Transgenic Fish Research Articles

Assessing Ecological and Evolutionary Consequences of Growth-Accelerated Genetically Engineered Fishes

Genetically engineered fish containing growth hormone (GH) transgenes have been synthesized for more than 25 years, now with modifications made in multiple aquacultured species. Despite significant improvements in production characteristics being realized, these fish have not yet entered commercial production. The very strong enhancement of growth rates that can arise from GH transgenesis in fish has generated public and scientific concern regarding ecological and food safety. Little ecological risk is anticipated from engineered strains kept in fully contained facilities, so the concern is largely directed toward the reliability of containment measures and determining whether robust ecological data, pertinent to nature, can be generated within research facilities to minimize uncertainty and allow reliable risk-assessment predictions. This article summarizes the growth, life history, and behavioral changes observed in GH-transgenic fish and discusses the environmental and evolutionary factors affecting the adaptation, plasticity, and fitness of transgenic fish and their potential consequences on natural ecosystems.

Effects of somatotrophic axis (GH/GHR) double transgenesis on structural and molecular aspects of the zebrafish immune system.

The development of growth hormone (GH) transgenic fish has been shown to be a promising method to improve growth rates. However, the role of GH is not restricted only to processes involved in growth. Several others physiological processes, including immune function, are impaired due to GH imbalances. Given the importance of generating GH transgenic organisms for aquaculture purposes, it is necessary to develop strategies to reduce or compensate for the collateral effects of GH. We hypothesized that the generation of double transgenic fish that overexpress GH and growth hormone receptor (GHR) in the skeletal muscle could be a possible alternative to compensate for the deleterious effects of GH on the immune system. Specifically, we hypothesized that increased GHR amounts in the skeletal muscle would be able to reduce the level of circulating GH, attenuating the GH signaling on the immune cells while still increasing the growth rate. To test this hypothesis, we evaluated the size of the immune organs, T cell content in the thymus and head kidney, and expression of immune-related genes in double-transgenic fish. Contrary to our expectations, we found that the overexpression of GHR does not decrease the deleterious effect of GH excess on the size of the thymus and head kidney, and in the content of CD3(+) and CD4(+) cells in the thymus and head kidney. Unexpectedly, the control GHR transgenic group showed similar impairments in immune system parameters. These results indicate that GHR overexpression does not reverse the impairments caused by GH and, in addition, could reinforce the damage to the immune functions in GH transgenic zebrafish.

Fate mapping of ptf1a-expressing cells during pancreatic organogenesis and regeneration in zebrafish.

Pancreas development in zebrafish shares many features with mammals, including the participation of epithelial progenitor cells expressing pancreas transcription factor 1a (ptf1a). However, to date it has remained unclear whether, as in mammals, ptf1a-expressing zebrafish pancreatic progenitors are able to contribute to multiple exocrine and endocrine lineages. To delineate the lineage potential of ptf1a-expressing cells, we generated ptf1a:creER(T2) transgenic fish and performed genetic-inducible lineage tracing in developmental, regenerating, and ptf1a-deficient zebrafish pancreas. In addition to their contribution to the acinar cell lineage, ptf1a-expressing cells give rise to both pancreatic Notch-responsive-cells (PNCs) as well as small numbers of endocrine cells during pancreatic development. In fish with ptf1a haploinsufficiency, a higher proportion of ptf1a lineage-labeled cells are traced into the PNC and endocrine compartments. Further reduction of ptf1a gene dosage converts pancreatic progenitor cells to gall bladder and other non-pancreatic cell fates. Our results confirm the presence of multipotent ptf1a-expressing progenitor cells in developing zebrafish pancreas, with reduced ptf1a dosage promoting greater contributions towards non-acinar lineages. As in mammals, loss of ptf1a results in conversion of nascent pancreatic progenitor cells to non-pancreatic cell fates, underscoring the central role of ptf1a in foregut tissue specification.

Food intake and appetite control in a GH-transgenic zebrafish.

The biological actions of growth hormone (GH) are pleiotropic, including growth promotion, energy mobilization, gonadal development, appetite, and social behavior. The regulatory network for GH is complex and includes many central and peripheral endocrine factors as well as that from the environment. It is known that GH transgenesis results in increased growth, food intake, and consequent metabolic rates in fishes. However, the manner in which GH transgenesis alters the energetic metabolism in fishes has not been well explored. In order to elucidate these consequences, we examined the effect of GH overexpression on appetite control mechanisms in a transgenic zebrafish (Danio rerio) model. To this, we analyzed feeding behavior and the expression of the main appetite-related genes in two different feeding periods (fed and fasting) in non-transgenic (NT) and transgenic (T) zebrafish as well as glycaemic parameters of them. Our initial results have shown that NT males and females present the same feeding behavior and expression of main appetite-controlling genes; therefore, the data of both sexes were properly grouped. Following grouped data analyses, we compared the same parameters in NT and T animals. Feeding behavior results have shown that T animals eat significantly more and faster than NT siblings. Gene expression results pointed out that gastrointestinal (GT) cholecystokinin has a substantial contribution to the communication between peripheral and central control of food intake. Brain genes expression analyses revealed that T animals have a down-regulation of two strong and opposite peptides related to food intake: the anorexigenic proopiomelanocortin (pomc) and the orexigenic neuropeptide Y (npy). The down-regulation of pomc in T when compared with NT is an expected result, since the decrease in an anorexigenic factor might keep the transgenic fish hungry. The down-regulation of npy seemed to be contradictory at first, but if we consider the GH's capacity to elevate blood glucose, and that NPY is able to respond to humoral factors like glucose, this down-regulation makes sense. In fact, our last experiment showed that transgenics presented elevated blood glucose levels, confirming that npy might responded to this humoral factor. In conclusion, we have shown that GT responds to feeding status without interference of transgenesis, whereas brain responds to GH transgenesis without any effect of treatment. It is clear that transgenic zebrafish eat more and faster, and it seems that it occurs due to pomc down-regulation, since npy might be under regulation of the humoral factor glucose.

TOWARD THE ESTABLISHMENT OF THE TOOLS FOR MONITORING COASTAL ENVIRONMENTS UTILIZING GENE RESPONSE IN ORYZIAS FISHES

Southeast Asia is known as a center of biodiversity of the earth. As economic growth of this region is remarkable, conservation of biodiversity is a top priority issue. For this purpose, it is important to monitor the environmental condition in effective ways. We are trying to detect pollutants in coastal and estuary waters through the expression level of pollutant-responsive genes of rice fishes of the genus Oryzias, widely distributed in Asia. Japanese medaka O. latipes is a useful model because whole genome sequence is available. Javanese medaka O. javanicus and Indian medaka O. dancena, both of which are widely distributed in Southeast Asia and adaptable to seawater, are also potential models. One possible method for pollution monitoring is the use of transgenic fish bearing artificialgene construct containing the pollutant-responsive promoter and a reporter gene. For example, transgenic strains that can detect estrogen-like substances have already been established. It is also probable to detect pollutants by quantifying mRNA or proteins expressed from the pollutant-responsive gene. In any case, the most important point is to identify the gene that responds to specific pollutants. Comprehensive transcriptomic analyses are powerful tool for this purpose. Organotin-responsive genes are being screened at present.

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic “drivers” among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.

Mosaic zebrafish transgenesis for functional genomic analysis of candidate cooperative genes in tumor pathogenesis.

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic "drivers" among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.

Concatemer-Associated Transgene Expression Patterns in Transgenic Marine Medaka Oryzias dancena Strains

To examine the interrelationship between transgenic insertion patterns and transgene expression profiles in established transgenic fish lines, four s table transgenic marine medaka Oryzias dancena germlines harboring β-actin regulator-driven RFP reporter constructs were selected. The established transgenic strains were characterized with regard to their transgenic genotypes (insertion pattern, concatemer formation, and transgene copy number based on genomic Southern blot hybridization and qPCR assay) and expression characteristics at the mRNA (qRT-PCR), protein (western blot), and phenotypic (fluorescent appearance) levels. From comparative examinations, it was found that transgenic expression at both the transcription and translation levels could be signifi cantly downregulated in transgenic strains, potentially through methylation-mediated transgene silencing that was particularly associated with the formation of a long tail-to-head tandem concatemer in the chromosomal integration site(s). When this occurred, an inverse relationship between the transgene copy number and fluorescence intensity was observed in the resultant transgenic fish. However, with the other transgenic genotype, transgenic individuals with an identical Southern blot hybridization pattern, containing a tandem concatemer(s), had very different expression levels (highly robust vs. low expression strengths), which was possibly related to the differential epigenetic modifications and/or degrees of methylation. The concatemer-dependent downregulation of transgene activity could be induced in transgenic fish, but the overall pattern was strain-specific. Our data suggest that neither a low (or single) transgene copy number nor tandem transgene concatemerization is indicative of strong or silenced transgene expression in transgenic fish carrying a ubiquitous transgene. Hence, a sufficient number of transgenic lineages, with different genotypes, should be considered to ensure the establishment of the best-performance transgenic line(s) for practical applications.

Early life-history consequences of growth-hormone transgenesis in rainbow trout reared in stream ecosystem mesocosms.

There is persistent commercial interest in the use of growth modified fishes for shortening production cycles and increasing overall food production, but there is concern over the potential impact that transgenic fishes might have if ever released into nature. To explore the ecological consequences of transgenic fish, we performed two experiments in which the early growth and survival of growth-hormone transgenic rainbow trout (Oncorhynchus mykiss) were assessed in naturalized stream mesocosms that either contained predators or were predator-free. We paid special attention to the survival bottleneck that occurs during the early life-history of salmonids, and conducted experiments at two age classes (first-feeding fry and 60 days post-first-feeding) that lie on either side of the bottleneck. In the late summer, the first-feeding transgenic trout could not match the growth potential of their wild-type siblings when reared in a hydrodynamically complex and oligotrophic environment, irrespective of predation pressure. Furthermore, overall survival of transgenic fry was lower than in wild-type (transgenic = 30% without predators, 8% with predators; wild-type = 81% without predators, 31% with predators). In the experiment with 60-day old fry, we explored the effects of the transgene in different genetic backgrounds (wild versus domesticated). We found no difference in overwinter survival but significantly higher growth by transgenic trout, irrespective of genetic background. We conclude that the high mortality of GH-transgenic trout during first-feeding reflects an inability to sustain the basic metabolic requirements necessary for life in complex, stream environments. However, when older, GH-transgenic fish display a competitive advantage over wild-type fry, and show greater growth and equal survival as wild-type. These results demonstrate how developmental age and time of year can influence the response of genotypes to environmental conditions. We therefore urge caution when extrapolating the results of GH-transgenesis risk assessment studies across multiple life-history or developmental stages.

Time point-based integrative analyses of deep-transcriptome identify four signal pathways in blastemal regeneration of zebrafish lower jaw.

There has been growing interest in applying tissue engineering to stem cell-based regeneration therapies. We have previously reported that zebrafish can faithfully regenerate complicated tissue structures through blastemal cell type conversions and tissue reorganization. To unveil the regenerative factors and engineering arts of blastemal regeneration, we conducted transcriptomal analyses at four time points corresponding to preamputation, re-epitheliation, blastemal formation, and respecification. By combining the hierarchical gene ontology term network, the DAVID annotation system, and Euclidean distance clustering, we identified four signaling pathways: foxi1-foxo1b-pou3f1, pax3a-mant3a-col11/col2, pou5f1-cdx4-kdrl, and isl1-wnt11 PCP-sox9a. Results from immunohistochemical staining and promoter-driven transgenic fish suggest that these pathways, respectively, define wound epidermis reconstitution, cell type conversions, blastemal angiogenesis/vasculogenesis, and cartilage matrix-orientation. Foxi1 morpholino-knockdown caused expansions of Foxo1b- and Pax3a-expression in the basal layer-blastemal junction region. Moreover, foxi1 morphants displayed increased sox9a and hoxa2b transcripts in the embryonic pharyngeal arches. Thus, a Foxi1 signal switch is required to establish correct tissue patterns, including re-epitheliation and blastema formation. This study provides novel insight into a blastema regeneration strategy devised by epithelial cell transdifferentiation, blood vessel engineering, and cartilage matrix deposition.

Sufficient Numbers of Early Germ Cells Are Essential for Female Sex Development in Zebrafish

The sex determination for zebrafish is controlled by a combination of genetic and environmental factors. The determination of sex in zebrafish has been suggested to rely on a mechanism that is affected by germ cell-derived signals. To begin our current study, a simplified and efficient germ cell-specific promoter of the dead end (dnd) gene was identified. Utilizing the metrodinazole (MTZ)/ bacterial nitroreductase (NTR) system for inducible germ cell ablation, several stable Tg (dnd:NTR-EGFP-3'UTR) and Tg (dnd:NTR-EGFP+3'UTR) zebrafish lines were then generated with the identified promoter. A thorough comparison of the expression patterns and tissue distributions of endogenous dnd and ntr-egfp transcripts in vivo revealed that the identified 2032-bp zebrafish dnd promoter can recapitulate dnd expression faithfully in stable transgenic zebrafish. The correlation between the levels of the germ cell-derived signals and requirement for maintaining the female fate has been also explored with different durations of the MTZ treatments. Our results revealed the decreasing ratios of female presented in the treated transgenic group are fairly associated with the reducing levels of the early germ cell-derived signals. After the juvenile transgenic fish treated with 5 mM MTZ for 20 days, all MTZ-treated transgenic fish exclusively developed into males with subfertilities. Taken together, our results identified here a simplified and efficient dnd promoter, and provide clear evidence indicating that it was not the presence but the sufficiency of signals derived from germ cells that is essential for female sex development in zebrafish. Our model also provides a unique system for sex control in zebrafish studies.

The role of Sox6 in zebrafish muscle fiber type specification.

BackgroundThe transcription factor Sox6 has been implicated in regulating muscle fiber type-specific gene expression in mammals. In zebrafish, loss of function of the transcription factor Prdm1a results in a slow to fast-twitch fiber type transformation presaged by ectopic expression of sox6 in slow-twitch progenitors. Morpholino-mediated Sox6 knockdown can suppress this transformation but causes ectopic expression of only one of three slow-twitch specific genes assayed. Here, we use gain and loss of function analysis to analyse further the role of Sox6 in zebrafish muscle fiber type specification.MethodsThe GAL4 binary misexpression system was used to express Sox6 ectopically in zebrafish embryos. Cis-regulatory elements were characterized using transgenic fish. Zinc finger nuclease mediated targeted mutagenesis was used to analyse the effects of loss of Sox6 function in embryonic, larval and adult zebrafish. Zebrafish transgenic for the GCaMP3 Calcium reporter were used to assay Ca2+ transients in wild-type and mutant muscle fibres.ResultsEctopic Sox6 expression is sufficient to downregulate slow-twitch specific gene expression in zebrafish embryos. Cis-regulatory elements upstream of the slow myosin heavy chain 1 (smyhc1) and slow troponin c (tnnc1b) genes contain putative Sox6 binding sites required for repression of the former but not the latter. Embryos homozygous for sox6 null alleles expressed tnnc1b throughout the fast-twitch muscle whereas other slow-specific muscle genes, including smyhc1, were expressed ectopically in only a subset of fast-twitch fibers. Ca2+ transients in sox6 mutant fast-twitch fibers were intermediate in their speed and amplitude between those of wild-type slow- and fast-twitch fibers. sox6 homozygotes survived to adulthood and exhibited continued misexpression of tnnc1b as well as smaller slow-twitch fibers. They also exhibited a striking curvature of the spine.ConclusionsThe Sox6 transcription factor is a key regulator of fast-twitch muscle fiber differentiation in the zebrafish, a role similar to that ascribed to its murine ortholog.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-014-0026-2) contains supplementary material, which is available to authorized users.

Myc-induced liver tumors in transgenic zebrafish can regress in tp53 null mutation.

Hepatocellular carcinoma (HCC) is currently one of the top lethal cancers with an increasing trend. Deregulation of MYC in HCC is frequently detected and always correlated with poor prognosis. As the zebrafish genome contains two differentially expressed zebrafish myc orthologs, myca and mycb, it remains unclear about the oncogenicity of the two zebrafish myc genes. In the present study, we developed two transgenic zebrafish lines to over-express myca and mycb respectively in the liver using a mifepristone-inducible system and found that both myc genes were oncogenic. Moreover, the transgenic expression of myca in hepatocytes caused robust liver tumors with several distinct phenotypes of variable severity. ~5% of myca transgenic fish developing multinodular HCC with cirrhosis after 8 months of induced myca expression. Apoptosis was also observed with myca expression; introduction of homozygous tp53-/- mutation into the myca transgenic fish reduced apoptosis and accelerated tumor progression. The malignant status of hepatocytes was dependent on continued expression of myca; withdrawal of the mifepristone inducer resulted in a rapid regression of liver tumors, and the tumor regression occurred even in the tp53-/- mutation background. Thus, our data demonstrated the robust oncogenicity of zebrafish myca and the requirement of sustained Myc overexpression for maintenance of the liver tumor phenotype in this transgenic model. Furthermore, tumor regression is independent of the function of Tp53.

Transgenic modification of the n-3 HUFA biosynthetic pathway in nibe croaker larvae: improved DPA (docosapentaenoic acid; 22:5n-3) production

Marine fish are generally unable to produce sufficient quantities of n-3 highly unsaturated fatty acid (n-3 HUFA) such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). Consequently, the seed production of marine fish requires careful nutritional enrichment of live feeds such as rotifers and brine shrimp Artemia to meet n-3 HUFA requirements for normal growth. Another strategy for improving n-3 HUFA availability is modifying the biosynthetic pathway of marine fish using transgenic technology. In this study, we conducted a feeding trial with non-transgenic and transgenic nibe croaker Nibea mitsukurii carrying the elongation of very long-chain fatty acids protein 2 (Elovl2) gene isolated from masu salmon Oncorhynchus masou and three groups of Artemia (non-enriched and enriched with two products). For all Artemia groups, docosapentaenoic acid (DPA, 22:5n-3), which is a direct product of Elovl2, was significantly higher in the transgenic fish than that in non-transgenic fish, despite the absence of DPA in all diets. Thus, applying transgenic techniques to marine fish at the larval stage are a powerful strategy for modifying n-3 HUFA biosynthetic pathways.

A controllable on-off strategy for the reproductive containment of fish

A major impediment to the commercialization and cultivation of transgenic fish is the potential ecological risks they pose to natural environments: a problem that could be solved by the production of sterile transgenic fish. Here, we have developed an on-off reproductive containment strategy for fish that renders the offspring sterile but leaves their parents fertile. TG1 (Tol2-CMV-GFP-pA-CMV-gal4-pA-Tol2) and TG2 (Tol2-CMV-RFP-pA-5 × UAS-as/dnd-pA-Tol2) zebrafish lines were established using a GAL4/UAS system. While the parental lines remained fertile, in the hybrid offspring, GAL4 induced 5 × UAS to drive the transcription of antisense dnd, which significantly down-regulated endogenous dnd expression. This disrupted the migration of primordial germ cells (PGCs), led to their apoptosis, and resulted in few or no PGCs migrating to the genital ridge. This process induced sterility or reduced fertility in adult fish. This on-off strategy is a potentially effective means of generating sterile fish for commercialization while retaining fertility in brood stocks, and offers a novel method to mitigate the ecological risks of fish introductions.

Importance of Transgenic Fish to Global Aquaculture: A Review

Many faster growing transgenic fish including both cold water (salmon, trout) and warm water (tilapia, carp) species have been produced. The development of transgenic fish can serve as excellent experimental models for basic scientific investigations, environmental toxicology and in biotechnological applications. The fast growth characteristic of GH gene transgenic fish will be of great importance to support aquaculture production and economic efficiency. The transgenic fish are efficient feed converter, thus more economical for the fish farmer as the feed accounts for 60-70% of total cost in aquaculture.

The β-actin gene promoter of rohu carp (Labeo rohita) drives reporter gene expressions in transgenic rohu and various cell lines, including spermatogonial stem cells.

We previously characterized the β-actin gene promoter of Indian domesticated rohu carp (Labeo rohita) and made a reporter construct via fusion to green fluorescence protein (GFP) cDNA. In this study, the same construct was used to breed transgenic rohu fish. About 20% of the transgenic offspring showed ubiquitous expression of the reporter GFP gene. In a few of the transgenic fish, we documented massive epithelial and/or muscular expression with visible green color under normal light. The expression of GFP mRNA was higher in the muscle tissue of transgenic fish than in that of non-transgenic fish. A highly efficient nucleofection protocol was optimized to transfect proliferating spermatogonial cells of rohu using this reporter construct. The β-actin promoter also drove expressions in HEK293 (derived from human embryonic kidney cells), K562 (human leukemic cells) and SF21 (insect ovarian cells) lines. These findings imply conserved regulatory mechanisms of β-actin gene expression across eukaryotes. Furthermore, the isolated β-actin promoter with consensus regulatory elements has the potential to be used in generating transgenic carp with genes of interest and in basic biology research.

A Cyan Fluorescent Protein Gene (cfp)-Transgenic Marine Medaka Oryzias dancena with Potential Ornamental Applications

Abstract To evaluate their potential utility as an ornamental organism, novel transgenic marine medaka Oryzias dancena strains with a highly vivid fluorescent phenotype were established through transgenesis of a cyan fluorescent protein gene ( cfp ) driven by the endogenous fast skeletal myosin light chain 2 gene ( mlc2f ) promoter. The transgenic marine medaka strains possessed multiple copies of transgene integrants and passed their fluorescent transgenes successfully to subsequent generations. Transgenic expres-sion in skeletal muscles at both the mRNA and phenotypic levels was, overall, dependent upon transgene copy numbers. In the external phenotype, an authentic fluorescent color was dominant in the skeletal muscles of the transgenic fish and clearly visible to the unaided eye. The phenotypic fluorescent color presented differentially in response to different light-irradiation sources; the transgenics displayed a yellow–green color under normal daylight or white room light conditions, a strong green-glowing fluores-cence under ultraviolet light, and a cyan-like fluorescence under blue light from a light-emitting diode.

Responsible techno-innovation in aquaculture: Employing ethical engagement to explore attitudes to GM salmon in Northern Europe

The rapid growth of the aquaculture sector over the past forty years can be closely linked to advances in science and technology. Some innovations in biotechnology have proven more controversial than others, and none more so than proposals for transgenic fish. Such innovations introduce concerns for the responsible governance of research and innovation for aquaculture, particularly in recognition of the significant uncertainties and high stakes characterising these technologies. This paper argues in favour of a participatory approach to the governance of contentious aquaculture technologies that includes an ‘upstream’ regard for their ethical and social implications. From this point of departure, the paper presents empirical research that employed one particular approach to engage aquaculture stakeholders from Northern Europe in deliberation over the ethical and social considerations of transgenic growth-enhanced salmon, using the ‘ethical matrix’ tool. This research explores the complexity of considerations raised by these small groups of diverse and knowledgeable stakeholders across workshops in Germany, Norway and the United Kingdom, and links them with wider ethical discussions in the bioethics literature. The dual ambitions of the paper are to first provide insights into an applied approach for incorporating ethical reasoning in processes for responsible aquaculture techno-innovation, and second to provide an appreciation for the plurality of considerations that decision-makers need to navigate and carefully weigh when reaching a decision on the appropriateness of the particular transgenic growth-enhanced salmon.

Multiple Roles for the Zebrafish Homologue of the Murine Evi1 Gene during Primitive Myelopoiesis and HSC Development

The Evi1 locus was originally identified as a common site of retroviral integration in murine myeloid tumors. Several reports associate Evi1 expression with aggressiveness in myeloid leukemia. Since developmental pathways often reactivate in cancer, we hypothesized that Evi1 also plays critical roles during developmental hematopoiesis. Here, we employ the zebrafish model to study how evi1 modulates early blood development. We find that indeed zebrafish evi1 co-localizes with the hematopoietic markers scl, gata1, pu.1 and gata2 in the posterior lateral mesoderm and the rostral blood islands, indicating involvement in primitive hematopoiesis. Knockdown of evi1 via three independent Morpholino Oligonucleotides impairs embryonic myelopoiesis as shown by reduced pu.1, mpo and l-plastin staining, while not affecting hemangioblast formation and primitive erythropoiesis. Additionally, we observe reduced levels of cd41 expression upon evi1 knockdown, indicating that megakaryopoiesis is also impaired.Notably, at later time-points, evi1 is also expressed in the AGM region and evi1 morphants show strong reduction of runx1/c-myb expression in this region, demonstrating an additional role in hematopoietic stem cell (HSC) formation. Consistently, evi1 morphants lack ikaros+ lymphocyte precursor cells and rag1+ T-lymphocytes, and less circulating globin+, lyz+ and cd41+ cells are detected in transgenic fish analyzed by flow cytometry at 5 days post fertilization. To dissect the mechanisms by which evi1 regulates HSC formation, we furthermore analyzed genes specifically expressed in the dorsal aorta, where HSCs emerge from the hemogenic endothelium. We hypothesize that evi1 is indeed regulating HSC specification from hemogenic endothelial cells, since detailed analyses show defective aortic expression of efnb2a and dlc. How this effect is mediated is currently being investigated. Furthermore, TUNEL, anti-activated Caspase-3, anti-phospho Histone H3 and BrdU assays show both increased apoptosis and decreased proliferation in the AGM region of evi1 morphants as compared to control injected fish, suggesting important additional effects of evi1 on HSC biology beyond the specification step. Similar effects are seen in the rostral blood islands and are possibly responsible for the observed reduction of primitive myeloid progenitors.We also examined potential downstream targets of evi1. Previous reports in adult murine hematopoietic cells suggest that Evi1 affects HSC proliferation through regulation of Gata2. Indeed, gata2 co-injection enhances hematopoietic cell viability also in zebrafish embryos, and co-injection of gata2 mRNA is able to fully rescue both primitive myelopoiesis and HSC formation. We are currently investigating whether gata2 expression can compensate evi1 requirement during HSC specification or, alternatively, might rescue HSC numbers by amplifying residual HSCs that escape evi1 inhibition.Taken together, our data demonstrate that evi1 plays multiple roles during hematopoietic development, critically regulating the biology of primitive myeloid progenitors and pre-formed HSCs, as well as HSC specification from the hemogenic endothelium. Currently, we analyze the molecular mechanisms that mediate evi1 effects during these different regulatory steps and use the CRISPR/Cas9 system to generate evi1 mutant fish for further validation of our findings. DisclosuresNo relevant conflicts of interest to declare.