Transgenic Fish Research Articles

Adult Zebrafish as a Model System for Cutaneous Wound-Healing Research

Upon injury, the skin must quickly regenerate to regain its barrier function. In mammals, wound healing is rapid and scar-free during embryogenesis, whereas in adults it involves multiple steps including blood clotting, inflammation, re-epithelialization, vascularization, and granulation tissue formation and maturation, resulting in a scar. We have established a rapid and robust method to introduce full-thickness wounds onto the flank of adult zebrafish, and show that apart from external fibrin clot formation, all steps of adult mammalian wound repair also exist in zebrafish. Wound re-epithelialization is extremely rapid and initiates with no apparent lag-phase, subsequently followed by the immigration of inflammatory cells and the formation of granulation tissue, consisting of macrophages, fibroblasts, blood vessels and collagen. The granulation tissue later regresses, resulting in minimal scar formation. Studies after chemical treatment or with transgenic fish further suggest that wound re-epithelialization occurs independently of inflammation and Fibroblast growth factor (FGF) signaling, whereas both are essential for fibroblast recruitment and granulation tissue formation. Together these results demonstrate that major steps and principles of cutaneous wound healing are conserved among adult mammals and adult zebrafish, making zebrafish a valuable model for studying vertebrate skin repair.

Gut Microbiota Contributes to the Growth of Fast-Growing Transgenic Common Carp (Cyprinus carpio L.)

Gut microbiota has shown tight and coordinated connection with various functions of its host such as metabolism, immunity, energy utilization, and health maintenance. To gain insight into whether gut microbes affect the metabolism of fish, we employed fast-growing transgenic common carp (Cyprinus carpio L.) to study the connections between its large body feature and gut microbes. Metagenome-based fingerprinting and high-throughput sequencing on bacterial 16S rRNA genes indicated that fish gut was dominated by Proteobacteria, Fusobacteria, Bacteroidetes and Firmicutes, which displayed significant differences between transgenic fish and wild-type controls. Analyses to study the association of gut microbes with the fish metabolism discovered three major phyla having significant relationships with the host metabolic factors. Biochemical and histological analyses indicated transgenic fish had increased carbohydrate but decreased lipid metabolisms. Additionally, transgenic fish has a significantly lower Bacteroidetes:Firmicutes ratio than that of wild-type controls, which is similar to mammals between obese and lean individuals. These findings suggest that gut microbiotas are associated with the growth of fast growing transgenic fish, and the relative abundance of Firmicutes over Bacteroidetes could be one of the factors contributing to its fast growth. Since the large body size of transgenic fish displays a proportional body growth, which is unlike obesity in human, the results together with the findings from others also suggest that the link between obesity and gut microbiota is likely more complex than a simple Bacteroidetes:Firmicutes ratio change.

Development of Cre–loxP technology in zebrafish to study the regulation of fish reproduction

One cannot seek permission to market transgenic fish mainly because there is no field test or any basic research on technological developments for evaluating their biosafety. Infertility is a necessary adjunct to exploiting transgenic fish unless completely secure land-locked facilities are available. In this study, we report the generation of a Cre transgenic zebrafish line using a cytomegalovirus promoter. We also produced fish carrying the Bax1 and Bax2 plasmids; these genes were separated by two loxP sites under a zona pellucida C promoter or were driven by an anti-Müllerian hormone promoter. We inserted a red fluorescent protein gene between the two loxP sites. After obtaining transgenic lines with the two transgenic fish crossed with each other (Cre transgenic zebrafish x loxP transgenic zebrafish), the floxed DNA was found to be specifically eliminated from the female or male zebrafish, and apoptosis gene expressions caused ovarian and testicular growth cessation and degeneration. Overexpression of the Bax1 and Bax2 genes caused various expression levels of apoptosis-related genes. Accordingly, this transgenic zebrafish model system provides a method to produce infertile fish and may be useful for application to genetically modified fish.

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express(1) . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons(2, 3) .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property(4-6), the frequency of which is modulated by visual cues from conspecifics(2) and the neuropeptide kisspeptin 1(5). In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein(7) to show you how to identify neurons and monitor their electrical activity in the whole brain preparation(6).

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

DEVELOPMENTAL AND MOLECULAR GENETICS IN FISH

ABSTRACT Fish are the oldest vertebrates and the most divergent group, and therefore studies on developmental and molecular genetics in fish can provide a framework and reference for other vertebrates. The zebrafish embryo combines the advantages of experimental embryology and cell fate commitment as in amphibians with the genetic analysis of development as in mammalian systems. The zebrafish has very rapidly become a center of attention because of the thousands of induced developmental mutants that are currently in the process of isolation, characterization, mapping, and cloning. Together with more traditional approaches, like chromosomal and sex manipulation, the use of transgenic fish, and the attempts to produce targeted mutants in pluripotential fish embryonic stem cells, the field of molecular genetics of the fish embryo is now becoming the focus of studies on vertebrate development.

A method to measure an indicator of viraemia in Atlantic salmon using a reporter cell line

RTG-P1 is a transgenic fish cell line producing luciferase under the control of the IFN-induced Mx rainbow trout gene promoter. This cell line was used to measure viraemia of Salmonid alphavirus (SAV), the cause of Salmon Pancreas Disease (SPD), a serious disease in farmed Atlantic salmon. Two SAV genotype 1 (SAV1) isolates were used in this study, F93-125 (tissue-culture adapted, from Ireland) and 4640 (from a field case in Scotland). The kinetics and magnitude of luciferase activity were monitored versus the time of infection. During a direct infection experiment, the induction of luciferase significantly increased 16- and 4-fold after incubation for 6 days with F93-125 at 15 and 20°C, respectively. Filtration and heat treatment experiments demonstrated that the luciferase induction in RTG-P1 was dependent on viral replication. Unlike many cell lines used in fish viral diagnostic, RTG-P1 is not sensitive to salmonid serum, therefore, viraemia could be successfully monitored on serum collected from fish during a cohabitation challenge with 4640 isolate. A peak of viraemia could be detected 16 days post IP inoculation of the shedders. This novel cost-effective method to measure viraemia does not rely on development of cytopathic effect (CPE) in culture, is compatible with non-lethal blood collections in fish and can be used to assign emerging diseases to a viral aetiology.

Abstract 1579: Establishment of transgenic medaka as a stable tumor model for in vivo screening of anticancer drugs.

Abstract The development of anticancer drugs would be facilitated by the availability of an in vivo screening system based on a multicellular organism. We intended to construct an animal model which can be stable and easily detected tumors without autopsy for chemical screening and assessment. In this study, the medaka which is a small egg-laying freshwater fish native to East Asia was applied. The medaka has proved suitable for toxicology and carcinogenesis in the past studies. Furthermore most of molecular technology is available to the medaka and the size of the medaka genome (∼800 Mb) is about half that of the zebrafish genome. To construct a tumor model using transgenic medaka fish, a promoter sequence suitable for the tissue specific tumorigenesis was selected. There are some genes having specific expression in melanocyte. Melanocyte specific expression of oncogenes may induce progression to melanoma. Therefore we selected a promoter sequence encoding Tyrosinase, one of the melanocyte specific genes. The recombinant plasmids for microinjection are consisting of the medaka tyrosinase promoter and human oncogenes (cMyc, BRAF mutant V600E and HRAS mutant G12V). One cell stage embryos were applied for microinjection. We obtained three stable transgenic individuals derived from one cMyc and two BRAF mutant (BRAF600E) injected F0, but none from HRAS mutant (HRAS12V). The transgenic (Tg)-cMyc and the Tg-BRAF600E produced tumor in internal organs with more than 50 % of all fish, but in eyes or skins (outer detectable organs) with less than 10 %. The Tg-HRAS12V animals died before achieving adulthood. We therefore developed a conditional gene expression system in which HRAS12V is expressed in response to the induction of Cre recombinase by heat treatment, given that the Cre gene was placed under the control of a medaka heat shock promoter. One of the stable transgenic lines developed abnormal pigment cell proliferation in the eyes and epidermis with 100% penetrance by 6 months postfertilization. Sorafenib, an inhibitor of RAS signaling, was administered to the transgenic fish and was found both to reduce the extent of melanophore proliferation and to improve survival. This Tg-HRAS12V medaka established here thus represents a promising in vivo system with which to screen potential anticancer drugs that target RAS signaling, and this system can readily be adapted for the screening of agents that target other oncogenes. Citation Format: Yuriko Matsuzaki, Haru Hosokai, Yukiyo Mizuguchi, Atsushi Shimizu, Hideyuki Saya. Establishment of transgenic medaka as a stable tumor model for in vivo screening of anticancer drugs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1579. doi:10.1158/1538-7445.AM2013-1579

Abstract 356: Role of the LMO1 oncogene in neuroblastoma pathogenesis.

Abstract Neuroblastoma, an embryonic tumor of the peripheral sympathetic nervous system (PSNS), accounts for 10% of all childhood cancer deaths. We recently developed a robust zebrafish model of neuroblastoma and demonstrated that activated ALK synergizes with MYCN by inhibiting a developmentally-timed apoptotic response that is otherwise induced by MYCN (Zhu et.al. Cancer Cell, 2012). We have now used this model to provide evidence in support of the results of a neuroblastoma genome-wide association study (GWAS) showing that common variation within the LIM domain-only 1 (LMO1) gene locus is highly associated with the development of advanced neuroblastoma and may function as an oncogene in established disease (Wang K et.al. Nature, 2011). We are collaborating because our zebrafish system provides a robust in vivo tumor model to investigate the underlying mechanisms through which LMO1 overexpression contributes to malignant transformation. Here we developed transgenic lines in which LMO1 is overexpressed in the PSNS under control of the dopamine-beta-hydroxylase (dβh) promoter. We observed that overexpression of LMO1 in three individual transgenic lines synergized with MYCN to accelerate the onset of neuroblastoma in the interrenal gland (IRG), the zebrafish analogue of the adrenal medulla. Tumors began to appear at 13 weeks in transgenic fish overexpressing MYCN alone and this latency was shortened dramatically to 5 weeks (p=0.02) and the penetrance was increased more than three-fold in the transgenic fish coexpressing both MYCN and LMO1. In addition, we found that coexpression of LMO1 with activated ALK induced neuroblastoma, which is the first time in our model system that neuroblastoma has been induced without MYCN overexpression. Thus, the zebrafish model system appears to be robust for “functional genomics analysis” to provide in vivo evidence and investigate mechanisms and pathways underlying new associations emerging from GWAS, tumor genome resequencing and other genome-wide technologies that are currently under intense investigation in human cancers. Citation Format: Shizhen Zhu, Andrew Wood, Shuning He, Rebecca Stanton, Feng Guo, John Maris, A. Thomas Look. Role of the LMO1 oncogene in neuroblastoma pathogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 356. doi:10.1158/1538-7445.AM2013-356

Abstract SY16-01: Transcriptional regulation and epigenetics in zebrafish cancer.

Abstract Melanoma is a devastating disease for which few therapies lead to long term remission after metastasis has occurred. We have been using the zebrafish to model melanoma. A transgenic fish carrying human V600E BRAF mutation in combination with p53 mutation leads to a rapid melanoma model in the zebrafish. Using a transposon based approach, we have been able to overexpress particular potential oncogenes to look for acceleration of the melanoma. One such oncogene (SETDB1) is amplified in 30% of human melanomas and accelerates tumor rate in the zebrafish. Using a library of chromatin factors that are full length, we have been able to overexpress over 80 factors and found several new factors that activate and induce more melanoma. This approach has led to a fundamentally new insight into melanoma and epigenetic regulation. We also have found a chemical (leflunomide) that lowers nucleotide levels and induces a transcriptional pause of neural crest genes. Leflunomide and a BRAF inhibitor is now in clinical trial for patients with metastatic melanoma. To understand the mechanism of action, we have focused on PTEFb that is a kinase that regulates the elongation step in transcription. We found that the HEXIM complex is able to sequester PTEFb into an inactive state. HEXIM is a new tumor suppressor gene for melanoma. Our studies have led to a better understanding of transcription in melanoma and have developed a novel therapeutic that is being tested in the clinic. Citation Format: Leonard I. Zon. Transcriptional regulation and epigenetics in zebrafish cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY16-01. doi:10.1158/1538-7445.AM2013-SY16-01

Def Functions as a Cell Autonomous Factor in Organogenesis of Digestive Organs in Zebrafish

Digestive organs originate from the endoderm. Morphogenesis of the digestive system is precisely controlled by multiple factors that dictate the cell fate and behavior so that the specific digestive organs are timely formed in the right place and develop into right size and structure. We showed previously that digestive organ expansion factor (def) is a gene whose expression is enriched in the liver, pancreas and intestine. Loss-of-function of def in the defhi429 mutant confers hypoplastic digestive organs partly due to alteration of expression of genes related to the p53 pathway. However, the molecular mechanism for the involvement of Def in the organogenesis of digestive organs is still largely unknown. For example, it is not known whether Def regulates specific pathways in a specific organ. To address this question, we generated four independent Tg(fabp10a:def) transgenic fish lines which over-expressed Def specifically in the liver. We characterized Tg-I, one of the transgenic lines, in detail with genetic, molecular and histological approaches. We found that Tg-I restored the liver but not exocrine pancreas and intestine development in the defhi429 mutant. However, Tg-I adult fish in the wild type (WT) background exhibits reduced liver-to-body ratio and all four transgenic lines conferred abnormal intrahepatic structure. Microarray data analysis showed that certain specific functional pathways were affected in the liver of Tg-I. These results demonstrate that Def functions in a cell autonomous manner during early liver development and aberrant Def protein expression might lead to disruption of the structural integrity of a normal adult liver.

Transgenic labeling of higher order neuronal circuits linked to phospholipase C‐β2–expressing taste bud cells in medaka fish

The sense of taste plays a pivotal role in the food-selecting behaviors of vertebrates. We have shown that the fish ortholog of the phospholipase C gene (plc-β2) is expressed in a subpopulation of taste bud cells that transmit taste stimuli to the central nervous system to evoke favorable and aversive behaviors. We generated transgenic medaka expressing wheat germ agglutinin (WGA) under the control of a regulatory region of the medaka plc-β2 gene to analyze the neuronal circuit connected to these sensory cells. Immunohistochemical analysis of the transgenic fish 12 days post fertilization revealed that the WGA protein was transferred to cranial sensory ganglia and several nuclei in the hindbrain. WGA signals were also detected in the secondary gustatory nucleus in the hindbrain of 3-month-old transgenic fish. WGA signals were observed in several diencephalic and telencephalic regions in 9-month-old transgenic fish. The age-dependent increase in the labeled brain regions strongly suggests that labeling occurred at taste bud cells and progressively extended to cranial nerves and neurons in the central nervous system. These data are the first to demonstrate the tracing of higher order gustatory neuronal circuitry that is associated with a specific subpopulation of taste bud cells. These results provide insight into the basic neuronal architecture of gustatory information processing that is common among vertebrates.

Disease resistance and health parameters of growth-hormone transgenic and wild-type coho salmon, Oncorhynchus kisutch

To extend previous findings regarding fish health and disease susceptibility of growth-enhanced fish, hematological and immunological parameters have been compared between growth hormone (GH) transgenic and wild-type non-transgenic coho salmon (Oncorhynchus kisutch). Compared to non-transgenic coho salmon, transgenic fish had significantly higher hematocrit (Hct), hemoglobin (Hb), mean cellular hemoglobin (MCH), mean cellular volume (MCV), and erythrocyte numbers, and lower white cell numbers. In addition, resistance to the bacterial pathogen Aeromonas salmonicida (causal agent of furunculosis) has been assessed between the strains. Higher susceptibility of transgenic fish to this disease challenge was observed in two separate year classes of fish. The present findings provide fundamental knowledge of the disease resistance on GH enhanced transgenic coho salmon, which is of importance for assessing the fitness of transgenic strains for environmental risk assessments, and for improving our understanding effects of growth modification on basic immune functions.

Homologous Rearranged DNA can Change Phenotype and Genotype of the Host by Transgenic Method and a QTL Related to Weight was Obtained from it

The research study aim at looking for a simple way to obtain mutant while know what change in the genome of the host. We rearrange carp genomic DNA by digestion, ligation and addition of adaptor and then transferred the homologous rearranged DNA into carp eggs. The results showed that introduction of the homologous rearranged DNA slightly decreased the hatchability of fertilized eggs. PCR products with primers against adaptors amplified from offspring carps had different sizes compared with those amplified from the parent carps, indicating that shuffled genomic DNA has been incorporated into the genomes of offspring. Different size of PCR fragments were obtained after amplification of DNA from two small-size carps that has ceased to develop. Four clones of introducing DNA were sequenced and most of them were microsatellite DNA. Based on one of these sequences, we designed three forward and one reverse primer to amplify the genomic DNA from normal carps and we found that the amplified sequences were homologous rearranged DNA. Four transgenic fish with large body weight were selected as the father and hybridized with common female carp. We gained four groups of offspring. The muscle tissue was chosen as the sample for amplification of introducing DNA fragments. The separation of introducing DNA in three groups is confusing but clear in one group. Further analysis on the group with clear separation shows that the introducing sequence can make the weight of the host drift to the large direction and lower the differentiation between individuals with such sequence. The sequence has no coding function and no region similar to the known regulatory sequence. The study shown that the homologous rearranged DNA can be integrated into the genome of the host and make impact on the host both in genotype and phenotype.

Hepatitis B virus X antigen and aflatoxin B1 synergistically cause hepatitis, steatosis and liver hyperplasia in transgenic zebrafish

Aflatoxin B1 (AFB1) and the hepatitis B virus X antigen (HBx) are linked to the formation of liver diseases and hepatocellular carcinoma (HCC). The aim of this study was to investigate the synergistic effects between HBx and AFB1 in causing liver disorders using a transgenic zebrafish animal model. Histopathology, Periodic acid-Schiff (PAS) staining, Sirius red staining, TdT-mediated dUTP Nick End Labeling (TUNEL) assay, immunohistochemistry, and quantitative reverse transcriptase-polymerase chain reaction (Q-RT-PCR) were used to examine the livers of the HBx transgenic fish injected with AFB1. We found that HBx and AFB1 synergistically promoted steatosis as indicated by histopathological examinations and the increased expression of lipogenic factors, enzymes, and genes related to lipid metabolism. Moreover, treatment of AFB1 in HBx transgenic fish accelerated the development of liver hyperplasia and enhanced the expression of cell cycle related genes. PCNA was co-localized with active caspase 3 protein expression in HBx zebrafish liver samples and human HBV positive HCC samples by double fluorescence immunostaining. Finally, we found that in human patients with liver disease, significant glycogen accumulated in the inflammation, cirrhosis stage, and all cases of hepatocellular and cholangiocellular carcinoma showed a moderate cytoplasmic accumulation of glycogen. Our data demonstrated a synergistic effect of AFB1 and HBx on the regulation of lipid metabolism related genes and cell cycle/division-related genes which might contribute to enhanced steatosis and hyperplasia at 5.75months.

Characterization of transgenic zebrafish lines that express GFP in the retina, pineal gland, olfactory bulb, hatching gland, and optic tectum

Transgenic animals are powerful tools to study gene function invivo. Here we characterize several transgenic zebrafish lines that express green fluorescent protein (GFP) under the control of the LCRRH2-RH2-1 or LCRRH2-RH2-2 green opsin regulatory elements. Using confocal immunomicroscopy, stereo-fluorescence microscopy, and Western blotting, we show that the Tg(LCRRH2-RH2-1:GFP)pt112 and Tg(LCRRH2-RH2-2:GFP)pt115 transgenic zebrafish lines express GFP in the pineal gland and certain types of photoreceptors. In addition, some of these lines also express GFP in the hatching gland, optic tectum, or olfactory bulb. Some of the expression patterns differ significantly from previously published similar transgenic fish lines, making them useful tools for studying the development of the corresponding tissues and organs. In addition, the variations of GFP expression among different lines corroborate the notion that transgenic expression is often subjected to position effect, thus emphasizing the need for careful verification of expression patterns when transgenic animal models are utilized for research.

Cftr controls lumen expansion and function of Kupffer’s vesicle in zebrafish

Regulated fluid secretion is crucial for the function of most organs. In vertebrates, the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) is a master regulator of fluid secretion. Although the biophysical properties of CFTR have been well characterized in vitro, little is known about its in vivo role during development. Here, we investigated the function of Cftr during zebrafish development by generating several cftr mutant alleles using TAL effector nucleases. We found that loss of cftr function leads to organ laterality defects. In zebrafish, left-right (LR) asymmetry requires cilia-driven fluid flow within the lumen of Kupffer's vesicle (KV). Using live imaging we found that KV morphogenesis is disrupted in cftr mutants. Loss of Cftr-mediated fluid secretion impairs KV lumen expansion leading to defects in organ laterality. Using bacterial artificial chromosome recombineering, we generated transgenic fish expressing functional Cftr fusion proteins with fluorescent tags under the control of the cftr promoter. The transgenes completely rescued the cftr mutant phenotype. Live imaging of these transgenic lines showed that Cftr is localized to the apical membrane of the epithelial cells in KV during lumen formation. Pharmacological stimulation of Cftr-dependent fluid secretion led to an expansion of the KV lumen. Conversely, inhibition of ion gradient formation impaired KV lumen inflation. Interestingly, cilia formation and motility in KV were not affected, suggesting that fluid secretion and flow are independently controlled in KV. These findings uncover a new role for cftr in KV morphogenesis and function during zebrafish development.

The Critical Role of Protein Arginine Methyltransferase prmt8 in Zebrafish Embryonic and Neural Development Is Non-Redundant with Its Paralogue prmt1

Protein arginine methyltransferase (PRMT) 1 is the most conserved and widely distributed PRMT in eukaryotes. PRMT8 is a vertebrate-restricted paralogue of PRMT1 with an extra N-terminal sequence and brain-specific expression. We use zebrafish (Danio rerio) as a vertebrate model to study PRMT8 function and putative redundancy with PRMT1. The transcripts of zebrafish prmt8 were specifically expressed in adult zebrafish brain and ubiquitously expressed from zygotic to early segmentation stage before the neuronal development. Whole-mount in situ hybridization revealed ubiquitous prmt8 expression pattern during early embryonic stages, similar to that of prmt1. Knockdown of prmt8 with antisense morpholino oligonucleotide phenocopied prmt1-knockdown, with convergence/extension defects at gastrulation. Other abnormalities observed later include short body axis, curled tails, small and malformed brain and eyes. Catalytically inactive prmt8 failed to complement the morphants, indicating the importance of methyltransferase activity. Full-length prmt8 but not prmt1 cRNA can rescue the phenotypic changes. Nevertheless, cRNA encoding Prmt1 fused with the N-terminus of Prmt8 can rescue the prmt8 morphants. In contrast, N-terminus- deleted but not full-length prmt8 cRNA can rescue the prmt1 morphants as efficiently as prmt1 cRNA. Abnormal brain morphologies illustrated with brain markers and loss of fluorescent neurons in a transgenic fish upon prmt8 knockdown confirm the critical roles of prmt8 in neural development. In summery, our study is the first report showing the expression and function of prmt8 in early zebrafish embryogenesis. Our results indicate that prmt8 may play important roles non-overlapping with prmt1 in embryonic and neural development depending on its specific N-terminus.

Regeneration of cone photoreceptors when cell ablation is primarily restricted to a particular cone subtype.

We sought to characterize the regenerated cells, if any, when photoreceptor ablation was mostly limited to a particular cone subtype. This allowed us to uniquely assess whether the remaining cells influence specification of regenerating photoreceptors. The ability to replace lost photoreceptors via stem cell therapy holds promise for treating many retinal degenerative diseases. Zebrafish are potent for modelling this because they have robust regenerative capacity emanating from endogenous stem cells, and abundant cone photoreceptors including multiple spectral subtypes similar to human fovea. We ablated the homolog of the human S-cones, the ultraviolet-sensitive (UV) cones, and tested the hypothesis that the photoreceptors regenerating in their place take on identities matching those expected from normal cone mosaic development. We created transgenic fish wherein UV cones can be ablated by addition of a prodrug. Thus photoreceptors developed normally and only the UV cones expressed nitroreductase; the latter converts the prodrug metronidazole to a cell-autonomous neurotoxin. A significant increase in proliferation of progenitor cell populations (p<0.01) was observed when cell ablation was primarily limited to UV cones. In control fish, we found that BrdU primarily incorporated into rod photoreceptors, as expected. However the majority of regenerating photoreceptors became cones when retinal cell ablation was predominantly restricted to UV cones: a 2-fold increase in the relative abundance of cones (p = 0.008) was mirrored by a 35% decrease in rods. By primarily ablating only a single photoreceptor type, we show that the subsequent regeneration is biased towards restoring the cognate photoreceptor type. We discuss the hypothesis that, after cone death, the microenvironment formed by the remaining retinal cells may be influential in determining the identity of regenerating photoreceptors, though other interpretations are plausible. Our novel animal model provides control of ablation that will assist in identifying mechanisms required to replace cone photoreceptors clinically to restore daytime vision.

Molecular characterization of fast skeletal muscle-specific myosin light chain 2 gene (mlc2f) in marine medaka Oryzias dancena

The genomic structure including the 5′-upstream regulatory region of the fast skeletal myosin light chain 2 gene (mlc2f) was characterized in the marine medaka (Oryzias dancena; Beloniformes). Molecular phylogenic analysis inferred that the marine medaka mlc2f should belong to the teleost mlc2fa group characterized by seven-exon organization. Bioinformatic analysis of the regulatory region represented the various transcription factor binding motifs especially including myogenic regulatory factor binding sites such as myocyte enhancer factor-2 site and E-box. Real-time RT-PCR assays revealed that the mlc2f mRNA was highly predominant in skeletal muscles and also that the muscular mlc2f expression was little modulated by environmental salinity ranging from 0 to 30 ppt. The mlc2f mRNA expression was differentially modulated during embryonic development of this species, in which the expression of mlc2f was stimulated from the retinal pigmentation stage and then markedly activated until hatching. From the microinjection-based introduction of a green fluorescent protein gene (gfp) driven by a 2.95-kb marine medaka mlc2f promoter into the marine medaka embryos, the onset of transgenic GFP expression was in accordance with that of endogenous mlc2f gene. Although all the microinjected embryos and resultant F0 fish showed a mosaic distribution of GFP expression with some ectopic expression pattern, the overall expression of transgenic GFP was enriched in the skeletal muscles. However, transgenic hemizygous F1 fish produced from the germline positive founders showed uniform, fast-skeletal muscle-specific expression of GFP, which resembled the pattern of the endogenous mlc2f gene expression. The expression of transgenic GFP was observable mainly in head region and weakly in caudal peduncle of the hatched larvae. The GFP expression was gradually intensified in these sites and became spread over other skeletal muscle parts with larval ages, such that the fish at 21 days post hatching acquired uniform GFP expression over nearly whole skeletal muscles. Data from this study suggest that the mlc2f promoter-driven transgenesis holds promising potential to monitor the differentiation of the fast skeletal muscles of this species in a real-time fashion and also to drive efficiently the expression of foreign proteins in the marine medaka skeletal muscles.