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Keywords:

  • gata1;
  • hematopoiesis;
  • Tol2 transposable element;
  • transient transgenic rescue;
  • zebrafish

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Phenotypic rescue experiments have been commonly used in zebrafish since it is convenient to study the causality of mutant phenotypes just by injecting mRNA into embryos. However, this strategy is only effective for phenotypes at early embryonic stages due to mRNA instability. For later developmental stages, DNA constructs are used to express exogenous genes, while it is usually ineffective owing to the problem of mosaicism. This study attempted to solve the problem by using Tol2-mediated transgenesis. As a model case, we used vlad tepes (vlt), a zebrafish gata1 mutant, whose phenotypes have never been able to be rescued at later stages by transient rescue experiments. Blood cell-specific transgenic expression of gata1 was driven by its own promoter/enhancer elements. The co-injection of a Tol2-donor plasmid containing gata1 cDNA and transposase mRNA efficiently rescued the bloodless phenotypes of vlt even in day 12 larvae when definitive erythropoiesis took place with primitive erythropoiesis. This Tol2-mediated rescue is therefore considered to be a quick and easy method for analyzing the mutant phenotypes in zebrafish.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Phenotypic rescue experiments are promising approaches to identify the physiological roles, regulatory cascades and functional domains of interest genes and/or proteins. Zebrafish is a powerful tool for elucidating the gene functions during development and is expected to be a useful model for human diseases (Lieschke & Currie 2007). A number of zebrafish mutants with specific phenotypes have been isolated to date. In addition, the ease of gene knockdown by morpholino antisense oligonucleotides allowed the production of a variety of morphants with interesting phenotypes. Phenotypic rescue experiments have been commonly used in zebrafish since it is convenient to study the causality of mutant and morphant phenotypes just by injecting mRNA into embryos (Gilmour et al. 2002). However, this strategy is only effective for phenotypes during early embryonic stages due to mRNA instability, and sometimes leads to artificial additional phenotypes due to ectopic expression of injected mRNA. To overcome these limitations, DNA constructs instead of mRNA are used to express exogenous genes under the control of a tissue- and stage-specific promoter (Gilmour et al. 2002). However this approach is usually ineffective owing to the problem of mosaicism. This mosaic expression is not as serious when mutant phenotypes of early embryonic stages are analyzed because considerable amounts of injected DNA remain episomal and functional. It becomes critical at later developmental stages when episomal DNA disappears due to dilution and degradation. Preparing stable transgenic lines is a reliable method to solve this problem but it is extremely laborious and time-consuming.

The use of Tol2, a transposable element of medaka fish, enhances the transgenesis efficiency of injected DNA (Kawakami et al. 2004). The problem of mosaicism will be overcome by using Tol2 elements. Indeed, improvements of mosaicism in the transient GFP reporter analysis have been demonstrated (Fisher et al. 2006). It is therefore considered to be worth while to investigate whether the Tol2 system is applicable to the transient rescue experiments. In vlad tepes (vlt), the expression of gata1 downstream genes such as βe1-globin and alas2 are dramatically downregulated, thereby, hemoglobin is not produced, and little blood circulation is observed (Lyons et al. 2002). Previous experiments tried to rescue the vlt phenotypes by gata1 mRNA injection but failed. Exploitation of transient transgenic expression of gata1 under the transcriptional control of the gata1 hematopoietic regulatory domain (HRD) enabled the rescue of the expression of βe1-globin and alas2 in vlt embryos (Nishikawa et al. 2003). However, hemoglobin production which could be detected by o-dianisidine staining was not recovered, thus suggesting that the rescue was transient only during early developmental stages. In this study, we attempted to rescue the late stage vlt phenotypes by introducing Tol2 cis-sequences into the gata1 construct. The attempt was successful not only at the embryonic stages but also at the larval stages when definitive erythropoiesis took place with primitive erythropoiesis.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Zebrafish

Zebrafish embryos and larvae were obtained by natural mating. Embryos from vltm651 (Lyons et al. 2002) heterozygote intercrosses were used for the rescue experiments. Genomic DNA was extracted for genotyping the vltm651 mutants by incubating embryonic or larval tails in extraction buffer [10 mmol/L Tris-HCl pH 8.2, 10 mmol/L EDTA pH 8.0, 200 mmol/L NaCl, 0.5% SDS, 800 μg/mL proteinase K (Sigma-Aldrich, St. Louis, MO, USA)] at 55°C for more than 4 hours, amplified by PCR using specific primers 5′-gtgagtatacacaattacac and 5′-ctgaagctgcattctttttgc, and directly sequenced using a primer 5′-cactacgtttaccagaagg.

Plasmid Construction

A 3-kb internal region in the gata1 HRD of p8.1kG1-eGFP (Kobayashi et al. 2001) was deleted by NsiI digestion and re-ligation to construct p8.1kG1Δ3-eGFP. The pT8.1gata1Δ3-eGFP was constructed by subcloning a 5.1-kb XhoI-NcoI fragment from p8.1G1Δ3-eGFP containing the internal-deleted gata1 HRD between XhoI and NcoI sites of pT2AL200R200G (Urasaki et al. 2006), followed by a partial NotI digestion, filling in by T4 DNA polymerase and ligation. pCS2FLgata1 was constructed by subcloning an EcoRI-Bsp120I fragment from pCS2zGATA1 (Kobayashi et al. 2001) into pCS2FL (Li et al. 2008). pT8.1gata1Δ3-FLgata1 was constructed by subcloning both an NcoI-BglII fragment containing the open reading frame of FLAG-tagged gata1 in pCS2FLgata1 amplified by PCR using primers 5′-aaaccatggg-ccatggactacaaagaccatg and 5′-aaaagatctttaaaaaacc-tcccacacctcc and an XhoI-NcoI fragment from p8.1G1Δ3-eGFP between NcoI and BglII sites and between XhoI and NcoI sites of pT2AL200R200G, respectively. All constructs were verified by DNA sequencing. A plasmid pCS-TP was described previously (Kawakami et al. 2004).

Microinjection

12.5 pg of pT8.1gata1Δ3-GFP or pT8.1gata1Δ 3-FLgata1 was co-injected into a blastmere of early one-cell stage embryos with 25 pg of Tol2-transposase mRNA synthesized using SP6 mMESSAGE mMACHINE in vitro transcription kit (Ambion, Austin, TX, USA). The IM300 microinjector (Narishige, Tokyo, Japan) was used for the injection. GFP expression was examined under a GFP2 (480 nm excitation, 510 nm barrier) filter of an MZFLIII microscope (Leica, Wetzler, Germany) equipped with a 600CL-CU digital camera (Pixera, Los Galos, CA, USA). Expression of FLAG-tagged gata1 proteins was analyzed by whole mount immunostaining as described previously (Kobayashi et al. 1998) using anti-FLAG antibodies (M2, peroxidase conjugate; Sigma-Aldrich). Staining of hemoglobin by o-dianisidine was performed as previously described (Detrich et al. 1995).

Observation of circulating blood cells

At 4 days per fertilization (dpf), each larva was placed individually into a well of 6-well tissue culture plates and was grown further. The number of circulating blood cells in 4-, 8- and 12-dpf larvae was counted under an MZ16 microscope (Leica). Time-lapse photographs of larvae were taken with a BIOZERO microscope (Keyence, Osaka, Japan), after anesthesia with 160 μg/mL tricaine (Sigma-Aldrich) and mounting in 3% methyl cellulose (Sigma-Aldrich).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

The Tol2system enhances the hematopoietic expression of exogenous genes linked togata1HRD

GFP expression driven by gata1 HRD recapitulates endogenous gata1 expression (Kobayashi et al. 2001; Long et al. 1997), thereby, it was suitable for use as a gene regulatory region in the Tol2-gata1 construct for the transgenic rescue experiments. The middle 3-kb region of the 8.1-kb gata1 HRD was deleted to shorten the construct, and this has no effects on the promoter activity (Fig. 1a). The GFP-positive embryos here were those contain more than 6 GFP-positive cells in the hematopoietic intermediate cell mass (ICM) (1 dpf) or 10 GFP-positive cells in the circulating blood (2 and 3 dpf).

image

Figure 1.  Exogenous gene expression using the Tol2-gata1 vector. (a) GFP expression directed from gata1 HRD reporter constructs. Values in the right panel show the percentages of embryos that contained more than 6 GFP-positive cells in the ICM (1 dpf) or 10 GFP-positive cells in the circulating blood (2 and 3 dpf). The numbers of embryos examined are indicated in the parentheses. dGATA, CACCC, Ex1 and Int1 denote the double GATA motif, CACCC box, Exon 1 and Intron 1, respectively. (b) Structure of pT8.1gata1Δ3-eGFP. It is noted that the upstream NotI site was disrupted. (c) GFP expression in embryos injected with pT8.1gata1Δ3-eGFP together with (TPase+) or without (TPase −) Tol2-transposase mRNA. Arrows denote ICM. (d) Percentages of embryos expressing GFP in almost all cells in the ICM at 24 dpf. Error bars indicate S.D. from four independent experiments. (e) Whole-mount immunostaining using anti-FLAG antibody. Expression of exogenous gata1 protein in embryos co-injected with pT8.1gata1Δ3-FLgata1 and Tol2-transposase mRNA was analyzed.

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The pT8.1gata1Δ3-eGFP was constructed by introducing gata1 HRD with an internal deletion into pT2AL200R200G (Urasaki et al. 2006) (Fig. 1b). An upstream NotI site was disrupted to make a downstream NotI site as an unique site for convenient subcloning. We injected pT8.1gata1Δ3-eGFP with or without mRNA for the Tol2 transposase, and observed GFP expression in the ICM of 24-hours per fertilization (hpf) embryos. About half of the embryos in which Tol2-transposase mRNA was co-injected showed an almost uniform GFP expression in the ICM region (Fig. 1c). The number of embryos expressing GFP in almost all cells in the ICM was counted (Fig. 1d) and this revealed that the co-injection of Tol2-transposase mRNA dramatically increased the number of GFP-positive cells. We next constructed pT8.1gata1Δ3-FLgata1 for transgenic rescue experiments by replacing the GFP gene of pT8.1gata1Δ3-eGFP with FLAG-tagged gata1. pT8.1gata1Δ3-FLgata1 was co-injected with Tol2-transposase mRNA and the presence of an almost uniform expression of exogenous gata1 proteins in the ICM was confirmed by immunostaining analysis using anti-FLAG antibody (Fig. 1e). The results suggest that a variety of exogenous genes as well as GFP and gata1 can also be expressed in almost all the hematopoietic cells using this Tol2-gata1 vector.

Phenotypic rescue of gata1mutantvlt

We examined the activity of Tol2-gata1 vector in vlt embryos using pT8.1gata1Δ3-eGFP and demonstrated that it was indistinguishable from that in wild-type embryos (Fig. 2a). The result was consistent with our previous observations using the conventional vector (Nishikawa et al. 2003).

image

Figure 2.  Phenotypic rescue experiments of gata1 mutant vlt using Tol2-gata1 vector. (a) GFP expression at 24 hpf in wild-type or vlt mutant embryos co-injected with pT8.1gata1Δ3-eGFP and Tol2-transposase mRNA. Arrows denote ICM. (b) o-dianisidine staining at 48 hpf in the blood islands of vlt embryos injected with pT8.1gata1Δ3-FLgata1 and Tol2-transposase mRNA and of uninjected wild-type embryos. (c) Percentages of o-dianisidine positive embryos at 48 dpf. Indicated numbers of vlt embryos were tested. (d) Percentages of survived homozygous vlt larvae. Numbers of circulating blood cells were counted at 4, 8 and 12 dpf in vlt larvae co-injected with or without pT8.1gata1Δ3-FLgata1 and Tol2-transposase mRNA. It is noted that the co-injection could rescue lethal phenotypes of vlt larvae at 12 dpf.

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pT8.1gata1Δ3-FLgata1 was next co-injected with Tol2-transposase mRNA to rescue hematopoietic expression of gata1 in vlt embryos and hemoglobin production was analyzed in 48-hpf embryos by o-dianisidine staining (Fig. 2b). Homozygous vlt embryos were identified by genotyping after the staining. Intense o-dianisidine staining over the surface of the yolk was observed in wild-type siblings, while no staining was evident in homozygous vlt embryos (data not shown). When we co-injected pT8.1gata1Δ3-FLgata1 with Tol2-transposase mRNA, some homozygous vlt embryos became o-dianisidine positive (26.3%, n = 80) (Fig. 2b, c). This suggests that hemoglobin production was rescued in least in one-fourth of injected embryos.

The recovery of blood circulation was further examined. vlt embryos were co-injected with pT8.1gata1Δ3-FLgata1 and Tol2-transposase mRNA and grown individually until 12-dpf larvae. Moving pictures of blood circulation in each larva at 4, 8 and 12 dpf were taken (Movies S1-S8). In the control uninjected homozygous vlt larvae, <3 blood cells were circulating. All these larvae died by 12 dpf in the present experimental conditions. In contrast, more than 30 blood cells were circulating in 82% (n = 22) of the vlt larvae co-injected with pT8.1gata1Δ3-FLgata1 and Tol2-transposase mRNA at 4 and 8 dpf (Fig. 2d, Movies S6–S8). About half of all the co-injected vlt larvae survived until 12 dpf. The results indicate exogenous gata1 expression was able to rescue the hematopoietic phenotypes of vlt. Importantly, blood circulation was observed even after 10 dpf in the rescued larvae when definitive erythroid cells was considered to replace primitive erythroid cells (Davidson & Zon 2004), thus suggesting that not only primitive but also definitive erythropoiesis was rescued by the Tol2-gata1 system.

Transient rescue system using the Tol2system

Rescue experiments are useful for not only confirmation of the responsible gene for the mutants but also for identifying the functional domains in proteins and determining hierarchies of gene cascades. Transgenic rescue experiments of mouse hematopoietic mutants using Gata1 HRD have contributed significantly to experimental hematology (Shimizu & Yamamoto 2005). In particular, the results of hematopoietic-specific rescue experiments using a variety of mutant mice were outstanding, e.g. Runx1 (Yokomizo et al. 2007), EpoR (Suzuki et al. 2002), MafG (Motohashi et al. 2000, 2006) and Alas-E knock-out mice (Nakajima et al. 2006). This system is quite useful to observe the physiological roles of genes in hematopoietic cells. However, it takes more than a half year to obtain the results due to the time required for establishing stable transgenic lines. In this study, we demonstrated that the introduction of the Tol2-gata1 vector develops transient rescue experiments to practical levels for a zebrafish hematopoietic mutant. This therefore considered to be an attractive protocol to analyze the molecular basis of zebrafish hematopoiesis, especially definitive hematopoiesis, which up to now still remains poorly understood.

TheTol2-mediated transient rescue system is also thought to be a powerful tool for analyzing the phenotypes of non-hematopoietic zebrafish mutants. Though not very many transcriptional drivers for tissue-specific expression of exogenous genes have been identified in zebrafish such as gata1 HRD in the present study, this problem is expected to be overcome by the use of bacterial artificial chromosome (BAC)-based transgenesis which reduces the labor of identifying and isolating regulatory elements for the specific expression (Higashijima 2008; Nakamura et al. 2008). The Tol2 system can also improve the low efficiency of BAC-transgenesis (Suster et al. 2009). The GAL4-UAS system will be another choice for driving the expression of exogenous genes (Asakawa & Kawakami 2008), that can make it possible to carry out conditional rescue experiments as well. Inducible drivers such as heat shock promoter may also be useful for conditional rescue (Shoji & Sato-Maeda 2008). The Tol2-mediated transient rescue system is also applicable for rescue experiments against the morphants which are caused by gene knockdown using morpholino antisense oligonucleotides. The use of zebrafish and the current vector system will therefore simplify and accelerate the analysis of many biological aspects.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

We thank T. Kinoshita, H. Niu and T. Shimokoube for help in fish maintenance, A. Urasaki and T. Tsujita for experimental helps and discussions. This work was supported by Grants-in-Aids from the Japan Society for the Promotion of Science (07J02836 to M.T.), the Japan Science and Technology Corporation (ERATO) (M.Y.) and the Ministry of Education, Science, Sports and Culture of Japan (20052007 and 11680670 to M.K.).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Movie S1. Circulating blood cells in the wild-type sibling at 4 dpf.

Movie S2. Circulating blood cells in the wild-type sibling at 8 dpf.

Movie S3. Circulating blood cells in the wild-type sibling at 12 dpf.

Movie S4. Circulating blood cells in uninjected homozygous vlt larvae at 4 dpf.

Movie S5. Circulating blood cells in uninjected homozygous vlt larvae at 8 dpf.

Movie S6. Circulating blood cells in the homozygous vlt larvae co-injected with pT8.1gata1&Dgr;3-FLgata1 and Tol2-transposase mRNA at 4 dpf.

Movie S7. Circulating blood cells in the homozygous vlt larvae co-injected with pT8.1gata1&Dgr;3-FLgata1 and Tol2-transposase mRNA at 8 dpf.

Movie S8. Circulating blood cells in the homozygous vlt larvae co-injected with pT8.1gata1&Dgr;3-FLgata1 and Tol2-transposase mRNA at 12 dpf.

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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DGD_1168_sm_MovS1.mov953KSupporting info item
DGD_1168_sm_MovS2.mov930KSupporting info item
DGD_1168_sm_MovS3.mov597KSupporting info item
DGD_1168_sm_MovS4.mov846KSupporting info item
DGD_1168_sm_MovS5.mov734KSupporting info item
DGD_1168_sm_MovS6.mov574KSupporting info item
DGD_1168_sm_MovS7.mov816KSupporting info item
DGD_1168_sm_MovS8.mov680KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.