A transgenic zebrafish for in vivo visualization of cilia

Cilia are organelles for cellular signalling and motility. Mutations affecting ciliary function are also associated with cilia-related disorders (ciliopathies). The identification of cilia markers is critical for studying their function at the cellular level. Due to the lack of a conserved, short ciliary localization motif, the full-length ARL13b or 5HT₆ proteins are normally used for cilia labelling. Overexpression of these genes, however, can affect the function of cilia, leading to artefacts in cilia studies. Here, we show that Nephrocystin-3 (Nphp3) is highly conserved among vertebrates and demonstrate that the N-terminal truncated peptide of zebrafish Nphp3 can be used as a gratuitous cilia-specific marker. To visualize the dynamics of cilia in vivo, we generated a stable transgenic zebrafish Tg (β-actin: nphp3N-mCherry)^sx1001. The cilia in multiple cell types are efficiently labelled by the encoded fusion protein from embryonic stages to adulthood, without any developmental and physiological defects. We show that the line allows live imaging of ciliary dynamics and trafficking of cilia proteins, such as Kif7 and Smo, key regulators of the Hedgehog signalling pathway. Thus, we have generated an effective new tool for in vivo cilia studies that will help shed further light on the roles of these important organelles.     

Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo

Endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme. Here, we created Tie2-Cre transgenic mice, in which expression of Cre recombinase is driven by an endothelial-specific promoter/enhancer. To analyze the lineage of Cre expressing cells, we used CAG-CAT-Z transgenic mice, in which expression of lacZ is activated only after Cre-mediated recombination. We detected pan-endothelial expression of the Cre transgene in Tie2-Cre;CAG-CAT-Z double-transgenic mice. This expression pattern is almost identical to Tie2-lacZ transgenic mice. However, interestingly, we observed strong and uniform lacZ expression in mesenchymal cells of the atrioventricular canal of Tie2-Cre;CAG-CAT-Z double-transgenic mice. We also detected lacZ expression in the mesenchymal cells in part of the proximal cardiac outflow tract, but not in the mesenchymal cells of the distal outflow tract and branchial arch arteries. LacZ staining in Tie2-Cre;CAG-CAT-Z embryos is consistent with endocardial-mesenchymal transformation in the atrioventricular canal and outflow tract regions. Our observations are consistent with previously reported results from Cx43-lacZ, Wnt1-Cre;R26R, and Pax3-Cre;R26R transgenic mice, in which lacZ expression in the cardiac outflow tract identified contributions in part from the cardiac neural crest. Tie2-Cre transgenic mice are a new genetic tool for the analyses of endothelial cell-lineage and endothelial cell-specific gene targeting.     

Fat-1 transgenic mice: a new model for omega-3 research

An appropriate animal model that can eliminate confounding factors of diet would be very helpful for evaluation of the health effects of nutrients such as n-3 fatty acids. We recently generated a fat-1 transgenic mouse expressing the Caenorhabditis elegans fat-1 gene encoding an n-3 fatty acid desaturase that converts n-6 to n-3 fatty acids (which is absent in mammals). The fat-1 transgenic mice are capable of producing n-3 fatty acids from the n-6 type, leading to abundant n-3 fatty acids with reduced levels of n-6 fatty acids in their organs and tissues, without the need of a dietary n-3 supply. Feeding an identical diet (high in n-6) to the transgenic and wild-type littermates can produce different fatty acid profiles in these animals. Thus, this model allows well-controlled studies to be performed, without the interference of the potential confounding factors of diet. The transgenic mice are now being used widely and are emerging as a new tool for studying the benefits of n-3 fatty acids and the molecular mechanisms of their action.     

Generation of a transgenic mouse model with chondrocyte-specific and tamoxifen-inducible expression of Cre recombinase

Postnatal cartilage development and growth are regulated by key growth factors and signaling molecules. To fully understand the function of these regulators, an inducible and chondrocyte-specific gene deletion system needs to be established to circumvent the perinatal lethality. In this report, we have generated a transgenic mouse model (Col2a1-CreER(T2)) in which expression of the Cre recombinase is driven by the chondrocyte-specific col2a1 promoter in a tamoxifen-inducible manner. To determine the specificity and efficiency of the Cre recombination, we have bred Col2a1-CreER(T2) mice with Rosa26R reporter mice. The X-Gal staining showed that the Cre recombination is specifically achieved in cartilage tissues with tamoxifen-induction. In vitro experiments of chondrocyte cell culture also demonstrate the 4-hydroxy tamoxifen-induced Cre recombination. These results demonstrate that Col2a1-CreER(T2) transgenic mice can be used as a valuable tool for an inducible and chondrocyte-specific gene deletion approach.     

Oestrogen-induced expression of a novel liver-specific aspartic proteinase in Danio rerio (zebrafish)

Aspartic proteinases are a group of endoproteolytic proteinases active at acidic pH and characterized by the presence of two aspartyl residues in the active site. They include related paralogous proteins such as cathepsin D, cathepsin E and pepsin. Although extensively investigated in mammals, aspartic proteinases have been less studied in other vertebrates. In a previous work, we cloned and sequenced a DNA complementary to RNA encoding an enzyme present in zebrafish liver. The sequence resulted to be homologous to a novel form of aspartic proteinase firstly described by us in Antarctic fish. In zebrafish, the gene encoding this enzyme is expressed only in the female liver, in contrast with cathepsin D that is expressed in all the tissues examined independently of the sex. For this reason we have termed the new enzyme liver-specific aspartic proteinase (LAP).Northern blot analyses indicate that LAP gene expression is under hormonal control. Indeed, in oestrogen-treated male fish, cathepsin D expression was not enhanced in the various tissues examined, but the LAP gene product appeared exclusively in the liver. Our results provide evidence for an oestrogen-induced expression of LAP gene in liver. We postulate that the sexual dimorphic expression of the LAP gene may be related to the reproductive process.     

PhOTO zebrafish: a transgenic resource for in vivo lineage tracing during development and regeneration

Background

Elucidating the complex cell dynamics (divisions, movement, morphological changes, etc.) underlying embryonic development and adult tissue regeneration requires an efficient means to track cells with high fidelity in space and time. To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo.

Methodology

PhOTO zebrafish ubiquitously express targeted blue fluorescent protein (FP) Cerulean and photoconvertible FP Dendra2 fusions, allowing for instantaneous, precise targeting and tracking of any number of cells using Dendra2 photoconversion while simultaneously monitoring global cell behavior and morphology. Expression persists through adulthood, making the PhOTO zebrafish an excellent tool for studying tissue regeneration: after tail fin amputation and photoconversion of a ∼100µm stripe along the cut area, marked differences seen in how cells contribute to the new tissue give detailed insight into the dynamic process of regeneration. Photoconverted cells that contributed to the regenerate were separated into three distinct populations corresponding to the extent of cell division 7 days after amputation, and a subset of cells that divided the least were organized into an evenly spaced, linear orientation along the length of the newly regenerating fin.

Conclusions/Significance

PhOTO zebrafish have wide applicability for lineage tracing at the systems-level in the early embryo as well as in the adult, making them ideal candidate tools for future research in development, traumatic injury and regeneration, cancer progression, and stem cell behavior.     

Generation of Cas9 transgenic zebrafish and their application in establishing an ERV-deficient animal model

Objectives

To investigate the effect of endogenous Cas9 on genome editing efficiency in transgenic zebrafish.

Results

Here we have constructed a transgenic zebrafish strain that can be screened by pigment deficiency. Compared with the traditional CRISPR injection method, the transgenic zebrafish can improve the efficiency of genome editing significantly. At the same time, we first observed that the phenotype of vertebral malformation in early embryonic development of zebrafish after ZFERV knockout.

Conclusions

The transgenic zebrafish with expressed Cas9, is more efficient in genome editing. And the results of ZFERV knockout indicated that ERV may affect the vertebral development by Notch1/Delta D signal pathway.

     

Neuron-specific expression of a chicken gicerin cDNA in transient transgenic zebrafish

Gicerin, a novel cell adhesion molecule which belongs to the immunoglobulin superfamily, is expressed temporally and spatially in the developing chick brain and retina. The previous in vitro experiments using transfected cells showed that gicerin can function as a cell adhesion molecule which has both homophilic and heterophilic binding activities. For the in vivo analyses of gicerin in neural development, we tried to utilize a zebrafish system, a vertebrate suitable for studying early development. We generated transient transgenic animals by microinjecting DNA constructs into zebrafish embryos. Chicken gicerin, under control of the neurofilament gene promoter, was preferentially expressed in neuronal cells and gicerin-expressing neurons exibited a fasciculation formation with neighboring gicerin-positive axons, which may be partly due to homophilic cell adhesion activity of gicerin. These experimental results suggest that this fast and efficient transgenic animal system is useful for studying the functional roles of neuron-specific genes during the development. Special issue dedicated to Dr. Kinya Kuriyama.     

In vivo synthesis of meganuclease for generating transgenic zebrafish Danio rerio

The authors show that co-injection at the one-cell stage of mRNA encoding a nuclear-targeted meganuclease I- Sce I together with expression cassettes flanked by cognate restriction sites results in efficient stable transgenesis in zebrafish Danio rerio .     

In vivo studies of liver-type fatty acid binding protein (L-FABP) gene expression in liver of transgenic zebrafish (Danio rerio)

Mammalian liver fatty acid binding protein (L-FABP) is a small cytosolic protein in various tissues including liver, small intestine and kidney and is thought to play a crucial role in intracellular fatty acid trafficking and metabolism. To better understand its tissue-specific regulation during zebrafish hepatogenesis, we isolated 5'-flanking sequences of the zebrafish L-FABP gene and used a green fluorescent protein (GFP) transgenic strategy to generate liver-specific transgenic zebrafish. The 2.8-kb 5'-flanking sequence of zebrafish L-FABP gene was sufficient to direct GFP expression in liver primordia, first observed in 2 dpf embryos and then continuously to the adult stage. This pattern of transgenic expression is consistent with the expression pattern of the endogenous gene. F2 inheritance rates of 42-51% in all the seven transgenic lines were consistent with the ratio of Mendelian segregation. Further, hhex and zXbp-1 morphants displayed a visible liver defect, which suggests that it is possible to establish an in vivo system for screening genes required for liver development.     

Laser-induced gene expression in specific cells of transgenic zebrafish

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.     

Light-dependent development of circadian gene expression in transgenic zebrafish

The roles of environmental stimuli in initiation and synchronization of circadian oscillation during development appear to vary among different rhythmic processes. In zebrafish, a variety of rhythms emerge in larvae only after exposure to light-dark (LD) cycles, whereas zebrafish period3 (per3) mRNA has been reported to be rhythmic from day 1 of development in constant conditions. We generated transgenic zebrafish in which expression of the firefly luciferase (luc) gene is driven by the zebrafish per3 promoter. Live larvae from these lines are rhythmically bioluminescent, providing the first vertebrate system for high-throughput measurement of circadian gene expression in vivo. Circadian rhythmicity in constant conditions was observed only after 5–6 d of development, and only if the fish were exposed to LD signals after day 4. Regardless of light exposure, a novel developmental profile was observed, with low expression during the first few days and a rapid increase when active swimming begins. Ambient temperature affected the developmental profile and overall levels of per3 and luc mRNA, as well as the critical days in which LD cycles were needed for robust bioluminescence rhythms. In summary, per3-luc zebrafish has revealed complex interactions among developmental events, light, and temperature in the expression of a clock gene.