Zebrafish Germline Research Articles

Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases.

Direct genomic manipulation at a specific locus is still not feasible in most vertebrate model organisms. In vertebrate cell lines, genomic lesions at a specific site have been introduced using zinc-finger nucleases (ZFNs). Here we adapt this technology to create targeted mutations in the zebrafish germ line. ZFNs were engineered that recognize sequences in the zebrafish ortholog of the vascular endothelial growth factor-2 receptor, kdr (also known as kdra). Co-injection of mRNAs encoding these ZFNs into one-cell-stage zebrafish embryos led to mutagenic lesions at the target site that were transmitted through the germ line with high frequency. The use of engineered ZFNs to introduce heritable mutations into a genome obviates the need for embryonic stem cell lines and should be applicable to most animal species for which early-stage embryos are easily accessible.

Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line

S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.

Expression analysis of the rap55 homolog in the zebrafish germline

The zebrafish is a model organism that reproduces sexually, then requires to produce specialized germ cells. Oocytes are loaded with ribonucleo-proteins and mRNA of maternal origin clustered in germ granules. During the first 10 cell cycles in early zebrafish development, transcription is absent and maternal mRNA clustered at the germ granules, is the main source for protein translation. In zebrafish some germ granules components have been described such as the protein or mRNA of the genes vasa, gcl, dead end and nanos. Recently the protein Rap55/Car1/Trailer hitch/Lsm14/Rap55 was found to be another germ granule component in organisms like Drosophila melanogaster, Caenorhabditis elegans and Xenopus laevis. This is the Scd6 homolog described in Saccharomyces cerevisiae, that is located in P-bodies (Processing bodies), therefore Rap55 is possible involved in mRNA processing. We have found three zebrafish homologous genes for Rap55 located in chromosomes 13, 23 and 25 respectively. These genes have highly conserved domains, as the Lsm domain in the amino-terminal region and the FDF domains: DFDF, FFD and TFG. They contain also RGG boxes located in glycine-rich regions in the c-termini. RGG boxes are RNA binding domains. RT-PCR analysis showed that the three homologs were expressed in all developmental stages from two cells to 5 days post-fertilization. We are currently studying the expression pattern of a GFP-fusion protein from one isoform that express in ovary, besides analyzing the effect of blocking its expression by morpholino antisense injections.

Enhancer detection in the zebrafish using pseudotyped murine retroviruses

Vectors based on murine retroviruses are among the most efficient means to insert reporter constructs into the context of a vertebrate chromosome with the aim to visualize cis-regulatory information available to a basal promoter at the site of insertion. In combination with using the zebrafish embryo as a readout for the activity of regulatory elements, enhancer detection becomes a powerful technique for gene discovery and for the mapping of the extent of regulatory domains in a vertebrate genome. Our laboratory has performed the only large-scale enhancer detection screen to date in any vertebrate and we describe in this paper the methods we developed to generate viral particles, to insert reporter constructs into the zebrafish germ line, the screening of detection events in heterozygous F1 embryos, and the isolation of genomic sequence flanking the inserted vector for the purpose of genomic mapping. Given sufficient scale, the technology described here can be used to obtain cis-regulatory information across the entire zebrafish genome for any given basal promoter.

Zebrafish Embryo Cells Remain Pluripotent and Germ-Line Competent for Multiple Passages in Culture

Mouse embryonic stem (ES) cell lines are routinely used to introduce targeted mutations into the genome, providing an efficient method to study gene function. Application of similar gene knockout techniques to other organisms has been unsuccessful due to the lack of germ-line competent ES cell lines from non-murine species. Previously, we reported the production of zebrafish germ-line chimeras using short-term primary embryo cell cultures. Here we demonstrate that zebrafish embryo cells, maintained for several weeks and multiple passages in culture, remain pluripotent and germ-line competent. Zebrafish germ-line chimeras were generated from passage 5 and 6 cultures initiated from blastula- and gastrula-stage embryos. In addition to the germ line, the cultured cells contributed to multiple tissues of the host embryo, including muscle, liver, gut, and fin. To facilitate the identification of germ-line chimeras, ES cells expressing the green fluorescent protein (GFP) were introduced into host embryos, and germ-line contribution was detected by the presence of GFP+ cells in the region of the gonad. The germ-line competent embryo cell cultures will be useful for the development of a gene targeting strategy that will increase the utility of the zebrafish model for studies of gene function.

Development of Cell Cultures with Competency for Contributing to the Zebrafish Germ Line

Development of Cell Cultures with Competency for Contributing to the Zebrafish Germ Line

Development of Cell Cultures with Competency for Contributing to the Zebrafish Germ Line

The zebrafish is an established model for the genetic analysis of vertebrate development. Forward-genetic screens have generated thousands of mutations, and antisense-based methods have been used to transiently knockdown gene expression during embryogenesis. Although these methods have made the zebrafish a valuable system for the identification and functional characterization of developmentally important genes, one deficiency of the zebrafish model is the absence of methods to introduce targeted mutations to generate knockout lines of fish. Application of gene-targeting methods has been limited in nonmurine species due to the absence of germ-line competent embryonic stem (ES) cell lines. Recently, progress was made in addressing this problem by the derivation of zebrafish embryo cell lines that remain pluripotent and germ-line competent for multiple passages in culture. Zebrafish germ-line chimeras were generated using cultures derived from embryos at two different developmental stages, and targeted insertion of vector DNA by homologous recombination was demonstrated in both cultures. Several strategies are being used to optimize the production and identification of germ-line chimeras. The zebrafish embryo cell culture system should provide the basis of a gene-targeting approach that will complement other genetic strategies and improve the utility of the zebrafish model for studies of development and disease.

Production of zebrafish germline chimeras from cultured cells.

Because of its many favorable characteristics, the zebrafish has become a popular model for studies of vertebrate development (1,2). To further enhance the utility of the zebrafish model for the genetic analysis of embryogenesis, we have been working to establish methods for cell-mediated gene transfer and targeted mutagenesis using pluripotent zebrafish embryonic stem (ES) cells (3–5). Successful ES cell-mediated gene transfer requires that the cultured pluripotent ES cells are able to produce viable germ cells after being introduced into a host embryo (6,7). ES cells are genetically altered in culture by the incorporation of foreign DNA, and, following in vitro selection and expansion, the cells are introduced into the host embryo where they participate in normal development and contribute to the germ cell lineage. When sexually mature, the germline chimeric animal possesses eggs or sperm derived from ES cells, making it possible to pass the genetic alteration on to subsequent generations to establish the transgenic or knockout line (6,7). This chapter describes a protocol for the derivation of germline-competent pluripotent zebrafish ES cell cultures, along with methods for introducing foreign DNA into cells by electroporation and for using the cultured ES cells to generate germline chimeric embryos.

Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish.

In response to the lack of a transgenic line of zebrafish labeled with heart-specific fluorescence in vivo to serve as a research model, we cloned a 1.6-kb polymerase chain reaction (PCR) -product containing the upstream sequence (-870 bp), exon 1 (39 bp), intron 1 (682 bp), and exon 2 (69 bp) of the zebrafish cardiac myosin light chain 2 gene, (cmlc2). A germ-line transmitted zebrafish possessing a green fluorescent heart was generated by injecting this PCR product fused with the green fluorescent protein (GFP) gene with ends consisting of inverted terminal repeats of an adeno-associated virus. Green fluorescence was intensively and specifically expressed in the myocardial cells located both around the heart chambers and the atrioventricular canal. Neither the epicardium nor the endocardium showed fluorescent signals. The GFP expression in the transgenic line faithfully recapitulated with the spatial and temporal expression of the endogenous cmlc2. Promoter analysis showed that the fragment consisting of nucleotides from -210 to 34 (-210/34) was sufficient to drive heart-specific expression, with a -210/-73 motif as a basal promoter and a -210/-174 motif as an element involved in suppressing ectopic (nonheart) expression. Interestingly, a germ-line of zebrafish whose GFP appeared ectopically in all muscle types (heart, skeletal, and smooth) was generated by injecting the fragment including a single nucleotide mutation from G to A at -119, evidence that A at -119 combined with neighboring nucleotides to create a consensus sequence for binding myocyte-specific enhancer factor-2.

Production of maternal-zygotic mutant zebrafish by germ-line replacement.

We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal-zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryological, genetic, genomic, and biochemical studies.

An Evolutionary Conserved Region in the vasa 3′UTR Targets RNA Translation to the Germ Cells in the Zebrafish

In many animals, germ cells are set aside from somatic cells early during development to give rise to sperm in males and eggs in females. One strategy to achieve this separation is to localize special cytoplasmic granules to the precursors of the germline. In Drosophila, the vasa gene has been shown to encode an essential component of these granules. While Vasa protein is directly targeted to the forming germ cells of Drosophila, Vasa protein expression in the germline of Xenopus and zebrafish is thought to be achieved by RNA localization. To analyze whether the machinery responsible for RNA localization is conserved among lower vertebrates, we tested different vasa homologs for their ability to localize in Xenopus oocytes. Reporter transcripts fused to the vasa 3'UTR of zebrafish are recruited to the germ plasm of injected Xenopus oocytes, although the 3'UTR shows no clear sequence similarity to the Xenopus vasa-like DEADsouth 3'UTR. However, isolation, expression pattern analysis, and sequence inspection of vasa genes from different teleosts indicate that RNA localization correlates with the presence of several conserved regions in the 3'UTR. Introduction of reporter transcripts fused to different vasa 3'UTR deletions into Xenopus and zebrafish demonstrates that one of these conserved regions is sufficient for RNA localization in either species. Moreover, these regions target GFP translation to the germline of transgenic fish. Our results suggest the existence of a common RNA localization machinery in lower vertebrates that uses a functionally conserved localization signal to target gene expression to the germline.

A zebrafish histone variant H2A.F/Z and a transgenic H2A.F/Z:GFP fusion protein for in vivo studies of embryonic development.

Abstract. We have generated transgenic zebrafish lines expressing a fusion of a histone variant, H2A.F/Z, to the green fluorescent protein (GFP) of the jellyfish Aequorea victoria. Here, we describe the molecular cloning, partial characterisation and expression of the zebrafish H2A.F/Z histone gene, as well as the construction of the transgene and its transformation into the zebrafish germ line. No abnormality can be detected in transgenic fish expressing the H2A.F/Z:GFP fusion protein. The nuclear localisation of the fusion protein correlates with the start of zygotic transcription, in that it is present in the unfertilised egg and in the cytoplasm of cells after the first cleavages, being found in some nuclei after the seventh or eighth cleavage, whereas all nuclei from the 1,000-cell stage on, i.e. after midblastula transition, contain protein. In addition to these data, we present a few examples of the many possible applications of this transgenic line for developmental studies in vivo. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00427-001-0196-x

Transgene Expression in Zebrafish: A Comparison of Retroviral-Vector and DNA-Injection Approaches

To assess alternative methods for introducing expressing transgenes into the germ line of zebrafish, transgenic fish that express a nuclear-targeted, enhanced, green fluorescent protein (eGFP) gene were produced using both pseudotyped retroviral vector infection and DNA microinjection of embryos. Germ-line transgenic founders were identified and the embryonic progeny of these founders were evaluated for the extent and pattern of eGFP expression. To compare the two modes of transgenesis, both vectors used the Xenopus translational elongation factor 1-α enhancer/promoter regulatory cassette. Several transgenic founder fish which transferred eGFP expression to their progeny were identified. The gene expression patterns are described and compared for the two modes of gene transfer. Transient expression of eGFP was detected 1 day after introducing the transgenes via either DNA microinjection or retroviral vector infection. In both cases of gene transfer, transgenic females produced eGFP-positive progeny even before the zygotic genome was turned on. Therefore, GFP was being provided by the oocyte before fertilization. A transgenic female revealed eGFP expression in her ovarian follicles. The qualitative patterns of gene expression in the transgenic progeny embryos after zygotic induction of gene expression were similar and independent of the mode of transgenesis. The appearance of newly synthesized GFP is detectable within 5–7 h after fertilization. The variability of the extent of eGFP expression from transgenic founder to transgenic founder was wider for the DNA-injection transgenics than for the retroviral vector-produced transgenics. The ability to provide expressing germ-line transgenic progeny via retroviral vector infection provides both an alternative mode of transgenesis for zebrafish work and a possible means of easily assessing the insertional mutagenesis frequency of retroviral vector infection of zebrafish embryos. However, because of the transfer of GFP from oocyte to embryo, the stability of GFP may create problems of analysis in embryos which develop as quickly as those of zebrafish.

Insertional mutagenesis in zebrafish identifies two novel genes, pescadillo and dead eye, essential for embryonic development.

Recently our laboratory described an efficient method for generating retroviral provirus insertions in the zebrafish germ line, and we showed that provirus insertions induce embryonic mutations at a frequency of roughly one mutant per 70 insertions. To date we have isolated four insertional mutants and, using the proviruses as a molecular tag, have cloned the genes disrupted in three of them. The proviruses in all three mutants lie within or just 5' of the first coding exon, point in the opposite transcriptional orientation from the gene, and disrupt transcription. Here we present a molecular characterization of two genes identified by this method and describe the associated mutant phenotypes. The pescadillo (pes) gene is predicted to encode a protein of 582 amino acids with no recognizable functional motifs, which is highly conserved from yeast to humans. pes mRNA is expressed widely and dynamically during the first 3 days of embryogenesis. Prominent sites of expression are the eyes and optic tectum on day 1, the fin buds, liver primordium, and gut on day 2, and the branchial arches on day 3. Beginning at day 3 of embryogenesis, pes mutant embryos exhibit small eyes, a reduced brain and visceral skeleton, shortened fins, and a lack of expansion of the liver and gut, and then die on the sixth day of development. The dead eye (dye) gene encodes a protein of 820 amino acids that is homologous to genes of unknown function in human, mouse, and Xenopus, and that has weak homology with the yeast NIC96 (nucleoporin-interacting component) gene. dye mutants can be recognized on day 2 of embryogenesis by the presence of necrotic cells in the tectum and eyes. dye mutants die on day 5 of development. These results demonstrate the power of insertional mutagenesis in zebrafish for rapidly finding and characterizing novel genes essential for embryonic development. Using our current methodology, we estimate that our laboratory could screen approximately 25,000 insertions in 2-3 years, identifying perhaps 250-350 embryonic lethal genes. Assuming that all genes are accessible to proviral insertion, the wider application of this approach could lead to the rapid identification of the majority of genes that are required for embryonic development of this vertebrate.

Efficient recovery of ENU-induced mutations from the zebrafish germline.

We studied the efficiency with which two chemical mutagens, ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) can induce mutations at different stages of spermatogenesis in zebrafish (Brachydanio rerio). Both EMS and ENU induced mutations at high rates in post-meiotic germ cells, as indicated by the incidence of F1 progeny mosaic for the albino mutation. For pre-meiotic germ cells, however, only ENU was found to be an effective mutagen, as indicated by the frequencies of non-mosaic mutant progeny at four different pigmentation loci. Several mutagenic regimens that varied in either the number of treatments or the concentration of ENU were studied to achieve an optimal ratio between the mutagenicity and toxicity. For the two most mutagenic regimens: 4 x 1 hr in 3 mM ENU and 6 x 1 hr in 3 mM ENU, the minimum estimate of frequencies of independent mutations per locus per gamete was 0.9-1.3 X 10(-3). We demonstrate that embryonic lethal mutations induced with ENU were transmitted to offspring and that they could be recovered in an F2 screen. An average frequency of specific-locus mutations of 1.1 X 10(-3) corresponded to approximately 1.7 embryonic lethal mutations per single mutagenized genome. The high rates of mutations achievable with ENU allow for rapid identification of large numbers of genes involved in a variety of aspects of zebrafish development.