Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a -/- mutants
© Sapetto-Rebow et al; licensee BioMed Central Ltd. 2011
Received: 23 December 2010
Accepted: 23 November 2011
Published: 23 November 2011
Genetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse Top2a knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish top2a mutants arises from maternal expression of top2a or compensation from its top2b paralogue.
Here, we describe bloody minded (blm), a novel mutant of zebrafish top2a. In contrast to mouse Top2a nulls, zebrafish top2a mutants survive to larval stages (4-5 day post fertilization). Developmental analyses demonstrate abundant expression of maternal top2a but not top2b. Inhibition or poisoning of maternal topoisomerase II delays embryonic development by extending the cell cycle M-phase. Zygotic top2a and top2b are co-expressed in the zebrafish CNS, but endogenous or ectopic top2b RNA appear unable to prevent the blm phenotype.
We conclude that maternal top2a enables zebrafish development before the mid-zygotic transition (MZT) and that zebrafish top2a and top2b are not functionally redundant during development after activation of the zygotic genome.
Topoisomerase (DNA) II alpha (TOP2A) is a nuclear protein which regulates DNA architecture during the mitotic phase of the cell cycle . Studies in Xenopus have shown that its function in chromatin condensation is tightly coupled to prior DNA replication [2–4]. The expression of TOP2A is cell cycle regulated, reaching a peak in the G2/M phase . Thus, up-regulated levels of TOP2A protein are found in proliferating cancer cells, and TOP2A is essential for the viability of these dividing cells. Consistent with its role in cell proliferation, genetic aberrations in TOP2A are linked to numerous human cancers . To facilitate proper separation of chromatids and DNA replication, TOP2A generates transient double-stranded breaks in DNA [7–10]. Pharmacological targeting of TOP2A, which is extensively applied in cancer treatment, exploits this mechanism .
Vertebrate genomes contain two topoisomerases II paralogues. Human TOP2A and TOP2B have similar molecular masses (180 and 170 kDa, respectively) and share ~70% amino acid similarity, with the greatest divergence occurring in the C-terminal domain . Despite structural similarities, these topoisomerases II isoforms have different expression patterns and functions . In contrast to the expression of mammalian Top2a which peaks at G2/M, mammalian Top2b is expressed in differentiated tissues and its expression is not cell cycle regulated [14–17]. Several in vitro models have been utilised to study the loss-of-function effects associated with topoisomerase II genes. However, the critical role of Top2a genes for cell proliferation and survival necessitates conditional knockout models [18, 19]. In vivo, genetic elimination of Top2a is dramatic, with mouse knockouts not developing beyond the 4-8 cell stage . Underlying a distinct role, Top2b knockouts are not embryonic lethal. Instead Top2b is required during neuronal differentiation, for survival of some neural cells and neurite outgrowth [9, 21, 22]. Top2b null mice have defects in cerebral stratification and motor axons fail to contact skeletal muscles resulting in death soon after birth due to breathing impairment [23, 24]. The topoisomerases II paralogues are also important as targets of anti-cancer drugs. ICRF-193, a catalytic inhibitor, blocks topoisomerase II-mediated ATP hydrolysis which is required to regenerate its active enzymatic form . Etoposide, a topoisomerase II poison, inhibits the ability of topoisomerase II to re-ligate DNA molecules and therefore stabilises cleavable complexes of the enzyme leading to extensive fragmentation of DNA and cell death . Topoisomerase II poisons are reported to mediate their cytotoxic effects through TOP2A, while double strand breaks and DNA rearrangements associated with secondary malignancies are due to TOP2B .
Two top2a mutant alleles have previously been documented in zebrafish: hi3635 generated by viral insertion  and can4 by ENU mutagenesis . These mutants present with similar phenotypes including brain necrosis, abnormal tail curvature and death at 4-5 dpf. The can4 mutants have been studied more thoroughly and display reduced cell proliferation, mitotic spindle defects and increased DNA content. Recently, a zebrafish top2b mutant was reported with a phenotype distinct to top2a mutants, including defects in neurite targeting within the retinal inner plexiform layer and tectal neuropil . These distinct phenotypes indicate separate functions of the zebrafish Top2a and Top2b isoforms.
Previous studies, however, do not explain the viability of zebrafish top2a mutants up to larval stages, in contrast to the early embryonic lethality of mouse Top2a knockouts. One explanation could relate to differential requirements of teleost and mammalian embryos for the timing of initiation of zygotic transcription, i.e. transcription from the embryo's own genome. The mid-blastula transition (MBT) refers to the moment during the blastula stage of embryonic development when expression of the zygotic genome starts, cell cycles lengthen, and cells acquire the ability to migrate . The original term MBT (still used in relation to Xenopus) was expanded to include a phase of elimination of maternal transcripts and proteins starting before the activation of zygotic transcription . This developmental event is referred as maternal-to-zygotic-transition (MZT) in recent studies . In zebrafish, zygotic transcription increases gradually from the 10th (~2.75 hpf) to 13th cycle (~4.75 hpf) [34, 35].
Here, we describe a novel zebrafish top2a mutant named "bloody minded" (blm) which was identified in an ENU mutagenesis screen. Mutant larvae can survive to ~5 dpf, although abnormal embryo morphology is discernable at ~27 hpf. We tested the hypotheses that the advanced development of zebrafish top2a mutants is due to either the presence of maternal Top2a or redundancy with the Top2b isoform. Our results are consistent with early embryonic development depending on maternal transcripts of zebrafish top2a. The absence of zygotic top2a is not fully compensated for by maternal, zygotic or ectopic top2b mRNA suggesting distinct functions of Top2a and Top2b in embryonic development.
blmis a lethal recessive zebrafish mutant
blm arises from a nonsense mutation in the top2agene
To confirm that the blm phenotype arises from a mutation in the top2a gene we performed complementation assays (Figure 2D). Matings of blm carriers and hi3635 carriers fail to complement. Matings of blm+/- with hi3635+/- generated ~30% embryos with the blm phenotype, whereas outcrosses to wildtype fish did not. Thus, genomic and genetic data confirm that the blm mutant results from a nonsense mutation in the zebrafish top2a gene.
top2amutants exhibit defects in cell cycle progression
Maternal top2a but not top2bis expressed pre-MZT
Real-time PCR also demonstrates a reduced level of top2a transcript in blm embryos compared to wildtype siblings at 1-3 dpf (Figure 4C). Zebrafish top2b transcript is negligible before 4 hpf, but is abundantly expressed at 10 hpf, a stage after MZT and before blm phenotypes are detected (Figure 4D). At 27 hpf, top2b levels are similar in blm mutants and widtype siblings. In siblings, top2b levels remain high at 2 dpf. In contrast, top2b levels decrease by ~50% in blm mutants by 2 dpf, consistent with the morphological defects observed at this developmental stage. In summary, maternal top2a could account for the extended pre- and/or post-MZT development of blm mutants whereas zygotic top2b could only account for the extended post-MZT development.
No evidence of functional redundancy between top2a and top2b in vivo
Chemical inhibition of Top2a delays development of pre-MZT embryos
Inhibition of XenopusTop2a results in an extended M-phase in cycling extracts
Top2a mutagenic susceptibility
Vertebrate topoisomerase II alpha genes appear particularly susceptible to somatic and germline mutations. Amplifications of the human TOP2A locus is associated with somatic mutations in subsets of breast, bladder and gastric cancers . Notably, three genetically-distinct zebrafish top2a mutant alleles have now been uncovered (hi3635, can4, blm) in independent mutagenesis screens, suggesting that the top2a locus is also particularly susceptible to germline DNA perturbations [28, 29]. All zebrafish top2a mutants display equivalent gross morphological phenotypes: small eye and brain, CNS necrosis, abnormal tail curvature and death at 4-5 dpf. Although, no further characterisation of the hi3635 insertion mutant has been reported, can4 and blm top2a -/- alleles display similar cell cycle defects, including accumulation of mutant cells in G2/M and altered phosphohistone H3 expression (Figure 3), .
Overall our data lead us to conclusion that zebrafish Top2a and Top2b are not functionally redundant in vivo. The zebrafish paralogs are ~65% identical at the protein level; both containing N-terminal ATP-binding sites and a central domain tyrosine required for DNA cleavage and ligation (Additional file 1). Only the C-terminal domains, containing nuclear localization sequences and phosphorylation sites, are highly divergent . Previous studies in human cell lines suggested that TOP2B can compensate for depleted TOP2A [37, 38]. In contrast, abundant zebrafish top2b transcript at stages post-MZT is not sufficient to prevent the blm phenotype, despite extensive temporal expression in tissues that develop morphological abnormalities in blm mutants. The onset of the blm phenotype correlates with depletion of maternal top2a, at a stage when zygotic top2b is abundant, and we identify embryos overexpressing ectopic top2b that retain the blm phenotype. Furthermore, in contrast to top2a mutants, the recently described top2b mutant displays a distinct phenotype in post-mitotic cells and not in proliferating cells . There is a potential compensatory role for maternal Top2b in pre-MZT stages that we cannot directly exclude. However, pre-MZT top2b expression is negligible  and we have not found evidence of maternal deposition of Top2b in the literature. Notably, the functional divergence we observe in vivo between zebrafish Top2a and Top2b is in agreement with other in vitro studies reporting that the unique cell cycle functions of the Top2a isoform are dependent on specific C-terminal sequences [5, 12, 38]. Overall, our in vivo studies support that top2a and top2b do not exhibit complete functional redundancy during zebrafish embryo development.
Role of maternal Top2a pre-MZT
Previous genetic approaches to analyse maternal top2a function using morpholino knockdowns have proven unsuccessful, probably because of the abundance of top2a/Top2a . Therefore, to confirm that maternal top2a enables pre-MZT development, we pharmacologically targeted maternal Top2a protein using ICRF-193, a topoisomerase II catalytic inhibitor and etoposide, a topoisomerase II poison .
Our data supports the conclusion that maternal top2a is needed for the normal progression of pre-MZT embryos through developmental divisions. Both ICRF-193 and etoposide delay the progression of embryos through hierarchical developmental stages. This delay is reversible as washing out the drug results in the vast majority of zebrafish embryos proceeding beyond the 24 hpf, segmentation stage. It is likely that we are only partially inhibiting Top2a protein as complete inhibition is expected to arrest development earlier.
To explore the mechanism underlying the impaired developmental cell divisions in ICRF-193 treated embryos we utilised Xenopus cycling extracts which recapitulate in vitro the cell cycle of pre-MBT embryos . Only topoisomerase II α is expressed in Xenopus eggs and embryos as demonstrated by the fact that depletion of Top2a from egg extracts abolishes topoisomerase II activity . Our molecular analysis of Xenopus extracts treated with ICRF-193 indicates a lengthening of M phase. This is consistent with the previous morphological studies that observed anaphase stalling of pre-MZT zebrafish treated with etoposide .
We propose that the mechanism underlying delayed pre-MZT development and M phase lengthening following Top2 inhibition reflects activation of a cell cycle decatenation checkpoint. In the pre-MZT embryos lacking gap phases, Top2a inhibition results in a stoichiometric decrease in DNA decatenation during cell division activating a DNA decatenation checkpoint [40, 41]. The M phase lengthening observed in ICRF-193 treated cycling extracts are consistent with defects in chromosome condensation and segregation most likely slowing the cycle and possibly activating a checkpoint in mitosis.
Inhibition of Top2a could be impacting on one or more phases of the cell division cycle and its effects in the simple early embryonic cell cycle and later somatic cycles where gap phases are present may be the result of different cell cycle processes being impeded. ICRF-193 is known to activate a G2 decatenation checkpoint in adult cells [42, 43]. Interestingly, Top2a, but not Top2b, is required for activation of the decatenation checkpoint . Catalytically inhibited Top2a exposes a C-terminal phospho-serine that recruits MDC1 to chromatin and activates the decatenation checkpoint . As shown by our data, etoposide, which induces DNA damage in adult cells, phenocopies ICRF-193 in pre-MZT embryos. The likely explanation is the absence of Top2b and the absence of a DNA damage checkpoint in pre-MZT embryos . This also suggests that etoposide alters Top2a topology to activate the decatenation checkpoint. In summary, we propose that inhibition of maternal Top2a activates a DNA decatenation checkpoint that delays mitosis and results in delayed embryonic development.
Phenotypic differences between mouse and zebrafish Top2amutants
Why does maternal Top2a enable zebrafish top2a -/- mutants to progress to stages comprising of hundreds of thousands of cells, whilst mouse Top2a -/- knockouts fail to divide beyond the 4-8 cell stage? One possibility is that maternal Top2a is not expressed in mammalian embryos. This is not the case, as studies show that catalytically active Top2a is functional at the 1-cell stage in mouse embryos . Alternatively, maternal topoisomerase II alpha may have differential stability in mice and zebrafish due to the timing of MZT which occurs between embryonic day 0.5 and 1.5 (2-cell stage) in mouse but between 2.75 and 4.75 hours after fertilization (from 1,024 cells stage) in zebrafish [34, 35, 46]. Although some maternal transcripts are still present in embryonic day 2.5 murine morulas , previous reports demonstrate that newly synthesised mouse Top2a at the 2-cell stage is essential for cell cycle progression .
The ability of zebrafish top2a -/- mutants to proceed through thousands of developmental cell divisions starkly contrasts with mammalian Top2a -/- knockouts which fail to divide beyond the 4-8 cell stage . Our analyses reveal that pre-MZT, maternal top2a is sufficient to enable zebrafish top2a-/- mutants to progress through early development. Post-MZT, zebrafish top2b expression is insufficient to fully compensate for the absence of top2a in vivo and blm mutants die at ~5 dpf (Figure 9). Top2a inhibition or loss results in clear cell cycle defects in early embryonic cell cycles of both zebrafish and Xenopus, as well as in later somatic cell cycles where blm mutants show evidence of G2/M decatenation checkpoint activation.
All animal experiments were conducted under licences from the Department of Health and Children (B100/3641; B100/3003) and according to protocols approved by the University College Dublin Animal Research Ethics Committee (AREC-P-08-54; AREC-P-10-68).
Zebrafish males of AB strain were mutagenised with ENU and mated to wildtype females to generate F1 founder fish . F1 founders were mated to wildtype fish to establish 370 F2 families. F3 offspring of random incrosses within 200 F2 families were screened for recessive mutations at 5 dpf. 25% of the offspring from family Y-008 carriers presented with blm phenotypes.
Larvae at 27 hpf, 2 dpf and 3 dpf were euthanized with benzocaine and fixed in Sorensen phosphate buffer (pH 7.3) containing 4% PFA and 2.5% glutaraldehyde. Larvae were embedded in Epon resin and semi-thin (900 nm) sections stained with toluidine blue were photographed using a Leica DMLB microscope and a Leica DFC 480 camera.
Carriers of blm (AB strain) mutants were map-crossed to wildtype Tubingen fish to generate hybrid carriers AB*/Tub. 96 blm and 96 normal siblings from map crosses were used for bulk segregation analysis. Linkage was found to chromosome 12 with the blm mutation flanked by Z-markers Z99217 and Z10806. Several new simple sequence repeat (SSR) markers were designed to further narrow the critical interval of which zC13B10-SSR1 was the closest (6/96 recombinants). Primers zC13B10-SSR1-F: CTCCAATCGAGAGTCCTCGT, zC13B10-SSR1-R: AGCTGAAGGCCTGCTGTAAA. The top2a gene was localised in the critical interval.
RNA from 3 dpf blm larvae was extracted using the Qiagen RNeasy Mini Kit and cDNA was synthesised using the Invitrogen Superscript III First Strand Synthesis for RT-PCR system. Six sets of primers were designed to amplify the whole reading frame of zebrafish top2a. The causative point mutation was sequenced using the following primers: forward 5'TGTTGCGCTACTGACTCGAC, reverse 5'TGGCCAGTTATGATGGATGA.
Hi3635 carriers were obtained from ZIRC. Complementation matings of blm +/- ♀ × hi3635 +/- ♂ and blm +/- ♂ × hi3635 +/- ♀ were performed. Phenotypes were recorded at 2 and 3 dpf.
blm and wildtype sibling larvae were collected at 27 hpf. Embryos were dechorionated, rinsed in PBS and incubated for 10 minutes at 37°C in trypsin (1 mg/ml) with DNase I (1 U/200 μl) followed by mechanical disruption with a 25 G needle. Trypsin inhibitor was added to a final concentration of 1 mg/ml. After centrifugation, the cell pellet was fixed with 70% ethanol. Cells were rinsed with PBS, treated with RNase (10 μg/ml) and DNA stained with propidium iodide (40 μg/ml). DNA content was analysed using a Coulter EPICS XL-MCL or Accuri C6 flow cytometer.
Quantification of mitotic cells in the eye
Embryos at 27 hpf were fixed at 4°C overnight in 4% PFA, then dechorionated, dehydrated in methanol and stored at -20°C. Prior to immunostaining embryos were rehydrated in PBS, permeabilised for 5 min in proteinase K (20 μg/ml) and post-fixed in 4% PFA for 30 min. Larvae were treated with blocking solution (2% normal goat serum, 1% Triton X-100, 1% Tween-20 in PBS) for 60 min followed by incubation in the primary antibody: anti-phospho-histone H3 at 1:200 dilution (Upstate Biotech) for 20 hours. Primary antibody binding was detected with Cy3-conjugated goat anti-rabbit secondary antibody (dilution 1:200). Nuclei were counter stained with DAPI. Z-stack images of whole larval eyes (optical sections at 2 μm interval) were taken with Zeiss LMS 510 Meta laser confocal microscope (with 63 × objective). Images were analysed with Imaris v7.2.3 software. To quantify the proportion of mitotic cells, embryos at 27 hpf were fixed in PFA, cryoprotected and sectioned (12 μm thickness). Mitotic nuclei were labelled with anti-phospho-histone H3 antibody (Upstate Biotech) and counter stained with DAPI. Images of sections through the eyes, head and spinal cord (at least 3 per animal) were taken with Zeiss AxioImager M1 microscope (objective 100×) and all PH3 positive and DAPI labelled nuclei from the field (89.5 μm × 67.1 μm) were counted. The percentage of mitotic cells in 5 mutant and 5 wild type larvae was compared.
Acridine orange staining
To visualise apoptotic cells at 24 hpf, dechorinated wildtype and blm larvae were incubated with 5 μg/ml acridine orange (Sigma) for 30 minutes and washed in embryo medium. Larvae were anaesthetised with tricaine and imaged with an Olympus SZX2-ILLT microscope using fluorescence lamp and GFP filter.
RT-PCR and Q-RT-PCR
RNA was extracted using the Qiagen RNeasy Mini Kit from pools (15 to 70 individuals) of 8-cell, 16-cell, 4 hpf, 10 hpf, 27 hpf, 2 dpf and 3 dpf embryos or dissected eyes and rest of the body of 2, 3, 4 and 5 dpf larvae obtained from wildtype incrosses or blm carrier incrosses. cDNA was synthesised by reverse transcription after priming with random hexamers using the Invitrogen Superscript III system. Primers' sequences for cell cycle markers : p21-likeF-CCGTAGACCATGAGGAGC; p21-likeR-GTCTCGTCCACTTCTTTCTTTC; ccnb1F-GAGTCACAGCAATAAACCAC; ccnb1R-AGGAAGGCTCAGACACAAC. Primers' sequences for top2a, top2b and βactin: top2aF-AACGAGACCATGCCTCACC; top2aR-CAAACCAGCCTCTTTCTTCG; top2bF-GCAGTTGGAGGAAACTCTGC; top2bR-AGCTTCACAGCCGCATCTAT; βactF-GAGAAGATCTGGCATCACAC; βactR-ATCAGGTAGTCTGTCAGGTC. Primers' sequences for atoh7 (ath5): atoh7F-CCGGAGAAGTTTGAGAGTGC and atoh7R- GCTCAGAGCCATCTGTAGGG. Real-Time PCR was performed with SYBR-Green chemistry. 18s rRNA amplification with TaqMan probe served as an internal reference. For the time course experiment an arbitrary value of 1 was assigned to the average expression at 8 cell stage and expression level at other time points was normalised to this denominator. Plots of quantitative RT-PCR are average from 2 or 3 replicas and error bars represent standard error of the mean.
Wholemount in situ hybridization
Full length top2b cDNA was amplified with AccuPrime Taq polymerase (Invitrogen) using forward and reverse primers binding to 5'UTR and 3'UTR (5UTRzfTop2b-TCTGGCCACACACAATAGAAA, 3UTRzfTop2b- CCAATCAGTTTTCTGGACCAA) and cDNA (made with random hexamers from total RNA of 1 dpf old larvae) as template. It was cloned into pGEM vector and used for synthesis of antisense and sense digoxigenin labelled probes with DIG in vitro transcription kit (Roche).
Larvae were fixed in 4% PFA (overnight at 4°C). 3 dpf specimens were treated for 20 mins with a bleach solution (30% H2O2, 50% formamide, 20% 20× SSC) to remove pigmentation and permeabilised with proteinase K (10 μg/ml, 22 minutes). Probe hybridisation was carried out at 62.5°C (overnight), followed by a series of formamide/SSC washes at the same temperature. Detection of hybridised probe was performed by incubation (overnight at 4°C) with AP-conjugated anti-digoxigenin Fab fragments (1:5,000; Roche) followed by colorimetric detection (NBT/BCIP, Roche). Whole larvae were imaged under 100% glycerol using an Olympus SZX2-ILLT stereo zoom microscope. 30 μm sections of stained larvae embedded in Tissue Tek (Sakura) were imaged using Zeiss Axioplan2 microscope equipped with AxioCam HRc camera.
Overexpression of top2b
The top2b coding region was amplified from cDNA of 1 dpf larvae using AccuPrime Taq polymerase (Invitrogen) and cloned into pGEM vector. This plasmid was used to synthesise full length top2b mRNA with the mMessage mMachine SP6 system (Albion Inc.). Offspring of blm carriers were microinjected at 1-2 cell stages with zebrafish top2b mRNA at 250 ng/μl (approximately 75 pg per embryo). Phenotypes were recorded at 33 hpf and eye diameter was measured with CellF software (Olympus Soft Imaging Solutions) after imaging with an Olympus SZX2-ILLT stereo zoom microscope. To verify overexpression of top2b, pools of embryos presenting with wildtype, blm or malformed phenotypes were collected at 33 hpf for RNA extraction. Q-RT-PCR was performed as described above (using primers top2bF-GCAGTTGGAGGAAACTCTGC and top2bR-AGCTTCACAGCCGCATCTAT). Expression of top2b mRNA in all samples was normalised to the expression level in uninjected wildtype controls.
For transient treatment freshly laid eggs from blm carrier incrosses were incubated in embryo medium containing 100 μM ICRF-193 (Sigma) or 1% DMSO for 3.5 hours at 28°C and then the drug was replaced by fresh embryo medium. At 27 hpf larvae with mutant and wildtype phenotypes were separated and used for cell cycle analysis by flow cytometry.
For pre-MZT treatment, freshly laid wildtype AB eggs were placed in multi-well plates. Excess embryo medium was removed and replaced by a drug solution. Drugs were diluted in 1% DMSO in embryo medium at final concentrations of: 1 μM, 100 μM and 1 mM of ICRF-193 (Sigma) and 10 μM, 100 μM and 200 μM of etoposide (Ebewe). Embryos were incubated with drug for ~1.5 hours and then fixed with 4% PFA. Control embryos were incubated in 1% DMSO. The percentage of embryos reaching each development stage was calculated. Statistical analysis was performed using the Chi-squared test in GraphPad Prism.
Cell cycle extracts
Cycling extracts were prepared from unfertilized Xenopus laevis eggs activated using calcium ionophore A23187 (Sigma; C7522), as described in . Extracts were treated with 20 μM ICRF-193 diluted in 1% DMSO or 1% DMSO alone at time zero. Samples were collected at 10 minute intervals and snap frozen in liquid nitrogen.
Protein samples were separated according to their apparent molecular mass under denaturing conditions on NuPAGE 4-12% Bis-Tris Gel, 1.0 mm × 17 well gels (Invitrogen), and transferred to PVDF membrane. After blocking in 5% Bovine Serum Albumin (BSA) in PBS-0.1% Tween-20, membranes were incubated with either anti-phospho-Histone H3 (Ser10) antibody from Millipore to detect mitosis (Cat. # 06-570) or GAPDH antibody from Cell Signaling (#3900), followed by incubation with horse-radish peroxidase-conjugated secondary antibodies. Proteins were visualized using Enhanced Chemiluminescence Western Blotting Substrate (Pierce; #32106). Densitometry was performed using the ImageJ program http://rsbweb.nih.gov/ij/.
days post fertilization
hours post fertilization
topoisomerase 2 alpha
topoisomerase 2 beta.
We thank members of BK lab for their involvement in the mutagenesis screen: Vincent Smyth, Maria Cederlund, Theresa Heffernan, Olaya Astudillo Fernandez, Fan Yang, Maria Morrisey, Antonino Glaviano, Victor Vendrell, Alison Reynolds, Lisa Shine. We thank Alfonso Blanco for assistance and advice with flow cytometry, Dimitri Scholz for assistance with confocal microscopy and advice on image analysis and Jun Yin for help with retrieving microarray data. We thank Michael R. Taylor for comments on the manuscript. This work was supported by Science Foundation Ireland grants: 04/IN3/B559, 06/RFP/BIM052, 07/SRC/B1156 (LOS).
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