npgrj_NCHEMBIO_749 366..370 - Nature

© 2005 Nature Publishing Group http://www.nature.com/naturechemicalbiology

LETTERS

Small molecules that delay S phase suppress a zebrafish bmyb mutant Howard M Stern1,2,4, Ryan D Murphey2,4, Jennifer L Shepard2, James F Amatruda2, Christian T Straub2, Kathleen L Pfaff 2, Gerhard Weber2, John A Tallarico3, Randall W King3 & Leonard I Zon2 Bmyb is a ubiquitously expressed transcription factor involved in cellular proliferation and cancer1–4. Loss of bmyb function in the zebrafish mutant crash&burn (crb) results in decreased cyclin B1 expression, mitotic arrest and genome instability5. These phenotypic observations in crb mutants could be attributed to the decreased expression of cyclin B1, a cell-cycle regulatory protein that is responsible for driving cell progression from G2 through mitosis. To identify small molecules that interact with the bmyb pathway, we developed an embryo-based suppressor screening strategy. In 16 weeks we screened a diverse B16,000 compound library, and discovered one previously unknown compound, persynthamide (psy, 1), that suppressed bmyb-dependent mitotic defects. Psytreated embryos showed an S-phase delay, and knockdown of the cell-cycle checkpoint regulator ataxia telangiectasia—and Rad-related kinase (ATR) abrogated the suppression of crb. The DNA synthesis inhibitors aphidicolin (2) and hydroxyurea (3) also suppressed crb. S-phase inhibition upregulated cyclin B1 mRNA, promoting the progression of cells through mitosis. Our study demonstrates that chemical suppressor screening in zebrafish can identify compounds with cell-cycle activity and can be used to identify pathways that interact with specific cell-cycle phenotypes. Carrying out phenotypic chemical screens with a whole organism has several advantages over similar screens in cultured cell lines. The cells in an organism are in their normal context of cell-cell and cell– extracellular matrix interactions and lack the extensive genetic alterations seen in cell lines. In addition, because many distinct tissue types are present, it is possible to identify compounds that have cell-lineageor organ-specific effects. Zebrafish are particularly amenable to whole-organism chemical screening because embryos fit readily into microwell plates and compounds can thus be added directly to the embryo medium6,7. Furthermore, many cell-cycle drugs known to affect mammalian systems are also active in zebrafish (R.D.M., H.M.S. and L.I.Z., unpublished data). We chose to focus on crb, a recently identified zebrafish line with a splice-donor mutation resulting in a frameshift that knocks out bmyb function5. This mutant was identified

in a genetic screen for zebrafish with cell-cycle defects. Homozygotes have increased numbers of mitotic cells, abnormal spindles, alterations in centrosome number, polyploidy and decreased cyclin B1 expression, and they die by 4–5 d after fertilization. A carcinogenesis study of crb heterozygotes showed an increase in MNNG-induced cancer susceptibility as compared to wild-type siblings. To identify compounds that interact with the bmyb cell-cycle regulatory pathway, we performed an embryo-based small-molecule screen for suppressors of crb (Supplementary Fig. 1 online). Mitotic cells were detected by immunohistochemical staining of whole embryos with an antibody to serine-10–phosphorylated histone H3 (pH3). Histone H3 is phosphorylated on serine 10 in late G2 to early M phase and is dephosphorylated in anaphase8. The DIVERSet E library (Chembridge Corp.), which contains 16,320 compounds, was screened in 16 weeks. Because crb is a recessive, embryonic-lethal mutation, the screen was performed by mating unaffected adult heterozygotes, which produces a 3:1 ratio of wild-type to mutant embryos. This screening strategy identified three classes of compounds. Class I included 19 small molecules that increased the number of mitotic cells in wild-type embryos; class II consisted of 10 chemicals that reduced the number of mitotic cells in wild-type embryos (R.D.M., H.M.S. and L.I.Z., unpublished data); and class III consisted of one compound that suppressed the mitotic arrest in crb embryos and had little effect on mitosis in wild-type embryos (Fig. 1a,b and data not shown). Structure-based searches did not reveal any reported biological activity for the suppressor (Fig. 1c). The chemical structure was confirmed by LC-MS and NMR. To further verify the identity of the compound, we resynthesized it (Supplementary Methods online). The LC-MS and NMR spectra (Supplementary Fig. 2 online) of the synthesized compound were identical to those of the compound in the chemical library, as was its potency in suppressing crb. We named the compound persynthamide (psy)—‘per’ for the Greek mythological hero Perseus who rescued Andromeda, ‘synth’ for the fact that it causes S-phase delay (see below) and ‘amide’ for the chemical group. A dose-response analysis revealed a half-maximal effective dose (ED50) of 5 mM for crb suppression. Temporally, psy must be present from 10 to 24 hours post fertilization (hpf) for complete suppression of crb to occur (Supplementary Fig. 3 online). crb embryos

1Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115, USA. 2Stem Cell Program and Division of Hematology and Oncology, Children’s Hospital, Dana-Farber Cancer Institute, Howard Hughes Medical Institute and Harvard Medical School, 1 Blackfan Circle, Boston, Massachusetts 02115, USA. 3Institute of Chemistry and Cell Biology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA. 4These authors contributed equally to this work. Correspondence should be addressed to L.I.Z. ([email protected]).

Received 22 July; accepted 19 October; published online 13 November 2005; doi:10.1038/nchembio749

366

VOLUME 1

NUMBER 7

DECEMBER 2005

NATURE CHEMICAL BIOLOGY

LETTERS

Untreated

WT

Figure 1 Persynthamide (psy) suppresses mitotic accumulation in crb mutants. (a) A chemical screen for suppressors of crb identified one compound that suppresses the mitotic accumulation seen with bmyb mutation. WT, wild type; Het, heterozygote; Mut, homozygous mutant. Embryos were treated with 8 mM psy or vehicle (DMSO control) overnight. 24-hpf embryos were antibody stained for pH3 and genotyped using a restriction fragment length polymorphism (RFLP). (b) Quantification of pH3-positive cells in the 24-hpf embryo tails, determined with at least five embryos per condition. (c) Structure of psy. Error bars, s.e.m.; scale bar, 250 mm.

Persynthamide

Het

Mut

WT

Het

WT

Mut

WT

Het

Mut

Mut

Het

WT allele Mutant allele

b

c

400 pH3-positive cells

OH 300 200

N

O

N H

100 0 WT

crb

WT + psy crb + psy

proportion of cells in crb mutants with abnormal centrosome numbers, another indicator of genome instability (Supplementary Fig. 4 online). Our data demonstrate that psy suppresses many of the defects evident in crb. Mitotic arrest is sometimes related to abnormalities in the mitotic spindle. To assess spindle integrity and better delineate several stages of mitotic progression, we carried out double staining for pH3 (which marks condensed chromatin) and a-tubulin (which marks the mitotic spindle). The percentage of cells in crb embryos with abnormalities in the mitotic spindle was reduced from 67% to 55% by psy treatment (Supplementary Fig. 4), a change

crb untreated

WT persynthamide

crb persynthamide

TUNEL

Head morphology

treated with psy from 4 hpf to 24 hpf and then rinsed free of drug retain wild-type levels of mitotic cells through at least 36 hpf. Psy-mediated suppression is not specific to the bmyb mutation underlying crb because the same result is observed when bmyb expression is reduced by antisense knockdown instead of mutation9 (data not shown). crb mutants show significant cell death, WT untreated a seen as graying of the head morphologically and as elevated apoptosis throughout the embryo on TUNEL staining (Fig. 2a). Psy treatment reduced crb-related cell death. Polyploidy, a form of genome instability, is also associated with crb mutants. DNA content of psy-treated embryos was analyzed by flow cytometry and indicated that psy suppresses polyploidy in crb mutants (Fig. 2a). Treatment with psy also suppressed the

400

Figure 2 Psy suppresses many aspects of the crb phenotype. (a) Embryos were treated overnight with 8 mM psy, and the drug was washed away at 24 hpf. Cell death was analyzed at 30 hpf on the basis of morphological evidence of opacity in the brain and at 24 hpf by TUNEL staining for apoptosis. Psy-treated wild-type (WT) and crb embryos showed a range of TUNEL staining intensity, indicating that although psy can suppress crb-related apoptosis, a drug-related induction of apoptosis occurs in some wild-type and crb embryos. Control and psy-treated embryos were also disaggregated for analysis of DNA content by flow cytometry (below). Polyploidy was suppressed by overnight treatment with psy. (b) Embryos treated overnight with psy were double stained for a-tubulin and pH3. Photographs of double-stained embryos show a prometaphase arrest in crb mutants. Scale bars: a, 250 mm; b, 10 mm.


VOLUME 1

FACS cells


a

400

800

300

300 300

600

200

100

200 2n 4n

b

200

200

400

8n

DAPI

100

100 2n 4n pH3

8n

2n 4n α-Tubulin

8n

2n 4n

8n

Merge

WT

crb

NUMBER 7

DECEMBER 2005

367

368

a

WT persynthamide

WT untreated

pH3

G1 = 53.6% S = 32.3% G2/M = 11.2%

6,000

G1 = 47.9% S = 46.6% G2/M = 4.32%

4,000

4,000 2,000 2,000

2n

4n

2n

b

4n

c crb

crb + aphidicolin

d

e crb + HU

Percentage pH3+ cells with BrdU

that approaches statistical significance (P ¼ 0.05). The magnitude of the change, however, was small compared to the effect of psy on the number of mitotic cells (Fig. 1a,b). This apparent discrepancy is explained by analysis of embryos stained for both pH3 and a-tubulin, which revealed that crb mutants have a fourfold increase in mitotic cells with condensed chromatin but no associated spindle, signifying that the cells are accumulating in prometaphase (Fig. 2b and Supplementary Fig. 4). Our studies suggest that the morphological phenotype of crb is caused primarily by mitotic arrest before spindle formation rather than by arrest resulting from abnormal spindles. Although psy did not significantly affect mitotic cells in wild-type embryos with overnight treatment, it was possible that shorter-term treatment might reveal a cell-cycle effect in the wild type. We treated wild-type embryos at 24 hpf for 6 h with psy and then examined them for pH3 staining and DNA content. The number of mitotic cells decreased significantly, and this was accompanied by an accumulation of cells in early S phase (Fig. 3a). Examination of wild-type embryos treated earlier in development also demonstrated a decrease in mitotic cells (data not shown). To confirm that the FACS profile represents cells in S phase rather than fragmenting apoptotic cells, embryos were treated with BrdU and incorporation was assayed by whole-mount immunostaining and FACS analysis. In both cases, psy treatment resulted in increased numbers of BrdU-positive cells, indicating that an increased percentage of cells were in S phase (Supplementary Methods and data not shown). Thus, during overnight psy treatment, there is an initial drop in mitotic cells that recovers by the end of the assay period, indicating a delay in S phase. Several lines of evidence suggest the S-phase delay causes suppression of crb. First, the ED50 for psy to decrease pH3 staining in shortterm assays is 5 mM, the same as the ED50 for crb suppression (data not shown). Second, two known inhibitors of DNA synthesis, aphidicolin and hydroxyurea, also suppressed the mitotic accumulation in crb mutants (Fig. 3b–d). To further assess how S-phase delay affects G2-to-M progression, we used a BrdU pulse to mark S-phase cells in embryos at 16 hpf, the time of onset of elevated pH3 in crb mutants. Previous work has shown that 2 h after BrdU labeling, nearly all of the mitotic cells in untreated wild-type embryos are BrdU positive as compared to only 5% in untreated homozygous mutants5 (Fig. 3e), indicating a delay in cell progression from S phase to mitosis in the mutant. In crb homozygotes, the number of mitotic cells that show BrdU staining 2 h after a BrdU pulse increased from 5% in untreated to 60% in psy-treated embryos (Fig. 3e). These data indicate that cells in psy-treated embryos progress from S phase to mitosis and that cells in psy-treated mutants show more efficient S-to-M progression than do untreated mutants. Together with the statistically significant increase in the number of crb cells reaching late anaphase and telophase resulting from psy treatment (Supplementary Fig. 4, P ¼ 0.03), these data indicate that psy-induced S-phase delay allows the cells to cycle without accumulating in mitosis. Hydroxyurea and aphidicolin are known to slow S-phase progression by activating the intra–S-phase checkpoint10–12, an ATRdependent cascade that results in stabilization of replication forks and delay in firing of late origins of replication13,14. We treated crb embryos with aphidicolin in the presence or absence of caffeine, which is known to inhibit the intra–S-phase and G2/M checkpoints15–17. The presence of 2 mM caffeine completely abrogated the ability of aphidicolin to suppress mitotic accumulation in crb homozygotes (Fig. 4a). Addition of caffeine decreased the percentage of cells in S phase upon treatment of wild-type embryos with aphidicolin (data not shown), indicating that checkpoint activation is a significant component of the S-phase delay and mutant suppression. Similar

FACS cells


LETTERS

100 80 60 40 20 0

WT

crb

WT + psy

crb + psy

Figure 3 Psy suppresses crb by delaying cells in S phase. (a) Wild-type embryos were treated from 24 to 30 hpf with 8 mM psy, immunostained for pH3 and their DNA content analyzed by flow cytometry. (b–d) Aphidicolin (10 mg ml–1) and hydroxyurea (HU; 50 mM) also suppressed crb. (e) Wildtype and crb embryos were pulsed with BrdU at 16 hpf; this was followed by a 2-h chase. pH3-positive cells were evaluated for the appearance of BrdU staining within the mitotic population. At least 20 random pH3-positive cells were evaluated for BrdU staining in a minimum of 2 embryos per condition. Error bars, s.e.m.; scale bars, 250 mm.

experiments were performed with psy, but synergistic toxicity with caffeine precluded meaningful analysis. To assess the dependence of psy on the intra–S-phase checkpoint, we designed a 5¢ morpholino against the single zebrafish ATR ortholog. Of the psy-treated mutant embryos injected with the ATR morpholino, 20–40% showed elevated pH3 staining (Fig. 4a); injection with a control ATR mismatch morpholino did not abrogate crb suppression by psy. These findings support the idea that activation of the intra–S-phase checkpoint is the mechanism by which S-phase inhibitors suppress mitotic accumulation in crb mutants. In support of this conclusion, gene expression profiling of psy-, aphidicolin- and hydroxyurea-treated embryos revealed similar changes in the expression of many replication and checkpoint genes (Supplementary Methods and Supplementary Fig. 5 online). The earliest identified manifestation of bmyb loss of function in zebrafish is decreased cyclin B1 expression. There is evidence in Drosophila melanogaster and in mammalian cell lines that bmyb orthologs regulate cyclin B1 (refs. 18,19). These findings suggest that cyclin B1 could link S-phase delay with suppression of the mitotic phenotype in crb. Treatment with psy overnight increased cyclin B1 mRNA in crb mutants and wild type (Fig. 4b). Similar results were obtained with aphidicolin and hydroxyurea (data not shown). The in situ hybridization results are supported by quantitative RT-PCR, which showed that the abundance of cyclin B1 in mutants was two- to

VOLUME 1

NUMBER 7

DECEMBER 2005


LETTERS

a crb

crb + aph

crb + caff

crb + psy

crb + aph + caff


crb + psy + ATR MO

WT + caff

WT + aph + caff

WT + psy + ATR MO

b WT

crb

perturb specific developmental processes6,7,23,24, and here we present a zebrafish screen to identify chemicals that bypass the defect of a specific cell-cycle mutation. Through characterization of psy, we showed that the consequences of bmyb mutation are suppressed by activation of the intra–S-phase checkpoint and upregulation of cyclin B1 expression. Psy does not show activity on embryonic tissue lacking the yolk sac (data not shown) and thus may require metabolic activation. The possibility of discovering compounds that require in vivo activation illustrates a potential advantage of carrying out small-molecule screens on a whole organism such as the zebrafish. Given that most cell-cycle genes are conserved between zebrafish and humans, the approach described here could be applied to virtually any cell-cycle or cancer pathway. For example, a number of zebrafish cellcycle mutants have embryonic phenotypes and would be amenable to this approach5,25,26. Future challenges will include adapting this strategy to juvenile or adult phenotypes, thus opening the door to direct screens for cancer suppressors in transgenic zebrafish models27,28. METHODS Animal husbandry. All animal protocols were approved by the Children’s Hospital Institutional Animal Care and Use Committee (IACUC).

WT + psy

crb + psy

Figure 4 crb is suppressed by activation of the intra–S-phase checkpoint. (a) crb embryos were treated with 10 mg ml–1 aphidicolin (aph) overnight with or without 2 mM caffeine (caff). Caffeine had no effect on pH3 staining of mutant or wild type (WT) in the absence of aphidicolin, but caffeine abrogated the suppression of crb by aphidicolin. Similar results were obtained when using 8 mM psy in combination with a morpholino that targets the 5¢ UTR of ATR (ATR MO). Aph, aphidicolin; psy, persynthamide; caff, caffeine. (b) Wild-type and crb embryos were treated with 8 mM psy and then subjected to in situ hybridization for cyclin B1. Scale bars, 250 mm.

threefold lower in mutants as compared to wild type and was twofold higher in psy-treated as compared to untreated embryos (Supplementary Methods and Supplementary Fig. 6 online). Furthermore, injection of cyclin B1 mRNA into crb homozygotes suppressed the mutant phenotype5, demonstrating that increased cyclin B1 expression is sufficient to mitigate the effect of bmyb loss of function. Our data suggest a model (Supplementary Fig. 7 online) in which loss of bmyb function during S phase causes a decrease in cyclin B1 transcription, resulting in a G2 delay and subsequent problems with mitotic progression. Slowing S phase through activation of the intra– S-phase checkpoint allows more time for the accumulation of cyclin B1 mRNA. This accumulation of cyclin B1 prevents the G2 delay and the prometaphase arrest. The specificity of S-phase inhibitors for the suppression of crb is highlighted by three points: compounds that slow other stages of the cell cycle do not suppress crb; S-phase inhibitors are unable to suppress cease&desist, another cell-cycle mutant that shows polyploidy (data not shown); and the effect of S-phase inhibitors in decreasing the number of mitotic cells in crb embryos is distinct from the small change seen in wild-type embryos. Although we provide evidence that increased cyclin B1 expression can mitigate the bmyb mutant phenotype, there are likely to be additional genes with products acting downstream of bmyb that are important for regulating proper S-to-M progression. The zebrafish system presents a unique opportunity to identify small molecules that modify disease-relevant phenotypes20–22. Zebrafish embryos have been used to successfully identify compounds that


VOLUME 1

NUMBER 7

Suppressor screen. Chemicals from the 16,320 DIVERSet E library (Chembridge Corp.) were obtained from the Institute for Chemistry and Cell Biology. One microliter of each compound (5 mg ml–1 in DMSO) was pin transferred into 384-well plates with 80 ml of E3 supplemented with 1% DMSO, 20 mM metronidazole, 50 units per ml penicillin, 50 mg ml–1 streptomycin and 1 mM Tris, pH 7.4. Each 384-well plate in the library contained 320 compounds; the last four columns were left empty. To facilitate screening, four 8 10 matrices were defined from the 320-compound format. Each chemical was screened in a vertical and a horizontal pool of 8 and 10 compounds, respectively. Aliquots of 30 ml of each chemical dilution were placed in 48-well plates using a Tecan robot and diluted to a total volume of 300 ml with supplemented E3. Two 384well plates transferred to three 48-well plates. Approximately 5,000 embryos were collected weekly by crossing 100 heterozygote pairs of crb fish. Dead and malformed embryos were removed, and the remaining high-quality embryos were pooled. The E3 medium was aspirated and approximately 20 embryos were scooped into each well using a chemical spatula. Embryos were incubated with compounds at 28.5 1C from 4–6 hpf until 24 hpf. Two percent of the compounds caused death or severe developmental delay. Embryos were dechorionated, fixed with 4% paraformaldehyde and whole-mount antibody stained for pH3 in mesh-bottomed 48-well grids. Embryos were transferred to agarose-coated 48-well plates and scored for the crb phenotype manually with a dissecting microscope. Wells with no mutants were considered to contain putative suppressors. By cross-referencing chemicals that were scored as putative suppressor in both the 8- and 10-compound pools, we were able to deconvolute which chemicals had putative activity. With 20 embryos per well and a mendelian recessive inheritance pattern, statistically 0.3% of the wells would be expected not to contain mutants. Because each chemical was tested in two pools of 20 embryos each, the statistical false-positive rate for complete suppressors was 0.001%. Therefore, all putative hits were retested on at least 100 embryos. Chemical treatments. Overnight treatments were done by adding the compound at 4 hpf and incubating until 24 hpf in E3 with 1% DMSO at 28.5 1C. In experiments that required examining embryos later than 24 hpf, persynthamide was rinsed away at 24 hpf by washing three times in E3 to prevent toxicity. A similar rinse was performed to remove persynthamide at earlier time points when determining the minimum exposure to achieve suppression. Chemical treatments on 24-hpf embryos were also done in E3 with 1% DMSO. After addition of compound, embryos were incubated for 6 h at 28.5 1C. Except in the case of dose-response analysis, persynthamide was used at 8 mM. Aphidicolin dosage was 10 mg ml–1 and hydroxyurea dosage was 50 mM. Assessment of intra–S-phase checkpoint. Caffeine (2 mM) was added to 4-hpf embryos for 30 min, after which 10 mg ml–1 aphidicolin was added. Aphidicolin

DECEMBER 2005

369


LETTERS was chosen as a control because suppression of crb by hydroxyurea is much more sensitive to small changes in dose. Synergistic toxicity was seen when caffeine was combined with 8 mM persynthamide. This toxicity is likely to be due to catastrophic events related to cell-cycle progression in the presence of damaged or incompletely replicated DNA. In support of this hypothesis, shortterm experiments with higher doses of aphidicolin (100 mg ml–1), and persynthamide (80 mM) and caffeine (5 mM) revealed an accumulation of cells in mitosis and increased apoptosis, consistent with mitotic catastrophe. To address the status of the intra–S-phase checkpoint with persynthamide treatment, a morpholino was designed against the 5¢ untranslated region (5¢ UTR) of zebrafish ATR (5¢-TGACATTTCTAGTCCTTGCTCCATC-3¢). A 5-bp-mismatch morpholino (5¢-TGAgATTTgTAcTCCTTcCTCgATC-3¢) was designed as a control. Embryos were injected into the yolk sac at the 1–2-cell stage with 1 nl of 500 mM ATR morpholino. Messenger RNA levels were examined by semiquantitative PCR, and there was no evidence of any changes in message levels in the presence of the morpholino. Attempts were made to examine protein levels, but these were unsuccessful because no available antibodies cross-reacted with zebrafish ATR on western blots. Cell-cycle analysis and immunostaining. pH3, TUNEL, a-tubulin, cyclin B1 and BrdU staining, as well as FACS assessment for DNA content and bmyb morpholino injections, were done as described5. pH3-positive cells were quantified by counting all stained cells in the tail distal to the yolk extension. Quantification of DAPI-stained cells with condensed chromatin was performed by manual counts in one 100 field defined by the very tip of the tail. Counts of spindles were obtained from the embryo body distal to the yolk ball. Centrosomes were evaluated with a monoclonal antibody to g-tubulin (Sigma) used at a dilution of 1:1,000. Individual embryo FACS was performed as with groups of embryos, except that an aliquot of the cell suspension was removed for genotyping. To genotype embryos double stained for BrdU and pH3, a portion of the head was removed from each embryo just before the acid treatment, and the remainder of the embryo was placed into a 48-well staining grid. Note: Supplementary information is available on the Nature Chemical Biology website. ACKNOWLEDGMENTS We would like to thank D. Hayes, J. Follen and C. Shamu for help with smallmolecule screening, T. Mitchison for helpful advice, and C. Burns, J. DeCaprio, N. Dyson, I. Hariharan and W. Kaelin for critical comments on the manuscript. We also thank R. Finley for help with in situ hybridization, R. Wingert for providing reagents and C. Belair and B. Barut for expert laboratory management. This work was supported by NIH grants 5K08 DK061849 (H.M.S.), 1R01 DK55381 (L.I.Z.), 1R01 HD044930 (L.I.Z.) and 5K08 HL04082 (J.F.A.). L.I.Z. is an Investigator of the Howard Hughes Medical Institute. COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Published online at http://www.nature.com/naturechemicalbiology/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ 1. Sala, A. & Watson, R. B-Myb protein in cellular proliferation, transcription control, and cancer: latest developments. J. Cell. Physiol. 179, 245–250 (1999).

370

2. Oh, I.H. & Reddy, E.P. The myb gene family in cell growth, differentiation and apoptosis. Oncogene 18, 3017–3033 (1999). 3. Joaquin, M. & Watson, R.J. Cell cycle regulation by the B-Myb transcription factor. Cell. Mol. Life Sci. 60, 2389–2401 (2003). 4. Bessa, M., Joaquin, M., Tavner, F., Saville, M.K. & Watson, R.J. Regulation of the cell cycle by B-Myb. Blood Cells Mol. Dis. 27, 416–421 (2001). 5. Shepard, J.L. et al. A zebrafish bmyb mutation causes genome instability and increased cancer susceptibility. Proc. Natl. Acad. Sci. USA 102, 13194–13199 (2005). 6. Peterson, R.T., Link, B.A., Dowling, J.E. & Schreiber, S.L. Small molecule developmental screens reveal the logic and timing of vertebrate development. Proc. Natl. Acad. Sci. USA 97, 12965–12969 (2000). 7. Khersonsky, S.M. et al. Facilitated forward chemical genetics using a tagged triazine library and zebrafish embryo screening. J. Am. Chem. Soc. 125, 11804–11805 (2003). 8. Hendzel, M.J. et al. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348–360 (1997). 9. Nasevicius, A. & Ekker, S.C. Effective targeted gene ‘knockdown’ in zebrafish. Nat. Genet. 26, 216–220 (2000). 10. Feijoo, C. et al. Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing. J. Cell Biol. 154, 913–923 (2001). 11. Sorensen, C.S. et al. Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A. Cancer Cell 3, 247–258 (2003). 12. Zou, L. & Elledge, S.J. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300, 1542–1548 (2003). 13. Donaldson, A.D. & Blow, J.J. DNA replication: stable driving prevents fatal smashes. Curr. Biol. 11, R979–R982 (2001). 14. Tercero, J.A., Longhese, M.P. & Diffley, J.F. A central role for DNA replication forks in checkpoint activation and response. Mol. Cell 11, 1323–1336 (2003). 15. Shiloh, Y. ATM and ATR: networking cellular responses to DNA damage. Curr. Opin. Genet. Dev. 11, 71–77 (2001). 16. Costanzo, V. et al. An ATR- and Cdc7-dependent DNA damage checkpoint that inhibits initiation of DNA replication. Mol. Cell 11, 203–213 (2003). 17. Cortez, D. Caffeine inhibits checkpoint responses without inhibiting the ataxiatelangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases. J. Biol. Chem. 278, 37139–37145 (2003). 18. Okada, M., Akimaru, H., Hou, D.X., Takahashi, T. & Ishii, S. Myb controls G(2)/M progression by inducing cyclin B expression in the Drosophila eye imaginal disc. EMBO J. 21, 675–684 (2002). 19. Zhu, W., Giangrande, P.H. & Nevins, J.R. E2Fs link the control of G1/S and G2/M transcription. EMBO J. 23, 4615–4626 (2004). 20. Stern, H.M. & Zon, L.I. Cancer genetics and drug discovery in the zebrafish. Nat. Rev. Cancer 3, 533–539 (2003). 21. MacRae, C.A. & Peterson, R.T. Zebrafish-based small molecule discovery. Chem. Biol. 10, 901–908 (2003). 22. Pichler, F.B. et al. Chemical discovery and global gene expression analysis in zebrafish. Nat. Biotechnol. 21, 879–883 (2003). 23. Peterson, R.T., Mably, J.D., Chen, J.N. & Fishman, M.C. Convergence of distinct pathways to heart patterning revealed by the small molecule concentramide and the mutation heart-and-soul. Curr. Biol. 11, 1481–1491 (2001). 24. Peterson, R.T. et al. Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation. Nat. Biotechnol. 22, 595–599 (2004). 25. Berghmans, S. et al. tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors. Proc. Natl. Acad. Sci. USA 102, 407–412 (2005). 26. Amsterdam, A. et al. Many ribosomal protein genes are cancer genes in zebrafish. PLoS Biol. 2, E139 (2004). 27. Langenau, D.M. et al. Myc-induced T cell leukemia in transgenic zebrafish. Science 299, 887–890 (2003). 28. Patton, E.E. et al. BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma. Curr. Biol. 15, 249–254 (2005).

VOLUME 1

NUMBER 7

DECEMBER 2005
