Apoptosis in zebrafish development

Apoptosis (programmed cell death) is important in normal biological processes and in pathogenesis in vertebrates. This review focuses on some of the prominent features of apoptosis during fish development. Caspases and other apoptosis-regulating genes have been cloned from zebrafish (Danio rerio) and other fish species. Elucidation of in vivo functions of apoptosis is focused on development, morphogenesis and sex differentiation. In an attempt to elucidate cause and effect relationships between caspase and development, transgenic zebrafish overexpressing procaspase-3 were generated. Stress-induced apoptosis in zebrafish embryos can be monitored by whole mount TUNEL staining and caspase assay. Thus, zebrafish is a useful experimental model animal for investigation of apoptosis in vivo.     

Bioenergetic profiling of zebrafish embryonic development

Many debilitating conditions are linked to bioenergetic defects. Developing screens to probe the genetic and/or chemical basis for such links has proved intractable. Furthermore, there is a need for a physiologically relevant assay of bioenergetics in whole organisms, especially for early stages in life where perturbations could increase disease susceptibility with aging. Thus, we asked whether we could screen bioenergetics and mitochondrial function in the developing zebrafish embryo. We present a multiplexed method to assay bioenergetics in zebrafish embryos from the blastula period (3 hours post-fertilization, hpf) through to hatching (48 hpf). In proof of principle experiments, we measured respiration and acid extrusion of developing zebrafish embryos. We quantified respiratory coupling to various bioenergetic functions by using specific pharmacological inhibitors of bioenergetic pathways. We demonstrate that changes in the coupling to ATP turnover and proton leak are correlated with developmental stage. The multiwell format of this assay enables the user to screen for the effects of drugs and environmental agents on bioenergetics in the zebrafish embryo with high sensitivity and reproducibility.     

斑马鱼胚胎发育中细胞凋亡的研究进展

细胞凋亡是细胞在基因调控下发生的主动消亡过程,在脊椎动物胚胎发育过程中非常重要。斑马鱼作为一种十分理想的发育分子生物学研究模型,在有关细胞凋亡在诸如形态发生、性别分化等方面功能之活体在位研究中日益受到重视。目前,斑马鱼胚胎发育中主要凋亡通路研究已进行了不少工作,特别是caspase及其它凋亡调控基因在斑马鱼中已被成功克隆,通过转基因斑马鱼胚胎中胁迫诱导细胞凋亡并研究其信号通路以及斑马鱼胚胎形态发生的异常改变,为阐明这些凋亡调控基因与发育之间的关系提供了一个强有力的手段。     

Hsp27 is persistently expressed in zebrafish skeletal and cardiac muscle tissues but dispensable for their morphogenesis

Constitutive expression of Hsp27 has been demonstrated in vertebrate embryos, especially in developing skeletal and cardiac muscle. Results of several previous studies have indicated that Hsp27 could play a role in the development of these tissues. For example, inhibition of Hsp27 expression has been reported to cause defective development of mammalian myoblasts in vitro and frog embryos in vivo. In contrast, transgenic mice lacking Hsp27 develop normally. Here, we examined the distribution of Hsp27 protein in developing and adult zebrafish and effects of suppressing Hsp27 expression using phosphorodiamidate morpholino oligonucleotides (PMO) on zebrafish development. Consistent with our previous analysis of hsp27 messenger RNA expression, we detected the protein Hsp27 in cardiac, smooth, and skeletal muscle of both embryonic and adult zebrafish. However, embryos lacking detectable Hsp27 after injection of antisense hsp27 PMO exhibited comparable heart beat rates to that of control embryos and cardiac morphology was indistinguishable in the presence or absence of Hsp27. Loss of Hsp27 also had no effect on the structure of the skeletal muscle myotomes in the developing embryo. Finally, embryos injected with antisense hsp27 and scrambled control PMO displayed equal motility. We conclude that Hsp27 is dispensable for zebrafish morphogenesis but could play a role in long-term maintenance of heart and muscle tissues. Tucker and Ustyugov contributed equally to this work.     

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.     

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.     

An Apo-14 promoter-driven transgenic zebrafish that marks liver organogenesis

Several transgenic zebrafish lines for liver development studies had been obtained in the first decade of this century, but not any transgenic GFP zebrafish lines that mark the through liver development and organogenesis were reported. In this study, we analyzed expression pattern of endogenous Apo-14 in zebrafish embryogenesis by whole-mount in situ hybridization, and revealed its expression in liver primordium and in the following liver development. Subsequently, we isolated zebrafish Apo-14 promoter of 1763 bp 5'-flanking sequence, and developed an Apo-14 promoter-driven transgenic zebrafish Tg(Apo14: GFP). And, maternal expression and post-fertilization translocation of Apo-14 promoter-driven GFP were observed in the transgenic zebrafish line. Moreover, we traced onset expression of Apo-14 promoter-driven GFP and developmental behavior of the expressed cells in early heterozygous embryos by out-crossing the Tg(Apo14: GFP) male to the wild type female. Significantly, the Apo-14 promoter-driven GFP is initially expressed around YSL beneath the embryo body at 10 hpf when the embryos develop to tail bud prominence. In about 14-somite embryos at 16-17 hpf, a typical "salt-and-pepper" expression pattern is clearly observed in YSL around the yolk sac. Then, a green fluorescence dot begins to appear between the notochord and the yolk sac adjacent to otic vesicle at about 20 hpf, which is later demonstrated to be liver primordium that gives rise to liver. Furthermore, we investigated dynamic progression of liver organogenesis in the Tg(Apo14: GFP) zebrafish, because the Apo-14 promoter-driven GFP is sustainably expressed from hepatoblasts and liver progenitor cells in liver primordium to hepatocytes in the larval and adult liver. Additionally, we observed similar morphology between the liver progenitor cells and the GFP-positive nuclei on the YSL, suggesting that they might originate from the same progenitor cells in early embryos. Overall, the current study provides a transgenic zebrafish line that marks the through liver organogenesis.     

cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) assay is a classical method for screening cytotoxic anti-cancer agents. Candidate drugs from the MTT assay need in vivo models to test their efficiency and to assess the absorption, distribution, metabolism, excretion, and toxicity of the drugs. An in vivo screening model could increase the rate of development of anti-cancer drugs. Here, we used zebrafish to screen a library of 502 natural compounds and compared the results with those from an MTT assay of the MCF7 breast cancer cell line. We identified 59 toxic compounds in the zebrafish screen, 21 of which were also identified by the MTT assay, and 28 of which were already known for their anti-cancer and apoptosis-inducing effects. These compounds induced apoptosis and activated the p53 pathway in zebrafish within 3h treatment. Our results indicate that zebrafish is a simple, reliable and highly efficient in vivo tool for cancer drug screening, and could complement the MTT assay.     

Interplay of pu.1 and gata1 determines myelo-erythroid progenitor cell fate in zebrafish

The zebrafish is a powerful model system for investigating embryonic vertebrate hematopoiesis, allowing for the critical in vivo analysis of cell lineage determination. In this study, we identify zebrafish myeloerythroid progenitor cells (MPCs) that are likely to represent the functional equivalent of mammalian common myeloid progenitors. Utilizing transgenic pu.1-GFP fish, real-time MPC differentiation was correlated with dynamic changes in cell motility, morphology, and gene expression. Unlike mammalian hematopoiesis, embryonic zebrafish myelopoiesis and erythropoiesis occur in anatomically separate locations. Gene knockdown experiments and transplantation assays demonstrated the reciprocal negative regulation of pu.1 and gata1 and their non-cell-autonomous regulation that determines myeloid versus erythroid MPC fate in the distinct blood-forming regions. Furthermore, forced expression of pu.1 in the bloodless mutant cloche resulted in myelopoietic rescue, providing intriguing evidence that this gene can function in the absence of some stem cell genes, such as scl, in governing myelopoiesis.     

Zebrafish intestinal fatty acid binding protein (I-FABP) gene promoter drives gut-specific expression in stable transgenic fish

Mammalian intestinal fatty acid-binding protein (I-FABP) is a small cytosolic protein and is thought to play a crucial role of intracellular fatty acid trafficking and metabolism in gut. To establish an in vivo system for investigating its tissue-specific regulation during zebrafish intestinal development, we isolated 5'-flanking sequences of the zebrafish L-FABP gene and used a transgenic strategy to generate gut-specific transgenic zebrafish with green/red fluorescent intestine. The 4.5-kb 5'-flanking sequence of zebrafish I-FABP gene was sufficient to direct fluorescent expression in intestinal tube, first observed in 3 dpf embryos and then continuously to the adult stage. This pattern of transgenic expression is consistent with the expression pattern of the endogenous gene. In all five transgenic lines 45-52% of the F2 inheritance rates were consistent with the ratio of Mendelian segregation. These fish can also provide a valuable resource of labeled adult intestinal cells for in vivo or in vitro studies. Finally, it is possible to establish an in vivo system using these fish for screening genes required for gut development. genesis 38:26-31, 2004.     

Caspase-3 Induced Apoptosis in Transgenic Zebrafish

Zebrafish is an attractive model organism for studying apoptosis development because of its genetic accessibility. Here we describe the induction of clonally derived apoptosis in transgenic zebrafish expressing mouse caspase-3 (CASP3) under control of the zebrafish β-actin promoter (βp). Visualization of apoptotic cells, expressing a chimeric transgene encoding CASP3 fused to green fluorescent protein (GFP) gene, revealed that apoptosis arose in the thymus, spread locally into gill arches and retro-orbital soft tissue, and then disseminated into abdominal organs like testis, kidney. This transgenic model provides a platform for over-expression of caspase-3 induced extensive apoptosis in embryos and adult. An erratum to this article is available at .     

Carbonic anhydrase 5 regulates acid-base homeostasis in zebrafish

The regulation of the acid-base balance in cells is essential for proper cellular homeostasis. Disturbed acid-base balance directly affects cellular physiology, which often results in various pathological conditions. In every living organism, the protein family of carbonic anhydrases regulate a broad variety of homeostatic processes. Here we describe the identification, mapping and cloning of a zebrafish carbonic anhydrase 5 (ca5) mutation, collapse of fins (cof), which causes initially a collapse of the medial fins followed by necrosis and rapid degeneration of the embryo. These phenotypical characteristics can be mimicked in wild-type embryos by acetazolamide treatment, suggesting that CA5 activity in zebrafish is essential for a proper development. In addition we show that CA5 regulates acid-base balance during embryonic development, since lowering the pH can compensate for the loss of CA5 activity. Identification of selective modulators of CA5 activity could have a major impact on the development of new therapeutics involved in the treatment of a variety of disorders.     

Comprehensive application of an mtDsRed2-Tg mouse strain for mitochondrial imaging

Mitochondria are essential for many cellular functions such as oxidative phosphorylation and calcium homeostasis; consequently, mitochondrial dysfunction could cause many diseases, including neurological disorders. Recently, mitochondrial dynamics, such as fusion, fission, and transportation, have been visualized in living cells by using time-lapse imaging systems. The changes in mitochondrial morphology could be an indicator for estimating the activity of mitochondrial biological function. Here, we report a transgenic mouse strain, mtDsRed2-Tg, which expresses a red fluorescent protein, DsRed2, exclusively in mitochondria. Mitochondrial morphology could be clearly observed in various tissues of this strain under confocal microscope. Recently, many transgenic mouse strains in which enhanced green fluorescent protein (EGFP)-tagged proteins of interest are expressed have been established for physiological analysis in vivo. After mating these strains with mtDsRed2-Tg mice, red-colored mitochondria and green-colored proteins were detected simultaneously using fluorescent imaging systems, and the interactions between mitochondria and those proteins could be morphologically analyzed in cells and tissues of the F₁ hybrids. Thus, mtDsRed2-Tg mice can be a powerful tool for bioimaging studies on mitochondrial functions.     

Isolation of yellow catfish β-actin promoter and generation of transgenic yellow catfish expressing enhanced yellow fluorescent protein

Yellow catfish (Pelteobagrus fulvidraco Richardson) is one of the most important freshwater farmed species in China. However, its small size and slow growth rate limit its commercial value. Because genetic engineering has been a powerful tool to develop and improve fish traits for aquaculture, we performed transgenic research on yellow catfish in order to increase its size and growth rate. Performing PCR with degenerate primers, we cloned a genomic fragment comprising 5'-flanking sequence upstream of the initiation codon of β-actin gene in yellow catfish. The sequence is 1,017?bp long, containing the core sequence of proximal promoter including CAAT box, CArG motif and TATA box. Microinjecting the transgene construct Tg(beta-actin:eYFP) of the proximal promoter fused to enhanced yellow fluorescent protein (eYFP) reporter gene into zebrafish and yellow catfish embryos, we found the promoter could drive the reporter to express transiently in both embryos at early development. Screening the offspring of five transgenic zebrafish founders developed from the embryos microinjected with Tg(ycbeta-actin:mCherry) or 19 yellow catfish founders developed from the embryos microinjected with Tg(beta-actin:eYFP), we obtained three lines of transgenic zebrafish and one transgenic yellow catfish, respectively. Analyzing the expression patterns of the reporter genes in transgenic zebrafish (Tg(ycbeta-actin:mCherry)nju8/+) and transgenic yellow catfish (Tg(beta-actin:eYFP)nju11/+), we found the reporters were broadly expressed in both animals. In summary, we have established a platform to make transgenic yellow catfish using the proximal promoter of its own β-actin gene. The results will help us to create transgenic yellow catfish using "all yellow catfish" transgene constructs.     

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens ¹. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates ². In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling ³. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.     

Rapid BAC selection for tol2-mediated transgenesis in zebrafish

The generation of zebrafish transgenic lines that express specific fluorophores in a cell- or tissue-specific manner is an important technique that takes full advantage of the optical clarity of the embryo. Identifying promoter fragments that faithfully recapitulate endogenous expression patterns and levels is often difficult and using large genomic DNA fragments, such as bacterial artificial chromosomes (BACs), makes the process of transgenesis less reliable. Here we provide a detailed protocol that allows for BAC selection and subsequent rapid modification through recombineering in Escherichia coli, resulting in BACs that can be injected into zebrafish embryos and, aided by tol2-mediated transgenesis, reliably yield stable transgenic lines. A number of BACs can be prepared in parallel, and injection of the BACs containing CFP/YFP/RFP or Gal4 cassettes allows for immediate testing of whether a particular BAC will yield the desired result. Furthermore, since injected embryos often show widespread expression, recombineered BACs provide an alternative to two-color in situ hybridizations: BACs injected into embryos of a different transgenic reporter line thus enable in vivo colocalization studies. Using this protocol, we have generated 66 stable lines for 23 different genes, with an average transgenesis rate above 10%. Importantly, we provide evidence that BAC size shows no apparent correlation to the transgenesis rate achieved and that there are no severe position effects.     

Development of Novel Visual-Plus Quantitative Analysis Systems for Studying DNA Double-Strand Break Repairs in Zebrafish

The use of reporter systems to analyze DNA double-strand break(DSB) repairs,based on the enhanced green fluorescent protein (EGFP) and meganuclease such as I-Sce I,is usually carried out with cell lines.In this study,we developed three visual-plus quantitative assay systems for homologous recombination(HR),non-homologous end joining(NHEJ) and single-strand annealing(SSA) DSB repair pathways at the organismal level in zebrafish embryos.To initiate DNA DSB repair,we used two I-Sce I recognition sites in opposite orientation rather than the usual single site.The NHEJ,HR and SSA repair pathways were separately triggered by the injection of three corresponding I-Sce I-cut constructions,and the repair of DNA lesion caused by l-Sce I could be tracked by EGFP expression in the embryos.Apart from monitoring the intensity of green fluorescence,the repair frequencies could also be precisely measured by quantitative real-time polymerase chain reaction(qPCR).Analysis of DNA sequences at the DSB sites showed that NHEJ was predominant among these three repair pathways in zebrafish embryos.Furthermore,while HR and SSA reporter systems could be effectively decreased by the knockdown of rad51 and rad52,respectively,NHEJ could only be impaired by the knockdown of ligaseIV(lig4) when the NHEJ construct was cut by I-Sce I in vivo.More interestingly,blocking NHEJ with lig4-MO increased the frequency of HR,but decreased the frequency of SSA.Our studies demonstrate that the major mechanisms used to repair DNA DSBs are conserved from zebrafish to mammal,and zebrafish provides an excellent model for studying and manipulating DNA DSB repair at the organismal level.     

Zebrafish on a Chip: A Novel Platform for Real-Time Monitoring of Drug-Induced Developmental Toxicity

Pharmaceutical safety testing requires a cheap, fast and highly efficient platform for real-time evaluation of drug toxicity and secondary effects. In this study, we have developed a microfluidic system for phenotype-based evaluation of toxic and teratogenic effects of drugs using zebrafish (Danio rerio) embryos and larvae as the model organism. The microfluidic chip is composed of two independent functional units, enabling the assessment of zebrafish embryos and larvae. Each unit consists of a fluidic concentration gradient generator and a row of seven culture chambers to accommodate zebrafish. To test the accuracy of this new chip platform, we examined the toxicity and teratogenicity of an anti-asthmatic agent-aminophylline (Apl) on 210 embryos and 210 larvae (10 individuals per chamber). The effect of Apl on zebrafish embryonic development was quantitatively assessed by recording a series of physiological indicators such as heart rate, survival rate, body length and hatch rate. Most importantly, a new index called clonic convulsion rate, combined with mortality was used to evaluate the toxicities of Apl on zebrafish larvae. We found that Apl can induce deformity and cardiovascular toxicity in both zebrafish embryos and larvae. This microdevice is a multiplexed testing apparatus that allows for the examination of indexes beyond toxicity and teratogenicity at the sub-organ and cellular levels and provides a potentially cost-effective and rapid pharmaceutical safety assessment tool.     

BIM and other BCL-2 family proteins exhibit cross-species conservation of function between zebrafish and mammals

Here we investigate the function of zebrafish Bcl-2 family proteins and demonstrate important conservation of function across zebrafish and mammalian systems. We have isolated a zebrafish ortholog of mammalian BIM and show that it is the most toxic of the zebrafish BH3-only genes examined, sharing this characteristic with the mammalian BIM gene. The zebrafish bad gene shows a complete lack of embryonic lethality, but like mammalian BAD, its pro-apoptotic activity is regulated through phosphorylation of critical serines. We also found that the pattern of mitochondrial dysfunction observed by zebrafish BH3 domain peptides in a mammalian cytochrome c release assay recapitulates the pattern of embryonic lethality induced by the respective mRNA injections in vivo. In contrast to zebrafish Bim, Bid exhibited only weak binding to zebrafish Bcl-2 and moderate-to-weak overall lethality in zebrafish embryos and isolated mitochondria. Given that zebrafish Bcl-2 binds strongly to mammalian BID and BIM peptides and proteins, the protein identified as the zebrafish Bid ortholog has different properties than mammalian BID. Overall, our results demonstrate the high degree of functional conservation between zebrafish and mammalian Bcl-2 family proteins, thus validating the zebrafish as a model system to further dissect the molecular mechanisms that regulate apoptosis in future forward genetic and chemical modifier screens.