抑制Ligase4或Xrcc6活性增强斑马鱼原始生殖细胞中DNA同源重组的效率

利用斑马鱼作为体内模型, 研究旨在提高斑马鱼原始生殖细胞(Primordial germ cells, PGCs)中同源重组(Homologous recombination, HR)的效率。首先, 将UAS:mRFP-nos1载体显微注射到Tg (kop:KalTA4) 转基因胚胎中标记转基因PGCs, 结果表明筛选PGCs特异表达mRFP的胚胎能够相对提高转基因的生殖系传递效率。随后建立了PGCs中HR效率的评估体系, 并且证明抑制DNA ligase IV(Lig4)和Xrcc6(曾用名Ku70)的活性不但在全胚胎水平, 而且在PGCs水平都能够显著提高HR的效率。研究表明Tg (kop:KalTA4) 转基因品系是开展HR介导的基因打靶的一个有效平台。       

Inducible Sterilization of Zebrafish by Disruption of Primordial Germ Cell Migration

During zebrafish development, a gradient of stromal-derived factor 1a (Sdf1a) provides the directional cue that guides the migration of the primordial germ cells (PGCs) to the gonadal tissue. Here we describe a method to produce large numbers of infertile fish by inducing ubiquitous expression of Sdf1a in zebrafish embryos resulting in disruption of the normal PGC migration pattern. A transgenic line of zebrafish, Tg(hsp70:sdf1a-nanos3, EGFP), was generated that expresses Sdf1a under the control of the heat-shock protein 70 (hsp70) promoter and nanos3 3?UTR. To better visualize the PGCs, the Tg(hsp70:sdf1a-nanos3, EGFP) fish were crossed with another transgenic line, Tg(kop:DsRed-nanos3), that expresses DsRed driven by the PGC-specific kop promoter. Heat treatment of the transgenic embryos caused an induction of Sdf1a expression throughout the embryo resulting in the disruption of their normal migration. Optimal embryo survival and disruption of PGC migration was achieved when transgenic embryos at the 4- to 8-cell stage were incubated at 34.5°C for 18 hours. Under these conditions, disruption of PGC migration was observed in 100% of the embryos. Sixty-four adult fish were developed from three separate batches of heat-treated embryos and all were found to be infertile males. When each male was paired with a wild-type female, only unfertilized eggs were produced and histological examination revealed that each of the adult male fish possessed severely under-developed gonads that lacked gametes. The results demonstrate that inducible Sdf1a expression is an efficient and reliable strategy to produce infertile fish. This approach makes it convenient to generate large numbers of infertile adult fish while also providing the capability to maintain a fertile brood stock.     

Live image profiling of neural crest lineages in zebrafish transgenic lines

Zebrafish transgenic lines are important experimental tools for lineage tracing and imaging studies. It is crucial to precisely characterize the cell lineages labeled in transgenic lines to understand their limitations and thus properly interpret the data obtained from their use; only then can we confidently select a line appropriate for our particular research objectives. Here we profiled the cell lineages labeled in the closely related neural crest transgenic lines Tg(foxd3:GFP), Tg(sox10:eGFP) and Tg(sox10:mRFP). These fish were crossed to generate embryos, in which foxd3 and sox10 transgenic neural crest labeling could be directly compared at the cellular level using live confocal imaging. We have identified key differences in the cell lineages labeled in each line during early neural crest development and demonstrated that the most anterior cranial neural crest cells initially migrating out of neural tube at the level of forebrain and anterior midbrain express sox10:eGFP and sox10:mRFP, but not foxd3:GFP. This differential profile was robustly maintained in the different-tiating progeny of the neural crest lineages until 3.5dpf. Our data will enable researchers to make an informed choice in selecting transgenic lines for future neural crest research.     

Chicken β-globin insulators fail to shield the <Emphasis Type="Italic">nkx2.5</Emphasis> promoter from integration site effects in zebrafish

Genetic lineage tracing and conditional mutagenesis are developmental genetics techniques reliant on precise tissue-specific expression of transgenes. In the mouse, high specificity is usually achieved by inserting the transgene into the locus of interest through homologous recombination in embryonic stem cells. In the zebrafish, DNA containing the transgenic construct is randomly integrated into the genome, usually through transposon-mediated transgenesis. Expression of such transgenes is affected by regulatory features surrounding the integration site from general accessibility of chromatin to tissue-specific enhancers. We tested if the 1.2 kb cHS4 insulators derived from the chicken β-globin locus can shield a transgene from chromosomal position effects in the zebrafish genome. As our test promoters, we used two different-length versions of the zebrafish nkx2.5. We found that flanking a transgenic construct by cHS4 insulation sequences leads to overall increase in the expression of nkx2.5:mRFP. However, we also observed a very high degree of variability of mRFP expression, indicating that cHS4 insulators fail to protect nkx2.5:mRFP from falling under the control of enhancers in the vicinity of integration site.     

Generation and characterization of a zebrafish muscle specific inducible Cre line

Zebrafish transgenic lines provide valuable insights into gene functions, cell lineages and cell behaviors during development. Spatiotemporal control over transgene expression is a critical need in many experimental approaches, with applications in loss- and gain-of-function expression, ectopic expression and lineage tracing experiments. The Cre/loxP recombination system is a powerful tool to provide this control and the demand for validated Cre and loxP zebrafish transgenics is high. One of the major challenges to widespread application of Cre/loxP technology in zebrafish is comparatively small numbers of established tissue-specific Cre or CreERT2 lines. We used Tol2-mediated transgenesis to generate Tg(CrymCherry;-1.9mylz2:CreERT2) which provides an inducible CreERT2 source driven by muscle-specific mylz2 promoter. The transgenic specifically labels the trunk and tail skeletal muscles. We assessed the temporal responsiveness of the transgenic by screening with a validated loxP reporter transgenic ubi:Switch. Further, we evaluated the recombination efficiency in the transgenic with varying concentrations of 4-OHT, for different induction time periods and at different stages of embryogenesis and observed that higher recombination efficiency is achieved when embryos are induced with 10 μM 4-OHT from 10-somites or 24 hpf till 48 or 72 hpf. The transgenic is an addition to currently available zebrafish transgenesis toolbox and a significant tool to advance muscle biology studies in zebrafish.     

The Tol2-mediated Gal4-UAS method for gene and enhancer trapping in zebrafish

The Gal4-UAS system provides powerful tools to analyze the function of genes and cells in vivo and has been extensively employed in Drosophila. The usefulness of this approach relies on the P element-mediated Gal4 enhancer trapping, which can efficiently generate transgenic fly lines expressing Gal4 in specific cells. Similar approaches, however, had not been developed in vertebrate systems due to the lack of an efficient transgenesis method. We have been developing transposon techniques by using the madaka fish Tol2 element. Taking advantage of its ability to generate genome-wide insertions, we developed the Gal4 gene trap and enhancer trap methods in zebrafish that enabled us to create various transgenic fish expressing Gal4 in specific cells. The Gal4-expressing cells can be visualized and manipulated in vivo by crossing the transgenic Gal4 lines with transgenic lines carrying various reporter and effector genes downstream of UAS (upstream activating sequence). Thus, the Gal4 gene trap and enhancer trap methods together with UAS lines now make detailed analyses of genes and cells in zebrafish feasible. Here, we describe the protocols to perform Gal4 gene trap and enhancer trap screens in zebrafish and their application to the studies of vertebrate neural circuits.     

Radial glial cell‐specific ablation in the adult Zebrafish brain

The zebrafish brain can continue to produce new neurons in widespread neurogenic brain regions throughout life. In contrast, neurogenesis in the adult mammalian brain is restricted to the subventricular zone (SVZ) and dentate gyrus (DG). In neurogenic regions in the adult brain, radial glial cells (RGCs) are considered to function as neural stem cells (NSCs). We generated a Tg(gfap:Gal4FF) transgenic zebrafish line, which enabled us to express specific genes in RGCs. To study the function of RGCs in neurogenesis in the adult zebrafish brain, we also generated a Tg(gfap: Gal4FF; UAS:nfsB‐mcherry) transgenic zebrafish line, which allowed us to induce cell death exclusively within RGCs upon addition of metronidazole (Mtz) to the media. RGCs expressing nitroreductase were specifically ablated by the Mtz treatment, decreasing the number of proliferative RGCs. Using the Tg(gfap:Gal4FF; UAS:nfsB‐mcherry) transgenic zebrafish line, we found that RGCs were specifically ablated in the adult zebrafish telencephalon. The Tg(gfap:Gal4FF) line could be useful to study the function of RGCs. genesis 53:431–439, 2015. © 2015 Wiley Periodicals, Inc.     

Analysis of SDF-1/CXCR4 signaling in primordial germ cell migration and survival or differentiation in Xenopus laevis

Directional migration of primordial germ cells (PGCs) toward future gonads is a common feature in many animals. In zebrafish, mouse and chicken, SDF-1/CXCR4 chemokine signaling has been shown to have an important role in PGC migration. In Xenopus, SDF-1 is expressed in several regions in embryos including dorsal mesoderm, the target region that PGCs migrate to. CXCR4 is known to be expressed in PGCs. This relationship is consistent with that of more well-known animals. Here, we present experiments that examine whether chemokine signaling is involved in PGC migration of Xenopus. We investigate: (1) Whether injection of antisense morpholino oligos (MOs) for CXCR4 mRNA into vegetal blastomere containing the germ plasm or the precursor of PGCs disturbs the migration of PGCs? (2) Whether injection of exogenous CXCR4 mRNA together with MOs can restore the knockdown phenotype? (3) Whether the migratory behavior of PGCs is disturbed by the specific expression of mutant CXCR4 mRNA or SDF-1 mRNA in PGCs? We find that the knockdown of CXCR4 or the expression of mutant CXCR4 in PGCs leads to a decrease in the PGC number of the genital ridges, and that the ectopic expression of SDF-1 in PGCs leads to a decrease in the PGC number of the genital ridges and an increase in the ectopic PGC number. These results suggest that SDF-1/CXCR4 chemokine signaling is involved in the migration and survival or in the differentiation of PGCs in Xenopus.     

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ER^T2/UAS expression system¹ in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRAS^G12V expressing skin cells. The KalTA4-ER^T2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis^2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.     

Conserved Mechanisms for Germ Cell-Specific Localization of nanos3 Transcripts in Teleost Species with Aquaculture Significance

The importance of the aquaculture production is increasing with the declining global fish stocks, but early sexual maturation in several farmed species reduces muscle growth and quality, and escapees could have a negative impact on wild populations. A possible solution to these problems is the production of sterile fish by ablation of the embryonic primordial germ cells (PGCs), a technique developed in zebrafish. Cell-specific regulation of mRNA stability is crucial for proper specification of the germ cell lineage and commonly involves microRNA (miRNA)-mediated degradation of targeted mRNAs in somatic cells. This study reports on the functional roles of conserved motifs in the 3′ untranslated region (UTR) of the miRNA target gene nanos3 identified in Atlantic cod, Atlantic salmon, and zebrafish. The 3′UTR of cod nanos3 was sufficient for targeting the expression of green fluorescent protein (GFP) to the presumptive PGCs in injected embryos of the three phylogenetically distant species. 3′UTR elements of importance for PGC-specific expression were further examined by fusing truncated 3′UTR variants of cod nanos3 to GFP followed by injections in zebrafish embryos. The expression patterns of the GFP constructs in PGCs and somatic cells suggested that the proximal U-rich region is responsible for the PGC-specific stabilization of the endogenous nanos3 mRNA. Morpholino-mediated downregulation of the RNA-binding protein Dead end (DnD), a PGC-specific inhibitor of miRNA action, abolished the fluorescence of the PGCs in cod and zebrafish embryos, suggesting a conserved DnD-dependent mechanism for germ cell survival and migration.     

Isolation of teleost primordial germ cells using flow cytometry

Primordial germ cells (PGCs) generate gametes, the only cells that can transmit genetic information to the next generation. A previous report demonstrated that a fusion construct of green fluorescent protein (gfp) and zebrafish nos 1 3UTR mRNA could be used to label PGCs in a number of fish species. Here, we sought to exploit this labeling strategy to isolate teleost PGCs by flow cytometry (FCM), and to use these isolated PGCs to examine germ cell migration to the gonadal region. In zebrafish, medaka and goldfish, the PGCs were labeled by injecting the gfp-nos1 3UTR mRNA into 1- 4 cell embryos. When the embryos had developed to the somitogenesis or later stages, they were enzymatically disaggregated and GFP positive cells isolated using FCM. PGCs in the different species clustered in the same segments of the FCM scatter diagrams for total embryonic cells produced by plotting the forward scatter intensity against GFP intensity. In situ hybridization showed that the sorted zebrafish cells expressed vasa RNA in their cytoplasm, suggesting that they were PGCs. When the migration ability of the sorted cells from zebrafish was examined in an in vivo transplantation experiment, approximately 30% moved to the gonadal region of host embryos. These observations demonstrate that PGCs can be isolated without use of transgenic fishes and that the isolated PGCs retain the ability to migrate. Our data indicate that this technique will be of value for isolating PGCs from a range of fish species.     

荧光转基因斑马鱼Tg(ttn.2:EGFP)的构建

为了建立一种用于研究肌肉和心脏发育及其相关疾病的绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因斑马鱼品系,本研究使用斑马鱼ttn.2基因编码区上游启动子序列和绿色荧光蛋白基因编码序列构建了重组表达载体,并将该载体和Tol2转座酶的加帽mRNA显微共注射入斑马鱼1-细胞期胚胎,通过荧光检测、遗传杂交筛选和分子鉴定等方法,成功建立了能稳定遗传的Tg(ttn.2:EGFP)转基因斑马鱼品系。荧光表达分析及原位杂交分析结果表明,绿色荧光信号在斑马鱼肌肉和心脏组织中特异表达模式与ttn.2基因的mRNA表达一致。通过反向PCR鉴定转基因表达载体在F1代斑马鱼品系中的随机整合位点,结果表明:No.33转基因品系的EGFP基因整合在斑马鱼的4号和11号染色体上,No.34转基因品系则整合在1号染色体上。该荧光转基因斑马鱼品系Tg(ttn.2:EGFP)的成功构建为肌肉和心脏发育以及相关疾病研究提供了一个新的理想实验模型。此外,绿色荧光强烈表达的斑马鱼品系还可以作为一种新的观赏鱼。     

Characterisation and germline transmission of cultured avian primordial germ cells

Background

Avian primordial germ cells (PGCs) have significant potential to be used as a cell-based system for the study and preservation of avian germplasm, and the genetic modification of the avian genome. It was previously reported that PGCs from chicken embryos can be propagated in culture and contribute to the germ cell lineage of host birds.

Principal Findings

We confirm these results by demonstrating that PGCs from a different layer breed of chickens can be propagated for extended periods in vitro. We demonstrate that intracellular signalling through PI3K and MEK is necessary for PGC growth. We carried out an initial characterisation of these cells. We find that cultured PGCs contain large lipid vacuoles, are glycogen rich, and express the stem cell marker, SSEA-1. These cells also express the germ cell-specific proteins CVH and CDH. Unexpectedly, using RT-PCR we show that cultured PGCs express the pluripotency genes c-Myc, cKlf4, cPouV, cSox2, and cNanog. Finally, we demonstrate that the cultured PGCs will migrate to and colonise the forming gonad of host embryos. Male PGCs will colonise the female gonad and enter meiosis, but are lost from the gonad during sexual development. In male hosts, cultured PGCs form functional gametes as demonstrated by the generation of viable offspring.

Conclusions

The establishment of in vitro cultures of germline competent avian PGCs offers a unique system for the study of early germ cell differentiation and also a comparative system for mammalian germ cell development. Primary PGC lines will form the basis of an alternative technique for the preservation of avian germplasm and will be a valuable tool for transgenic technology, with both research and industrial applications.