present a model for the control of fin ray growth during development and regeneration, integrated with a model for joint pattern formation, which is in agreement with published, as well as new, experimental data. We propose that fin ray growth and joint patterning are coordinated through the interaction of three morphogens. When the model is extended to incorporate multiple rays across the fin, it also accounts for how the caudal fin acquires its shape during development, and regains its correct size and shape following amputation.     

A developmental transition in growth control during zebrafish caudal fin development

A long-standing question in developmental biology is how do growing and developing animals achieve form and then maintain it. We have revealed a critical transition in growth control during zebrafish caudal fin development, wherein a switch from allometric to isometric growth occurs. This morphological transition led us to hypothesize additional physiological changes in growth control pathways. To test this, we fasted juvenile and adult zebrafish. Juvenile fins continued allometric growth until development of the mature bi-lobed shape was completed. In contrast, the isometric growth of mature adult fins arrested within days of initiating a fast. We explored the biochemical basis of this difference in physiology between the two phases by assessing the sensitivity to rapamycin, a drug that blocks a nutrient-sensing pathway. We show that the nutrition-independent, allometric growth phase is resistant to rapamycin at 10-fold higher concentrations than are effective at arresting growth in the nutrition-dependent, isometric growth phase. We thus link a morphological transition in growth control between allometric and isometric growth mechanisms to different physiological responses to nutritional state of the animal and finally to different pharmacological responses to a drug (rapamycin) that affects the nutrition-sensing mechanism described from yeast to human.     

GH indirectly enhances the regeneration of transgenic zebrafish fins through IGF2a and IGF2b

The somatotropic axis, composed essentially of the growth hormone (GH) and insulin-like growth factors (IGFs), is the main regulator of somatic growth in vertebrates. However, these protein hormones are also involved in various other major physiological processes. Although the importance of IGFs in mechanisms involving tissue regeneration has already been established, little is known regarding the direct effects of GH in these processes. In this study, we used a transgenic zebrafish (Danio rerio) model, which overexpresses GH from the beta-actin constitutive promoter. The regenerative ability of the caudal fin was assessed after repeated amputations, as well as the expression of genes related to the GH/IGF axis. The results revealed that GH overexpression increased the regenerated area of the caudal fin in transgenic fish after the second amputation. Transgenic fish also presented a decrease in gene expression of the GH receptor (ghrb), in opposition to the increased expression of the IGF1 receptors (igf1ra and igf1rb). These results suggest that transgenic fish have a higher sensitivity to IGFs than to GH during fin regeneration. With respect to the different IGFs produced locally, a decrease in igf1a expression and a significant increase in both igf2a and igf2b expression was observed, suggesting that igf1a is not directly involved in fin regeneration. Overall, the results revealed that excess GH enhances fin regeneration in zebrafish through igf2a and igf2b expression, acting indirectly on this major physiological process.