A SIMPLE PARAFFIN EMBEDDED PROTOCOL FOR FISH EGG, EMBRYO, AND LARVAE

This paper describes a simple protocol of paraffin-embedded histological section for fish eggs, embryo and larvae of the hard-lipped barb and the giant gourami. The specimens were fixed in Bouin solution, washed in 70% ethanol, then were dehydrated in a series of ethanol solution of increasing concentration until absolute ethanol was reached. The specimens were cleared in graded xylene and were infiltrated with liquid paraffin then were embedded in pure paraffin. Upon sectioning, at 4–5 µm thick the specimens were attached to the gelatin-coated glass slide and let to dry at room temperature or 37°C overnight. The specimens were deparaffinized in xylene, rehydrated then were stained with hematoxylin and eosin. After being dehydrated in graded ethanol, the specimens were cleared in xylene and were mounted with an organic mounting agent. Any step in preparing histological section including samples collection, fixation, dehydration, infiltration and embedding might contribute to the quality of histological features. A proper knowledge of the tissues beeing processed, fixative solution and the histological techniques is essential to gain good results. Bouin fixative is preferable to fix fish larvae and produce a good histological feature. Decalcification is necessary to produce a good histological section on the specimens containing bone

Zebrafish Hagoromo mutants upregulate fgf8 post-embryonically and develop neuroblastoma

We screened an existing collection of zebrafish insertional mutants for cancer susceptibility by histologic examination of heterozygotes at 2 years of age. As most mutants had no altered cancer predisposition, this provided the first comprehensive description of spontaneous tumor spectrum and frequency in adult zebrafish. Moreover, the screen identified four lines, each carrying a different dominant mutant allele of Hagoromo previously linked to adult pigmentation defects, which develop tumors with high penetrance and that histologically resemble neuroblastoma. These tumors are clearly neural in origin, although they do not express catecholaminergic neuronal markers characteristic of human neuroblastoma. The zebrafish tumors result from inappropriate maintenance of a cell population within the cranial ganglia that are likely neural precursors. These neoplasias typically remain small but they can become highly aggressive, initially traveling along cranial nerves, and ultimately filling the head. The developmental origin of these tumors is highly reminiscent of human neuroblastoma. The four mutant Hagoromo alleles all contain viral insertions in the fbxw4 gene, which encodes an F-box WD40 domain–containing protein. However, although one allele clearly reduced the levels of fbxw4 mRNA, the other three insertions had no detectable effect on fbw4 expression. Instead, we showed that all four mutations result in the postembryonic up-regulation of the neighboring gene, fibroblast growth factor 8 (fgf8). Moreover, fgf8 is highly expressed in the tumorigenic lesions. Although fgf8 overexpression is known to be associated with breast and prostate cancer in mammals, this study provides the first evidence that fgf8 misregulation can lead to neural tumors. (Mol Cancer Res 2009;7(6):841–50)National Cancer Institute (U.S.) (Grant CA106416

Nkx2.7 and Nkx2.5 Function Redundantly and Are Required for Cardiac Morphogenesis of Zebrafish Embryos

Nkx2.7 is the tinman-related gene, as well as orthologs of Nkx2.5 and Nkx-2.3. Nkx2.7 and Nkx2.5 express in zebrafish heart fields of lateral plate mesoderm. The temporal and spatial expression patterns of Nkx2.7 are similar to those of Nkx2.5, but their functions during cardiogenesis remain unclear.Here, Nkx2.7 is demonstrated to compensate for Nkx2.5 loss of function and play a predominant role in the lateral development of the heart, including normal cardiac looping and chamber formation. Knocking down Nkx2.5 showed that heart development was normal from 24 to 72 hpf. However, when knocking down either Nkx2.7 or Nkx2.5 together with Nkx2.7, it appeared that the heart failed to undergo looping and showed defective chambers, although embryos developed normally before the early heart tube stage. Decreased ventricular myocardium proliferation and defective myocardial differentiation appeared to result from late-stage up-regulation of bmp4, versican, tbx5 and tbx20, which were all expressed normally in hearts at an early stage. We also found that tbx5 and tbx20 were modulated by Nkx2.7 through the heart maturation stage because an inducible overexpression of Nkx2.7 in the heart caused down-regulation of tbx5 and tbx20. Although heart defects were induced by overexpression of an injection of 150-pg Nkx2.5 or 5-pg Nkx2.7 mRNA, either Nkx2.5 or Nkx2.7 mRNA rescued the defects induced by Nkx2.7-morpholino(MO) and Nkx2.5-MO with Nkx2.7-MO.Therefore, we conclude that redundant activities of Nkx2.5 and Nkx2.7 are required for cardiac morphogenesis, but that Nkx2.7 plays a more critical function, specifically indicated by the gain-of-function and loss-of- function experiments where Nkx2.7 is observed to regulate the expressions of tbx5 and tbx20 through the maturation stage

Immunopathogenesis of chronic Mycobacterium marinum infection in adult zebrafish (Danio rerio)

Tuberculosis (TB) is still a global epidemic disease despite its discovery over 100 years ago. It is caused by Mycobacterium tuberculosis, which invades and replicates within macrophages, key cells of the innate immune system. The hallmark of tuberculosis is the granuloma which is an accumulation of Mycobacterium-infected cells surrounded by immune cells, and the containment of the bacteria is assured as long as the host immune response remains intact. Despite a well-developed immune response in the infected host, reactivation of latent tuberculosis infection (LTBI) may occur through the introduction of other bacterial pathogens, re-infection with M. tuberculosis or due to other immunosuppression, e.g. AIDS or cancer. The zebrafish–M. marinum model provides an ideal system for examining the pathogenesis of tuberculosis and the associated immune response of the host due to its vertebrate-like immune system, and the close phylogenetic relationship of M. marinum to M. tuberculosis. Granuloma formation and immune response to M. marinum have been investigated mainly in zebrafish embryos or larvae, which lack an adaptive immune response, and little work has been performed in adult fish. This complicates the transfer of findings in these models to chronic, latent or re-activated disease stages in humans, where adaptive immunity plays an important part. The aim of the research presented here was to investigate the immune response of the adult zebrafish to M. marinum infection, with the focus on the kidney as one of the major immune organs in fish. The results obtained support further use of the adult zebrafish-M. marinum model for human tuberculosis infections in the future. In the present study, adult zebrafish were infected with low doses of M. marinum (NCIMB 1297 or NCIMB 1298) and the kidney was investigated for histopathological changes in the form of granulomas over a period of two months(Chapter 3). No granulomas were detected in the fish infected with M. marinum NCIMB 1298 while in zebrafish infected with NCIMB 1297, macrophage aggregation and granuloma formation were detected as early as day 11 post-infection. Occurrence and severity of granulomas and the presence of replicating bacteria increased over time, resulting in a high density of non-caseating and caseating granulomas in the head and posterior kidney after two months of infection. Interleukin 1 beta (IL-1β), Interleukin-12 (IL-12), Tumor necrosis factor alpha (TNFα) and Interferon gamma (IFNγ) have been shown to be important cytokines functioning in defence against tuberculosis, especially IFNγ which is considered to play an important part in acute, chronic and latent tuberculosis. Changes in gene expression of these immune genes in adult zebrafish were investigated over the first two weeks of infection with M. marinum NCIMB 1298 and NCIMB 1297. The results obtained in the first week after infection were inconclusive for both strains investigated. In agreement with the results presented in Chapter 3, no specific immune response was detectable in fish infected with M. marinum NCIMB 1298. However, after 14 days, a high-fold change in IL-12 and TNFα expression were detected in fish infected with M. marinum NCIMB 1297, while IL-1β showed no changes compared to the control fish. Furthermore, no IFNγ expression was detectable over the first two weeks of infection. The delay in the expression of IL-12 and the lack of IFNγ expression can be explained by the ability of M. marinum to manipulate the host immune response, as described for M. tuberculosis and other intracellular bacteria. Besides in vivo investigations of the host-pathogen interactions, in vitro primary macrophage cultures from individual zebrafish kidneys were developed to investigate macrophage-specific gene expression to M. marinum infection (Chapter 4). Although the results looked promising, further optimization is required before the results of the in vitro assays can be fully compared to the in vivo results. Our understanding of reactivation in latent tuberculosis infection (LTBI) both in healthy and immune compromised individuals is insufficient and is delaying the development of treatments for the disease. Therefore, the transcriptome profile of long-term infections (26 weeks) with M. marinum NCIMB 1297 in adult zebrafish was investigated to determine whether the gene expression in this model is comparable to LTBI in humans or other vertebrate model organisms (Chapter 5). In addition, transcriptome profiling was investigated in a group of long-term infected zebrafish exposed to stress to induce re-activation of the disease. Expression profiles in the long-term infected fish and the infected plus stressed fish differed from each other and displayed similar gene profiles to those found in the latent or re-activated disease states, respectively, in human and other vertebrate models. Infected fish displayed a profile highlighted by IFNγ, TNFα, NOS2b and IL-8 expression alongside activating and regulatory T cell responses, including involvement of cytotoxic T cells (CTLs). The transcriptome profile of the group of fish that had been infected and then stressed was distinguished by the lack of IFNγ expression and reduction in TNFα and NOS2b expression, as well as a lack of T cell response compared to the infected fish. In conclusion, the results obtained from Chapters 3 and 4 showed that M. marinum NCIMB 1298 is non-pathogenic to zebrafish. Infection with M. marinum NCIMB 1297, on the other hand, resulted in a similar immune response to that described for human and other mammalian vertebrate models (Chapters 3-5). These results support the use of the adult zebrafish-M. marinum model to investigate LTBI and disease reactivation, and will aid our understanding host-pathogen interactions for tuberculosis in the future

Whole-Body Regeneration

This Open Access volume provides a comprehensive overview of the latest tools available to scientists to study the many facets of whole-body regeneration (WBR). The chapters in this book are organized into six parts. Part One provides a historical overview on the study of the WBR phenomena focusing on the primary challenges of this research. Parts Two and Three explore a series of non-vertebrate zoological contexts that provide experimental models for WBR, showing how they can be approached with cellular tools. Parts Four, Five, and Six discuss the future advancements of WBR, reporting about the cutting-edge techniques in genetics and omics used to dissect the underlying mechanisms of WBR, and systems biology approaches to reach a synthetic view of WBR. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and thorough, Whole-Body Regeneration: Methods and Protocols is a valuable resource for scientists and researchers who want to learn more about this important and developing field

Whole-Body Regeneration

This Open Access volume provides a comprehensive overview of the latest tools available to scientists to study the many facets of whole-body regeneration (WBR). The chapters in this book are organized into six parts. Part One provides a historical overview on the study of the WBR phenomena focusing on the primary challenges of this research. Parts Two and Three explore a series of non-vertebrate zoological contexts that provide experimental models for WBR, showing how they can be approached with cellular tools. Parts Four, Five, and Six discuss the future advancements of WBR, reporting about the cutting-edge techniques in genetics and omics used to dissect the underlying mechanisms of WBR, and systems biology approaches to reach a synthetic view of WBR. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and thorough, Whole-Body Regeneration: Methods and Protocols is a valuable resource for scientists and researchers who want to learn more about this important and developing field

Control sanitario en colonias de peces cebra (<i>Danio rerio</i>) de Argentina utilizados en experimentación

El pez cebra, Brachydanio rerio (también conocido como Danio rerio), es utilizado como modelo de desarrollo embrionario de vertebrados, para el análisis de la función génica, y mutagénesis. Previo al desarrollo del modelo del pez cebra en la década de 1970, genetistas del desarrollo experimentaban con modelos de invertebrados como Drosophila melanogaster (mosca de la fruta) y, más recientemente con Caenorhabditis elegans (nematodo) para la investigación del desarrollo embrionario temprano. La combinación de una prolífica capacidad reproductiva de Drosophila y la manipulación exitosa de sus embriones permite hacer ensayos de desarrollo embrionario y análisis genético de forma práctica en el laboratorio. Sin embargo, en relación al desarrollo embrionario, la aplicación de esta información a vertebrados fue limitada. Desde embriones de ratones que se desarrollan dentro de un útero hasta la rana de garras africana (Xenopus laevis) tienen una capacidad reproductiva lenta, y ninguno de estos modelos de vertebrados más populares poseen las características que hizo de Drosophila un modelo tan práctico (Kahn, 1994). Por su alta fecundidad y fertilización externa, el pez cebra poseía los atributos de estos modelos existentes, sin sus inconvenientes inherentes. Debido a que los mecanismos moleculares fundamentales del desarrollo de un embrión son similares para todos los vertebrados, posibilitó que el pez cebra, se haya convertido gradualmente en el modelo vertebrado inferior de elección.Facultad de Ciencias Veterinaria

The Use And Development Of Laser Microdissection To Separate Spermatozoa From Epithelial Cells For Str Analysis

Short Tandem Repeat (STR) analysis has become a valuable tool in identifying the source of biological stains, particularly from the investigation of sexual assault crimes. Difficulties in analysis arise primarily in the interpretation of mixed genotypes when cell separation of the sexual assailant\u27s sperm from the victim\u27s cells is incomplete. The forensic community continues to seek improvements in cell separation methods from mixtures for DNA typing. This report describes the use of laser microdissection (LMD) for the separation of pure populations of spermatozoa from two-donor cell mixtures. In this study, cell separation was demonstrated by microscopic identification of histologically stained spermatozoa and female buccal cell mixtures, and STR analysis of DNA obtained from the separated sperm cells. Clear profiles of the male donor were obtained with the absence of any additional alleles from the female donor. Five histological stains were evaluated for use with LMD and DNA analysis: hematoxylin/eosin, nuclear fast red/picroindigocarmine, methyl green, Wright\u27s stain, and acridine orange. Hematoxylin/eosin out-performed all other stains however nuclear fast red/picroindigocarmine could be used satisfactorily with STR analysis. In addition, three DNA isolation methods were evaluated for LMD collected cells: QIAamp (Qiagen), microLYSIS (Microzone Ltd.) and Lyse-N-Go (Pierce Chemical Co.). MicroLYSIS performed poorly, yielding low levels of PCR product. Lyse-N-Go extraction was effective for the recovery of DNA from LMD collected sperm cells while QIAamp isolation performed best for the recovery of DNA from LMD collected epithelial cells. LMD is shown to be an effective, low-manipulation separation method that enables the recovery of sperm while excluding epithelial cell DNA

Mass spectrometry techniques to assess the metabolism and toxicity of pharmaceutical and dietary nitrates

Nitric oxide (NO) is a signaling molecule that regulates blood pressure and vascular tone. Humans produce NO by endothelial nitric oxide synthase (eNOS), which is impaired in patients with cardiovascular disease leading to increased blood pressure, endothelial dysfunction, and an increased risk of adverse cardiovascular outcomes. Maintaining optimal levels of NO is critical for human health. Inorganic nitrate (NO3-) is reduced to NO and can be an alternate pathway to restore NO production. Diets high in nitrate reduce blood pressure and improve exercise performance in humans. In patients with advanced cardiovascular disease, organic nitrates such as nitroglycerin (glyceryl trinitrate, GTN) release NO and are essential for managing symptoms. This dissertation emphasizes mass spectrometry techniques to elucidate novel mechanisms for the effects of both organic and inorganic nitrate on cardiovascular health and exercise performance.Pharmaceutical Nitrate. Patients develop tolerance to GTN after several weeks of continuous use, limiting the potential for long-term therapy. The cause of nitrate tolerance is relatively unknown. I developed a cell culture model of nitrate tolerance that utilizes stable isotopes to measure metabolism of 15N3-GTN into 15N-nitrite. I performed global metabolomics to identify the mechanism of GTN-induced nitrate tolerance and to elucidate the protective role of vitamin C. Metabolomics demonstrated that GTN impaired purine metabolism and depleted intracellular ATP and GTP. GTN inactivated the enzyme xanthine oxidase (XO), an enzyme that is critical for the metabolism of GTN into NO. Vitamin C prevented inactivation of XO, resulting in increased NO production from GTN. Vitamin C supplementation should be further investigated as a simple and inexpensive strategy to prevent nitrate tolerance.Dietary Nitrate. Inorganic nitrate improves exercise performance by reducing the oxygen cost of exercise. Although previous research has suggested that nitrate improves mitochondrial efficiency and stimulates mitochondrial biogenesis, we have an incomplete understanding of the mechanism. In a zebrafish (Danio rerio) model, nitrate reduced the oxygen cost of exercise during a vigorous 2-hour exercise test. Although there were no significant differences in mitochondrial function between control and nitrate-treated zebrafish, nitrate treatment increased resting ADP, ATP, and cyclic AMP levels. Metabolomics analysis of whole fish illustrated that nitrate stimulated glycolysis and ketogenesis. I conclude that nitrate reduces the oxygen cost of exercise by favoring glycolysis for energy production, thereby producing more ATP while consuming less oxygen. Nitrate-stimulated alteration of metabolic fuels for energy production is a novel mechanism for the improvement in exercise performance.Summary. This dissertation is a significant contribution to scientific knowledge because of the development of a novel assay to measure 15N-labeled nitrate and nitrite, which I have applied to study the metabolism of pharmaceutical and dietary nitrates. Using metabolomics, I elucidated the novel mechanism that vitamin C prevents nitrate tolerance by protecting XO from inactivation. Furthermore, I demonstrated that nitrate alters the utilization of metabolic fuels, leading to reduced oxygen consumption during exercise.Keywords: pharmacology, zebrafish, toxicology, metabolomics, nitric oxide, nitrat