expression of h-rasv12 in a zebrafish model of costello ... · that fish carrying the integrated...

RESEARCH ARTICLE

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INTRODUCTIONRas proteins are small GTPases which are essential componentsof cellular signaling pathways, which are responsible for growth,migration, adhesion, cytoskeletal integrity, survival anddifferentiation. They have been intensely studied in the last 30 yearsfor their involvement in cancer; indeed, defects in Ras signalingmay result in malignant transformation (Downward, 1996;Malumbres and Barbacid, 2003). It is estimated that approximately30% of all human tumors have activating mutations in one of theRAS genes. In fact, somatic mutations, which lock Ras in an activeGTP-bound conformation resulting in constitutive activation of thedownstream pathways, are frequent in cancer – they are cataloguedby the Cancer Genome Project in the Catalogue Of SomaticMutations In Cancer (COSMIC) (www.sanger.ac.uk/genetics/CGP/Census/).

Moreover, the discovery of germline mutations in neurofibromin1 (NF1) and protein tyrosine phosphatase, non-receptor type 11(PTPN11) genes provided the first indication that aberrant Rassignaling might underlie a group of human developmentaldisorders, collectively known as cardio-facio-cutaneous syndromes(reviewed by Cichowski and Jacks, 2001; Tartaglia and Gelb, 2005).All of these disorders share similar phenotypic traits. Among them,it has emerged that Costello syndrome is caused by ‘de novo’germline mutations in the H-RAS gene (Aoki et al., 2005). Patientswith Costello syndrome have multiple congenital disorders, which,

all together, are diagnostic for the disease, including a characteristiccraniofacial dysmorphology, cardiac defects, mild mentalretardation, and high birth weight followed by a failure to thrive and developmental delays (Costello, 1977) (reviewed byRauen, 2007). These patients also have a tendency to developmultiple papillomas and various types of cancer, mainlyrhabdomyosarcomas, neuroblastomas and bladder carcinomas(Gripp et al., 2006).

It is still unclear how the expression of activated H-RAS caninduce oncogenic transformation and unrestricted growth as aconsequence of somatic mutations in cancer, and inducedevelopmental abnormalities affecting specific tissues in Costellopatients who have inherited the same mutations through germlinetransmission. Perhaps, by understanding how activation of the Raspathway is kept under control in Costello patients, we might beable to devise ways of controlling it in cancer. In contrast to theoncogenic activity of mutant H-RAS, the mutant protein can alsoinduce a potent antiproliferative response, i.e. cellular senescence(Serrano et al., 1997). Cellular senescence is characterized by anirreversible proliferation block, which is accompanied by theexpression of specific markers such as the upregulation ofsenescence-associated β-galactosidase (SA-βgal) activity (reviewedby Dimri and Campisi, 1994).

Oncogene-induced senescence (OIS) has been demonstrated incells that overexpress H-RAS (Serrano et al., 1997) and in severalbenign tumors (Collado et al., 2005). It is believed to represent aprotective mechanism against proliferation and transformation, andto be activated by H-RAS (Sun et al., 2007). In mouse models ofbreast and lung cancer, in which expression of the H-Ras or K-Rasoncogene is inducible (Sarkisian et al., 2007), the occurrence of OISwas found to be related to the level and duration of oncogenic Rasexpression. In this system, low levels of K-Ras expression stimulate

Disease Models & Mechanisms 2, 56-67 (2009) doi:10.1242/dmm.001016

Expression of H-RASV12 in a zebrafish model of Costellosyndrome causes cellular senescence in adultproliferating cellsCristina Santoriello1, Gianluca Deflorian1, Federica Pezzimenti1,2, Koichi Kawakami3, Luisa Lanfrancone4, Fabrizio d’Adda di Fagagna1 and Marina Mione1,*

SUMMARY

Constitutively active, ‘oncogenic’ H-RAS can drive proliferation and transformation in human cancer, or be a potent inducer of cellular senescence.Moreover, aberrant activation of the Ras pathway owing to germline mutations can cause severe developmental disorders. In this study we havegenerated transgenic zebrafish that constitutively express low levels, or can be induced to express high levels, of oncogenic H-RAS. We observedthat fish carrying the integrated transgene in their germline display several hallmarks of Costello syndrome, a rare genetic disease caused by activatingmutations in the gene H-RAS, and can be used as a model for the disease. In Costello-like fish, low levels of oncogenic H-RAS expression are associatedwith both reduced proliferation and an increase in senescence markers in adult progenitor cell compartments in the brain and heart, together withactivated DNA damage responses. Overexpression of H-RAS through a heat-shock-inducible promoter in larvae led to hyperproliferation, activationof the DNA damage response and tp53-dependent cell cycle arrest. Thus, oncogene-induced senescence of adult proliferating cells contributes tothe development of Costello syndrome and provides an alternative pathway to transformation in the presence of widespread constitutively activeH-RAS expression.

1IFOM, the FIRC Institute for Molecular Oncology Foundation, Milan, Italy2Cogentech Consortium for Genomic Technologies, Milan, Italy3Division of Molecular and Developmental Biology National Institute of Genetics,Mishima, Shizuoka, Japan4Department of Experimental Oncology, European Institute of Oncology, Milan,Italy*Author for correspondence (e-mail: [email protected])

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Disease Models & Mechanisms 57

DDR in a zebrafish Costello model RESEARCH ARTICLE

proliferation whereas high levels trigger an irreversible growtharrest (Sarkisian et al., 2007).

We investigated the effects of oncogenic H-RAS expression inzebrafish. Using germline integration, we generated zebrafish linescontaining a mutated form of H-RAS, carrying the activatingmutation G12V, that can be expressed in an inducible or constitutivemanner, and found that these fish display several developmentaldefects that are hallmarks of the Costello syndrome. We then usedthese fish to gain insights into the mechanisms by which oncogenicH-RAS expression leads to the Costello phenotype, and found thatit induces a DNA damage response (DDR) and initiates a senescenceprogram in subpopulations of adult proliferating cells in the heartand brain. The occurrence of OIS in a zebrafish model of Costellosyndrome suggests that OIS contributes to the severedevelopmental defects, and to the degenerative/premature agingphenotype, present in human patients.

RESULTSGeneration of zebrafish expressing oncogenic H-RASV12It has been documented that oncogenic Ras proteins can performdifferent functions according to the cellular context. We asked whatthe effect of oncogenic Ras expression would be, in vivo, duringthe development of different organs and tissues. In fish, as well as

in humans, three genes have been identified: h-ras, n-ras and k-ras (Rotchell et al., 2001). We decided to use the human H-RASgene and in particular a constitutively active form carrying amutation, in which a GGC codon is substituted with a GTC codon(G12V, or in short V12), because its roles in transformation andcellular senescence are well documented (Kraus et al., 1984;Barbacid, 1990; Downward, 1996; Di Micco et al., 2006).

In order to generate fish lines that express H-RASV12 in differentorgans and tissues, we took advantage of the Tol2 gene trap systemdeveloped by Kawakami (Kawakami et al., 2000). We generated achimeric H-RASV12 protein that was tagged with green fluorescentprotein (GFP) at the N-terminus (Fig. 1A) in order to trackdeveloping tumors. We verified that the GFP tag did not affect thefunction of H-RASV12 in vitro and in vivo. We transfected NIH3T3cells with the chimeric construct, or with an empty pEGFP vectoras a control, and performed a focus formation assay. We confirmedthat, both in vitro and in vivo, the oncogenic activity of H-RASV12was not affected by the presence of the GFP tag (supplementarymaterial Fig. S1A). Subsequently, we cloned the GFP-H-RASV12chimeric construct into a modified version of the T2KSAG plasmid(Kawakami et al., 2000). We injected fertilized eggs with thisplasmid (see Methods) and found that approximately 20% of theinjected fish developed some type of neoplasia (mainly nevi,

Fig. 1. Embryos and adults with germline integration and constitutive expression of GFP-H-RASV12 are healthy, whereas somatic integrations generatetumors. (A) Diagram of the gene trap T2KSAG:GFP-H-RASV12 construct used in this study, containing Tol2 sequences, a splice acceptor (SA) and the sequencecoding for the fusion protein GFP-H-RASV12. This construct is injected in combination with mRNA encoding the Tol2 transposase at the one-cell stage.(B) Formation of a malignant fibrosarcoma in injected 5-week-old fish. The presence of GFP-positive tumors in injected fish indicates that the GFP tag does notaffect the oncogenic activity of H-RASV12. Hematoxylin and eosin (H&E) staining of a paraffin section of the tumor (upper right panel) and GFP expression in acryostat section of the tumor (lower right panel). Bars, 40μm. (C) GFP-H-RASV12 expression in the P1 line at 24 hpf, lateral view, head to the left.(D) Immunostaining with anti-GFP of cultured cells derived from P1 embryos shows that GFP-H-RASV12 localizes to the cell membrane and the Golgi complex.The nuclei are stained with Topro (red). Bar, 10μm. (E) Southern blot analysis using genomic DNA extracted from 1-month-old wild-type (WT) zebrafish. Analysisof heterozygous (+/–) P1 fish DNA reveals that a single insertion occurred in the line. RV (EcoRV) and KpnI are restriction enzymes. We used the T2KSAG:GFP-H-RASV12 plasmid as a control.

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DDR in a zebrafish Costello modelRESEARCH ARTICLE

melanomas and lymphomas) (Fig. 1B), probably owing to somaticintegration and expression of the GFP-H-RASV12 transgene, asdemonstrated by the expression of GFP in the tumors (Fig. 1B).

When fish reached adulthood we screened for germlineintegrations. We screened 142 genomes, by analyzing the progenyfor GFP expression under a fluorescent stereomicroscope and byPCR (supplementary material Fig. S1B), and found that 22%(n=32/142) of the injected fish had integrated the transgene intotheir germline, but only 6.4% (n=2/32) of germline integrationsgave rise to GFP-H-RASV12-positive progeny. The two differenttransgenic lines, full names: Gt(GFP-H-RASV12)io1 and Gt(GFP-H-RASV12)io2, were called P1 and P2, respectively. We analyzedthe expression pattern of the fusion protein both in the embryosand in the juvenile fish from the two lines. Both P1 and P2embryos display ubiquitous expression of the fusion protein (Fig.1C; and data not shown), mainly confined to the cell membraneand Golgi apparatus, as visualized in fibroblast cultures derivedfrom fluorescent embryos at 24 hours postfertilization (hpf ) (Fig.1D). As multiple insertions could have occurred, we evaluatedthe integration number in the P1 and P2 lines. Southern blotanalysis showed that P1 and P2 carry single germline insertionsat different genomic locations (Fig. 1E; supplementary materialFig. S1C).

Characterization of a zebrafish model of Costello syndromeHemizygous fish from the two lines were healthy and fertile. Inthe progeny of hemizygous crosses, approximately 25% of the fishshowed an abnormal morphology that was visible by 5 weeks ofage. The morphological phenotype includes: shorter body length,flattened head with increased interocular distance (Fig. 2A; Table1), small heart (Fig. 2B) and scoliotic spine (Fig. 2A,E). Othervisible phenotypes include an enlarged gill area (Fig. 2C) andswimming near the water surface, both of which are indicative ofreduced blood oxygenation, and sterility. These features areindicative of the Costello phenotype and we refer to theseabnormal fish as Costello-like fish (CS-like). Consistent with thehuman disease, we observed that the phenotype worsened withincreasing age.

We performed an analysis of the craniofacial and spinalabnormalities of CS-like fish at 5-6 weeks of age using cartilageand bone staining. The analysis of several bones and cartilages atthis stage showed extensive alterations in ossification, withprecocious ossification in almost all examined bones including bothendochondral and intramembranous bones [for classification, seeElizondo et al. (Elizondo et al., 2005)]. Examples of the ossificationstate of the skull and Weberian complex in CS-like fish and normalsiblings are shown in Fig. 2D. In the spine, we observed a progressivecompression/disappearance of some intervertebral cartilagineousdiscs, leading to fusion of vertebrae and severe scoliosis between5 and 12 weeks of age (Fig. 2E).

Since Costello patients are prone to developing tumors, weanalyzed the predisposition of the CS-like fish to tumor formation.In older fish (5-12 months of age), we noticed a higher frequencyof tumor formation (n=12/200 P1 fish – corresponding to 1 in 16)compared with wild-type populations (1 in 500) and that tumorsdeveloped at an earlier stage than in wild-type fish (5 months vs>2 years). Half of the tumors (n=6/12) were lymphatic infiltrationsof the gut, liver or interstitial tissue; however, two transgenic fish

developed metastatic melanomas, one had gut carcinomas, two hadhepatocarcinoma and one developed rhabdomyosarcoma (Fig. 2F-H).

To show that the phenotype observed in CS-like fish was notthe result of disruption of a gene at the insertion site, we mappedthe insertion site in the P1 line using inverse PCR (Detrich, 2004).The location of the insertion was in a gene-free region inchromosome 15 (supplementary material Fig. S1D) (see Methods).Moreover, semiquantitative Southern blot analysis of genomic DNAcopy number (Gabellini et al., 2006) showed that, in the CS-likefish genome, two copies of the transgene are present(supplementary material Fig. S1E). In addition, the same CS-likephenotype was also observed in the other gene trap line (P2), which,as shown in the supplementary material Fig. S1C, has a differentinsertion site.

There is a high frequency of heart defects in Costello patients;therefore, we investigated the occurrence of signs indicating aheart malformation in CS-like fish. We found that the mutants’hearts were smaller (Fig. 2B) and had thicker walls (supplementarymaterial Fig. S2D) compared with their normal siblings. Next, weinvestigated heart morphogenesis at 16 hpf in groups of fish fromhemizygous intercrosses using a heart-specific probe, tbx20(Szeto et al., 2002). We observed a delay in heart morphogenesisin approximately 25% of the embryos, as shown by a large andflat tbx20-positive heart disc that was centrally located as opposedto the majority of embryos which, at the same stage, had a left-oriented tbx20-positive heart tube (supplementary material Fig.S2A). Defects in heart morphogenesis were also visible at 32 hpf,with a significant reduction in the tbx20 expression area(supplementary material Fig. S2B,C). The defects in heartmorphogenesis led to myocardial hypertrophy and defectivefunctions including reduced oxygenation of peripheral tissues,which was seen at later stages. In conclusion, we propose that adelay in heart morphogenesis is an early sign of a CS-likephenotype.

Activation of RAS downstream pathways does not occur in CS-likefishThe G12V mutation of the H-RAS gene is known to lead to RAF1activation and to increased ERK and AKT phosphorylation(Barbacid, 1990). Thus, we investigated the state of these pathwaysin CS-like fish and compared the results with the amount of H-RASV12 present in the tissue.

We found that the level of GFP-H-RASV12 expression wasslightly higher (approximately 1.4 times) than the level ofendogenous Ras expression (Fig. 3A). We then evaluated thepresence of RAS in its active form (RAS-GTP) by testing its abilityto bind to Raf1. Using an immunoprecipitation protocol that usesprotein extracts from pools of 4-day-old fluorescent P1 larvae(including both heterozygous and homozygous transgenic larvae)(Fig. 3B), we detected the presence of active GTP-bound GFP-H-RASV12. In addition, we detected active GFP-H-RASV12 inprotein extracts prepared from the brains of 3-month-old CS-likefish (Fig. 3C). Next, we investigated the presence of phosphorylatedforms of ERK1/2 and Akt – the latter results from activation of theMek and PI3 kinase pathways by active RAS. Despite the presenceof active GTP-bound H-RASV12, overt phosphorylation of ERK1/2or Akt was not be detected in the samples (Fig. 3B,C). Therefore,

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active H-RASV12, present in CS-like fish, is unable to sustainERK1/2 or Akt phosphorylation, although it is possible that a localor temporary activation of these downstream pathways could haveoccurred in transgenic larvae or juveniles and escaped our analysis.

Cellular senescence and persistent activation of DDR markers inCS-like fishIt has been reported that cells respond to oncogenic H-RASactivation by engaging in a senescence program (Bartkova et al.,2006; Di Micco et al., 2006), which protects them fromproliferating in the presence of oncogene-induced DNA damage.We asked whether constitutive expression of the H-RAS oncogeneactivates a senescence program in CS-like fish. Thus, we analyzedthe proliferative state and the presence of SA-βgal activity inseveral organs from wild-type and CS-like fish at 4-5 weeks andat 2-3 months of age. Cell proliferation takes place in certaintissues and in progenitor niches throughout the body of adultzebrafish (Poss et al., 2002; Adolf et al., 2006; Grandel et al., 2006),which are well known for their ability to regenerate several tissues,including brain, heart and fin (Kaul et al., 2007; Becker et al., 2005;Koster and Fraser, 2006). We analyzed two areas in the brain thatare renowned for being sites of adult neurogenesis and neuralprogenitor proliferation, namely the preoptic/precommissuralarea of the ventral telencephalon and the valvula cerebelli. Inaddition, we examined the whole ventricular region in the heart.

We found that, at both ages, bromodeoxyuridine (BrdU)-positive(BrdU+) cells in the brain and heart of adult CS-like fish werereduced to approximately half of the original population (Fig. 4A;Fig. 5A,B; supplementary material Fig. S3A). In contrast, wenoticed that there was little or no change in BrdU uptake in theskin, gills (Fig. 4B) and gut, and an increase in BrdU+ cells in theliver and kidney (Fig. 4E; supplementary material Fig. S3B). Theincrease in cell proliferation in the kidney and liver of CS-likefish leads to a net expansion in organ size. We also detected anincreased number of SA-βgal-positive cells in the brain, heart,liver and bone of CS-like fish (Fig. 4C,D; supplementary materialFig. S3C-G).

The occurrence of OIS in CS-like fish suggests that it might bethe result of persistent DNA damage caused by prolonged exposure

Fig. 2. Homozygous P1 fish develop a Costello(CS)-like phenotype. Developmental defects in a6-week-old CS-like fish. (A) CS-like fish havereduced body size compared with normal siblingfish. They also have a smaller heart (B), an enlargedgill area (C) and craniofacial dysmorphogenesiswith increased ossification (Alizarin Red staining) ofthe Weberian complex (arrow) (D). (E) Alizarin Redstaining also reveals fusion of vertebrae(arrowheads). An increase in cancer development isobserved in transgenic fish. (F) Trunkrhabdomyosarcoma in a heterozygous P1 5-month-old fish. (G,H) H&E staining of a paraffin section ofthe tumor, showing the overall appearance (G) andthe mature striated muscle component (H). Bars,40 μm.

Table 1. Morphometric parameters (mean±s.d.) of CS-like fish at 3

months of age

Genotype Normal siblings CS-like fish

Number 10 10

Body length (cm) 3.03±0.22 2.46±0.12 (P<0.01)

Cranium length (cm) 0.32±0.01 0.31±0.03 (P=0.01)

Cranium width (cm) 0.13±0.01 0.18±0.01 (P<0.01)

Width/length 9.52±0.66 8.07±0.88 (P<0.01)

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to oncogenic H-RAS. We investigated the presence of markers ofan activated DDR in CS-like fish; as positive controls, we used wild-type fish of the same age that had been exposed to X-rays.Canonical DDR markers include activation of the upstream ataxiatelangiectasia mutated (Atm) kinase which autophosphorylates inresponse to damage, several substrates of pAtm, which can bedetected collectively with a phosphorylated S/TQ antibody(pS/TQ), and the phosphorylated histone variant H2AX known asγH2AX (Campisi and d’Adda di Fagagna, 2007). Massiveupregulation of γH2AX, phosphorylated Atm (pAtm) and pS/TQwere detected through western blot analysis (data not shown) andimmunohistochemistry, with fluorescent detection in tissuesections of irradiated fish (supplementary material Fig. S4E). Theseresults demonstrate that these DDR activation markers can bereliably used in vivo in adult zebrafish. In CS-like fish, but not in

wild-type controls, distinct nuclear foci containing γH2AX andpAtm were detected in various tissues (Fig. 5; supplementarymaterial Fig. S4D-F,I). Strikingly, we noticed that the nuclei thatstained for γH2AX and pAtm in the brain and heart of CS-like fishare localized in specific regions (Fig. 5A-C; supplementary materialFig. S4F,I) and throughout the heart ventricle (Fig. 5H),corresponding to areas enriched with adult proliferating cells(Adolf et al., 2006; Grandel et al., 2006; Poss, 2007). There was aclear inverse correlation between the decrease in BrdU+ cells andthe increase of SA-βgal and DDR markers in the brain and heartof CS-like fish (Table 2; Fig. 5A-C; supplementary material Fig. S4G-I). These results suggest that constitutive expression of oncogenicH-RAS in a developing vertebrate organism leads to DNA damagein subpopulations of adult progenitor cells in different organs.

OIS and apoptosis are dependent on tp53 activation (Schmitt etal., 2002; Sarkisian et al., 2007), which mediates many of the celldefenses in response to oncogenic stress. We investigated whetherCS-like fish exhibited activation of tp53 through tp53 target geneexpression. We found an increase in the expression of the tp53target gene cdkn1a/p21 in RNA extracted from whole CS-like fishand from specific tissues (supplementary material Fig. S5A), andtissue sections processed for in situ hybridization, from CS-like fish(supplementary material Fig. S5B-E).

Overexpression of GFP-H-RASV12 in an inducible transgenic lineleads to tp53-dependent OIS and DNA damage in vivoAs in humans, the CS-like phenotype develops over time and wecan distinguish affected fish at just 5-6 weeks of age. In order toelucidate the development of the Costello phenotype, we generatedan inducible H-RASV12 transgenic line Tg(hsp70I:GFP-H-RASV12)io3 (see Methods) (supplementary material Fig. S6), inwhich we can overexpress GFP-H-RASV12. This inducible linehelped us to address the question of how constitutive expressionof oncogenic H-RASV12 in CS-like fish leads to the developmentof DDR and OIS.

Fig. 3. Ras targets are not activated in embryos and adult P1/CS-likezebrafish. (A) Western blot analysis shows that, in heterozygous transgenic(+/–) fish, the expression level of GFP-H-RASV12 from the transgene (T) isslightly higher than the expression of endogenous (E) Ras proteins. Theantibodies used are indicated on the right of each blot and molecular weightsare indicated on the left. (B,C) Protein extracts from 4 dpf P1 larvae (B) andadult CS-like fish (C) show active Ras (immunoprecipitated with Raf1), butthere is no increase in ERK or Akt phosphorylation.

Fig. 4. DDR and cellular senescence inproliferating cells from the adult brain andheart of CS-like fish. CS-like fish show signs ofcellular senescence in areas of adultneurogenesis as indicated by reduced BrdUincorporation (A) and increased SA-βgalstaining (B) in the cerebellum. The area of thevalvula cerebelli analyzed here is shown in theline diagram (left panel). In contrast, no changesin BrdU incorporation (C) and SA-βgal staining(D) were observed in the gills of CS-like fish. Theimmunostaining and SA-βgal staining wereperformed on cryosections from 6-week-old CS-like fish and their sibling controls. Bars, 50 μm(B); 20 μm (D). (E) Quantification of proliferatingcells (BrdU+ cells) in the brain, heart, gills, skin,gut, liver and kidney. Black bars indicate wild-type adult control fish and gray bars representCS-like fish (n=5, mean±s.d.; analysis ofvariance).

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In this transgenic line, higher levels of oncogenic H-RASV12expression were obtained through the exposure of developingembryos, larvae and adults to 39°C for 30 minutes (Fig. 6;supplementary material Fig. S6B). We asked whether transgeniclarvae from this line developed traits of the CS-like phenotype and,if so, during what stage of development. We induced the expressionof the chimeric protein in embryos at 24 hpf and analyzed thephenotype over time. We observed that larvae display an abnormalphenotype at 4-6 days postfertilization (dpf), characterized by heartedema (Fig. 6A) and craniofacial abnormalities (Fig. 6B). Thisembryonic phenotype mimics the CS-like phenotype observed inhomozygous juveniles of the P1 line at 4-5 weeks of age, but is rathercommon in manipulated zebrafish embryos. To ensure that it wasthe result of H-RASV12 overexpression, we analyzed the activationof Ras downstream targets and observed a strong and promptphosphorylation of ERK1/2 and Akt, and an increase in the amountof active GTP-bound GFP-H-RASV12 at 2 and 6 hours followingheat shock (Fig. 6C).

We asked whether the overexpression of H-RASV12 inducedhyperproliferation, apoptosis or cellular senescence. BrdUincorporation in embryos subjected to heat activation at 24 hpfshowed an increase in the number of proliferating cells up to 3 dpf(Fig. 7A). However, by 5 dpf the number of BrdU+ cells droppedto less than half of that observed in controls (Fig. 7A,B). We alsochecked whether H-RASV12 expression induced apoptosis and at5 dpf we observed an increase in the number of TUNEL-positivecells (Fig. 7C). Together with the proliferation block and theincrease in apoptosis, we detected an increase in the number ofγH2AX- and pAtm-positive nuclei in sections from 5 dpf heat-shocked transgenic larvae (hs-fluo) (Fig. 7D), as well as in fibroblastcultures derived from these embryos (Fig. 7E). Next, we askedwhether high levels of GFP-H-RASV12 could have any impact ongenome stability and found that aneuploidy in mitotic spreads fromhs-fluo embryos at 32 hpf (Fig. 7F; supplementary material Fig. S7A)was similar to that observed in irradiated embryos (supplementarymaterial Fig. S7A). This suggests that cells which overexpress GFP-H-RASV12 in zebrafish transgenic lines are subjected to replicativestress that leads to genome instability.

Finally, we analyzed the contribution of tp53 to the DDR andthe proliferation block that occur in larvae overexpressing GFP-H-RASV12. Using a tp53 morpholino (mo) (Langheinrich et al., 2002),we were able to prevent the proliferation block, which occurred 4days after induction of oncogene expression (Fig. 8A,B). In contrast,

development of the heart and cranial cartilage defects was notprevented by tp53 mo injection (data not shown) suggesting thattp53 is not involved in the early response to H-RASV12 expression,but rather it controls the response to DNA damage that occursafter prolonged oncogene expression and, therefore, promotescellular senescence. In support of this hypothesis, hs-fluo embryosinjected with tp53 mo show increased γH2AX and pAtm nuclearstaining (Fig. 8C). Moreover, as expected, the absence of tp53completely prevented both upregulation of cdkn1a/p21, mdm2 andbax expression, and the decrease in ccnd1 expression, which areseen 4 days after heat shock in the transgenic larvae (supplementarymaterial Fig. S7B). Interestingly, embryos that express an activatedH-RAS oncogene in the absence of tp53 developed overgrowths ofthe central nervous system that are composed of proliferating BrdU-and phosphoH3-positive cells (Fig. 8D).

DISCUSSIONDespite the fact that Ras proteins have been studied for years, it isclear that a complete understanding of their function is still

Fig. 5. DDR markers in tissues of CS-like zebrafish. BrdU staining oncryosections from the valvula cerebelli region of the brain. The inset in (A)shows the plane of the sections in a lateral view of the adult zebrafish brain.BrdU+ cells in the valvula cerebelli of normal sibling (A) and CS-like (B) 2-month-old fish compared with γH2AX immunofluorescence in CS-like fish (C).(D-I) γH2AX (green) and pAtm (red) immunofluorescence in the followingtissues from adult CS-like and wild-type controls (insets): bone (D); skin (E); gut(F); liver (G); heart (H) and gills (I). Bar, 40 μm.

Table 2. Number of BrdU-positive cells, and SA- gal and DDR

markers in tissues of CS-like fish

Tissue BrdU SA- gal DDR

Brain – + ++

Heart – + ++

Gills +/– +/– +

Liver +/+ + +

Kidney + +/– –

Gut +/– +/– +

Skin +/– + +

Both cellular senescence and DNA damage responses occur in the brain and heart of CS-like fish, whereas in other tissues these parameters are not clearly correlated. Increase, + or ++; decrease, –; no change +/–.

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missing. The observation that oncogenic H-RAS can lead to tworather different cell behaviors, i.e. transformation and senescence,is particularly puzzling. Moreover, owing to germline activatingmutations, the same oncogenic H-RAS also leads to developmentaldisorders when constitutively expressed. In order to dissect the roleof oncogenic H-RAS in vivo, we generated transgenic zebrafish linesexpressing different levels of the oncogene. Our study shows that:(1) fish expressing oncogenic H-RAS in their germline display traitsof Costello syndrome; (2) low but prolonged exposure of adeveloping vertebrate organism to the H-RAS oncogene causes aDDR followed by senescence (OIS) in adult proliferating cells oftarget organs (heart and brain); (3) the process can be recapitulatedin embryos using a GFP-H-RASV12-inducible line, where OISdepends on the ability of tp53 to block H-RASV12hyperproliferation.

Zebrafish as a model for Costello syndromeDuring the generation of fish lines that express oncogenic H-RASin different organs or tissues, we identified fish that expressed GFP-H-RASV12 ubiquitously. We observed that these fish displayedhallmarks of the Costello syndrome, which is a rare, congenital,multiple abnormality syndrome associated with a failure to thriveand with developmental delays. The syndrome is caused by de novogermline activating mutations in the H-RAS gene (Aoki et al., 2005).Moreover, the appearance of this phenotype in fish carrying twocopies of both the transgene and the endogenous h-ras gene isconsistent with the genetic situation in affected humans, where thepresence of the mutated H-RAS gene is balanced by the presenceof a wild-type allele (Aoki et al., 2005). Recently, a mouse modelof Costello syndrome was reported (Schuhmacher et al., 2008); inthis model, the endogenous H-Ras allele was replaced by a mutantallele carrying the G12V mutation. The phenotype of the mouseCostello model resembles the human disorder, but with twoimportant differences. First, an increase in tumor formation wasnot observed in the mouse model, which, unlike human patients,developed angiotensin II-dependent systemic blood hypertension

(Schuhmacher et al., 2008). In the zebrafish model, we found aremarkable increase in tumor incidence, with tumor types similarto those reported to occur in human Costello patients. Surprisingly,in mouse and zebrafish models, phosphorylation of ERK or Akt,two of the most commonly activated Ras downstream targets, wasnot observed. We cannot exclude the possibility that ERK or Aktactivation could have occurred at certain times or in restrictedplaces in the transgenic fish and escaped our analysis; however, itis clear that if such activation occurs, it is not maintained. Whatis surprising is that in both studies we were able to detect GTP-bound GFP-H-RASV12 which did not lead to phosphorylation ofERK1/2 or Akt. The presence of active H-RAS suggests that otherRas downstream pathways should be investigated, because thesemight be responsible for the OIS phenotype detected in our fish.We also observed an increase in cdkn1a/p21 expression in differentorgans and tissues in the CS-like fish. This strongly correlates withthe fact that OIS requires cdkn1a/p21 to arrest cell cycleprogression; in fact, human fibroblasts lacking CDKN1A/p21 failto arrest in response to DNA damage (Brown, 1997).

The predominant view on the pathogenesis of Costello syndromewas that activation of the ERK and/or AKT pathways owing to H-RAS mutations could be the main molecular driver(s) of the diseasephenotype, a concept reinforced by the study of mutant H-RASactivity in vitro (Aoki et al., 2005; Rauen, 2007; Zampino et al., 2007).In this study, we propose a zebrafish model for this syndrome andshow that, like in the mouse model (Schuhmacher et al., 2008),sustained ERK and/or Akt phosphorylation are unlikely to be solelyresponsible for the abnormalities observed in Costello patients.

DNA damage and senescence may be responsible for the age-related worsening of the Costello phenotypeIt has already been documented that high levels of oncogenic RAScan cause hyperproliferation and increased DNA damage, followedby cellular senescence (Di Micco et al., 2006). We propose that verysimilar outcomes can be obtained with low but prolonged exposureto H-RASV12. In fact, the relatively low and ubiquitous expression

Fig. 6. Heat-shock-induced H-RASV12 expression causesa CS-like phenotype within 5 days and ERK/Aktphosphorylation. Embryos were subject to heat shock at24 hpf. At 5 days, fluorescent transgenic larvae (inset)display abnormal phenotypes, characterized by (A) strongpericardial edema (arrow) and (B) craniofacial defects asrevealed by Alcian Blue-stained splanchnocranium (right)and neurocranium (left). Bar, 50 μm. (C) Western blotanalysis revealed that, in 4 dpf larvae, the inducible andhigh expression of H-RASV12 causes an increase in activeRas and the phosphorylation of ERK/Akt at 2 and 6 hoursfollowing heat shock, respectively. ‘WT’ or ‘ctrl’ indicatesheat-shocked non-fluorescent sibling control fish and ‘hs-fluo’ indicates heat-shocked fluorescent transgenic fish.

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of oncogenic RAS leads to a DDR in cells that continue to proliferatethroughout adult stages. The cells that are more affected belong topools of adult progenitors in the brain and heart, where oncogenicH-RAS expression paralyzes cellular functions leading to OIS inresponse to DDR. The reason why some organs, although engagedin a hyperproliferative response to H-RASV12, do not undergosenescence even in the presence of elevated DNA damage (i.e. kidneyand liver) is not known. Perhaps the increase in organ size that wedetected in the liver and kidney of CS-like fish reflects defective tp53activation, which would allow proliferation to continue even in thepresence of massive DDR, or the specific functions of these organs.The zebrafish kidney serves as the primary site for adulthematopoiesis (de Jong and Zon, 2005), which may be increased inresponse to oncogenic H-RAS; whereas, in the liver, hepatocytes maybe involved in metabolic responses to H-RASV12 expression,including detox functions to deal with the increase in reactive oxygenspecies that is often reported in response to oncogenic RAS. So, ourmodel shows that the CS-like phenotype is the result of a low andprolonged expression of oncogenic H-RAS, which uncovers a specificweakness of adult progenitor cells. Interestingly, a similar abnormalphenotype was also observed in the inducible line where higher H-RASV12 expression leads to activation of ERK/Akt signaling and toa robust DDR in a shorter time. Usually these larvae do not survivefor more than 5-6 days following heat shock, and this prevented usfrom using them to study the appearance of cellular senescence

following DDR activation. However, a different protocol for H-RASV12 induction, involving multiple, short heat shocks injuveniles/adults, leads to a dose-dependent DDR with senescence ofadult progenitor cells, similar to what we observe in CS-like fish, anda little heart and bone phenotype (data not shown). Thus, we proposethat the Costello syndrome phenotype might result from twodifferent concerted and linked actions of oncogenic H-RAS:activation of tissue-specific pathways and OIS in progenitor/stemcells. We suggest that the age-related worsening of the CS-likephenotype might be the result of OIS affecting progenitors pools.When these cells undergo senescence they can no longer repairdamaged or apoptotic tissues, generate specialized cells, or maintainthe normal turnover in regenerating organs. As a consequence,tissues and organs carrying senescent progenitor cells undergoirreparable damage under normal conditions of stress and becomedysfunctional earlier.

We predict that the tumor suppressor tp53 orchestrates theoncogene-induced DDR and cell cycle arrest, but not the tissue-specific responses to H-RAS activation. Our results show that thehyperproliferation observed in the inducible line is not the resultof tp53 activation. Interestingly, we observed that in the absenceof tp53, the induction of oncogenic H-RAS causes overgrowths inthe CNS. Besides the frequent appearance of glioblastomas inhuman Costello patients (Gripp et al., 2006), it has recently beenshown that when activating H-RAS mutations and p53 inactivation

Fig. 7. Overexpression of H-RASV12 leads to OIS of DNA damaged cells in vivo. (A) 24 hpf larvae subjected to a 30-minute heat shock display an initialincrease in proliferation, represented by the green BrdU+ cells and shown here at 2 dpf in the skin (left panels), followed by a decrease in proliferation at 5 dpf inboth skin (middle panels) and brain (right panels). The percentage of mitotic, PH3-positive, cells (red) is unchanged. (B) Quantification of BrdU+ cells shows areduced number in 5-day-old transgenic larvae (n=5; mean±s.d.) following heat shock. Quantification of apoptotic cells revealed an increase in TUNEL-positivecells in heat-shocked transgenic larvae (n=5; mean±s.d.). Nuclear γH2AX immunostaining in the brain of control (wt) or heat-shocked transgenic (hs-fluo) 5 dpflarvae (C), and in cells dissociated from transgenic larvae and heat shocked in vitro (D). GFP-H-RASV12 expression induces aneuploidy, a sign of genomicinstability, in mitotic spreads from 32 hpf embryos (E). Bars, 40 μm (A,C,D); 10 μm (E).

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occur simultaneously, they promote the generation of brain cancerstem-like cells in vitro (Lee et al., 2008).

In summary, exhaustion of the replication abilities of adultprogenitor cells, owing to oncogene-induced DNA damage andsenescence, might lead to the age-related worsening of the Costellophenotype.

METHODSRaising fish and generation of the transgenic fish linesZebrafish were maintained and crossed according to standardmethods (Westerfield, 2000). For the generation of the gene traplines, we used a modified version of the T2KSAG plasmiddesigned by Kawakami (Kawakami et al., 2000), in which the GFPis replaced by the fusion protein eGFP-H-RASG12V (indicatedas GFP-H-RASV12) using the restriction enzymes BamHI andBglII. Fertilized eggs were injected with 2 nl of a mixturecontaining 25 ng/μl of the circular plasmid T2KSAG:GFP-H-RASV12 and 25 ng/μl of T2 transposase mRNA. Injected embryoswere raised to adulthood and crossed with either wild-type fishor fish injected with the same construct. F1 embryos wereobserved under a fluorescent dissecting microscope from day 1to 5 after fertilization. For generation of the inducible line, thesame GFP-H-RASG12V construct was cloned in a Tol2 vectorcontaining the Hsp70 promoter.

NIH3T3 cell transfection and focus formation assayNIH3T3 cells were transfected with the pEGFP-C2 vector thatcarries a neo selective marker (Clontech) containing the full-length

active H-RASV12 sequence or the empty pEGFP-C2 vector as aninternal control. The transfection was carried out by the calciumphosphate coprecipitation method, as described previously (Coxand Der, 1994), followed by drug selection with G418 at 0.8 mg/ml.The focus formation assay was performed as described previously(Kraus et al., 1984). Culture plates containing foci or colonies werefixed with ice-cold methanol and stained with Crystal Violet thenscanned on a flatbed scanner and quantified using ImageJ.

The two constructs generated comparable numbers of G418-resistant colonies; therefore, differences in focus-forming ability arenot the result of differences in transfection efficiency between theconstructs.

Screening for GFP-RAS insertionsFor PCR analysis of F1 embryos, sixty embryos at 24 hpf werepooled and used for DNA genomic extraction. Embryos weredigested in 300 μl of a solution containing 10 mM Tris, 2 mMEDTA, pH 8, 0.2% Triton and 200 μg/ml proteinase K, incubatedovernight at 50°C and heated for 5 minutes at 95°C. DNA wassubsequently purified by phenol-chloroform extraction andprecipitation with ethanol. The first round of PCR used 300 ngof DNA for each template and the following primers: eGFP-RASF (5�-AGCTGACCCTGAAGTTCATCT-3�) and eGFP-RAS R(5�- GTACTGGTGGATGTCCTCAAAAG-3�). Twenty cycles ofPCR were carried out as follows: 96°C for 30 seconds, 55°C for30 seconds, 72°C for 1 minute. 0.5 μl of DNA from the first PCRwas used as a template for the second round, which used theprimers eGFP-F2 (5�-CACATGAAGCAGCACGACTT-3�) and

Fig. 8. The OIS phenotype is rescued by a tp53 morpholino. (A) BrdU incorporation in the inducible GFP-H-RASV12 line injected with the tp53 mo. Persistenceof hyperproliferation is observed in hs-fluo embryos injected with the tp53 mo at 5 dpf (hs-fluo+tp53 mo), lateral view of the tail region. (B) Quantification ofBrdU+ cells in the skin of heat-shocked 5 dfp transgenic larvae (n=5; mean±s.d.) injected with the tp53 mo (gray bar, hs-fluo) compared with control wild-typelarvae (n=5; mean±s.d.) injected with the same morpholino (black bar, ctrl). (C) Immunofluorescence for γH2AX shows a diffuse DDR in the hindbrain of an hs-fluolarva at 5 dpf. (D) GFP-H-RASV12 is able to induce brain overgrowths in the absence of tp53 (left), lateral view, dorsal to the top. The right panel shows anenlargement of the boxed area stained for BrdU (green) and PH3 (red). Bars, 40μm.

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eGFP-R3 (5�-ATCAATGACCACCTGCTTCC-3�). Twenty-eightcycles of PCR were carried out as follows: 96°C for 30 seconds,55°C for 30 seconds, 72°C for 50 seconds. A DNA band of 725nucleotides was present when the construct was inserted into thegenome.

Southern blot analysisGenomic DNA was prepared according to Westerfield(Westerfield, 2000). About 7 μg of DNA per fish was digestedwith EcoRV, KpnI or a combination of the two enzymes. Followingelectrophoresis with 1% TAE-agarose gel, the DNA wastransferred to a Hybond N+ membrane (Amersham) andhybridized with a probe designed upon the GFP-HRAS sequence.The GFP-RAS probe was prepared for use in a PCR using thefollowing primers: forward (5�-CCACTACCTGAGCACCCAGT-3�) and reverse (5�-GTCTCC CCATCAATGACCAC-3�). We used10 ng of pT2SAG:GFP-HRAS plasmid as a template. The 337-nucleotide PCR product was gel purified and labeled with 32P-dCTP (Amersham) using the Rediprime II random primer labelingkit (GE healthcare, Amersham).

RT-PCR analysisRNA was purified using the Trizol extraction protocol. Afterpurification, RNA was digested with DNase I according to theRNeasy kit (Qiagen). 2 μg of RNA was used, in combination witholigo(dT) primers, for reverse transcription with Superscript III(Invitrogen). We used 2 μl of cDNA for each PCR reaction; theoligonucleotides used are described by Ghiselli (Ghiselli, 2006). β-actin oligonucleotides are as follows: forward (5�-ACC TCA TGAAGA TCC TGA CC-3�) and reverse (5�-TGC TAA TCC ACA TCTGCT GG-3�).

Inverse PCR strategyThe full T2KSAG:GFP-H-RASV12 plasmid was found integratedinto the zebrafish germline. We identified the break point in theplasmid, which occurred between nucleotides 6850 and 7221, andused a modified version of Kawakami’s protocol (Kawakami et al.,2000) in which genomic DNA was digested with XmaI and the self-ligation reaction was used directly as a PCR template. We used thefollowing primers: I-NR2 (5�-CTG TGT TCC GAA GAT GAACGG AGG TGT-3�), I-NR3 (5�-CCT CTA CAA ATG TGG TATGGC TGA TTA-3�), I-F1 (5�-CCC GTG GAC CTA ACG TTACCA ATT ACA-3�) and I-NF2 (5�-GCG GGA GAC AGA GGACAC ATT TCA TCT-3�).

Detection of apoptotic cellsApoptotic cells in 5 dpf embryos were detected using an ApopTagRed in situ apoptosis detection kit (S7165, Chemicon), which isbased on a terminal transferase dUTP nick-end labeling (TUNEL)assay. Samples were mounted in Vectashield (Vector Laboratories)with DAPI.

In situ hybridizationIn situ hybridization on whole-mounts and cryostat sections wasperformed as described previously (Costagli et al., 2002).

Zebrafish primary cell culturesZebrafish cell cultures from transgenic embryos of Gt(GFP-H-RASV12)io1 and Tg(hsp70I:GFP-H-RASV12)io3 lines weregenerated and maintained as described previously (Vallone et al.,2007).

Fish ionizing radiation treatmentAdult fish were exposed to 42 Gy using a Faxitron X-ray machine(Faxitron, Lincolnshire, Illinois). Protein extracts were preparedbetween 2 hours and 1 day after irradiation.

Paraffin embedding and H&E stainingThe fish were dehydrated in ethanol, decalcified in 0.25 M EDTA,cleared in xylene and infiltrated with paraffin. Briefly, slides weredeparaffinized in xylene, passed through a graded ethanol series,rinsed in water and briefly soaked in hematoxylin and then ineosin. Slides were dehydrated and mounted using EUKITT(GmbH).

Cartilage and bone staining for larvae and adults5-day-old larvae were fixed overnight in 4% paraformaldehyde,rinsed in distilled water and stained overnight in Alcian Bluesolution (1% HCl, 70% ethanol, 0.1% Alcian Blue). Larvae werethen cleared by washing in 3% hydrogen peroxide, dehydratedthrough an ethanol series and equilibrated in 85% glycerol-PBS.Splanchnocranium and neurocranium were manually dissectedfrom stained larvae and flat mounted in 85% glycerol-PBS.

Fixed juvenile/adult fish were eviscerated, skinned anddehydrated in 100% ethanol for 2 days. The carcasses wereincubated in Alcian Blue solution for 12 hours and then in 100%ethanol overnight. Fish were then cleared in 1% KOH for 6 hoursand stained in 0.05% Alizarin Red in 2% KOH for 3 hours. Thefish were cleared in 2% KOH overnight and stored in 25%glycerol.

MorphometryMeasurements were taken using a dissecting microscope equippedwith a micrometer lens.

Western blot analysis and Ras activation assayAdult fish were killed by anesthetic overdose (0.04% MESAB,Sigma), which was ground to a fine powder in liquid nitrogen andresuspended in sample buffer (2% SDS, 10% glycerol, 60 mM TrispH 6.8). Organs and larvae were lysed in sample buffer; 50 μg oftotal extracts were resolved by SDS-PAGE, transferred tonitrocellulose and tested with the following antibodies: Ras (1:500,BD Bioscience), GFP (1:2000, Torrey Pines Biolabs), phospho-p44/42 (1:1000, Cell Signaling), p44/42 (1:1000, Cell Signaling),phospho-Akt (1:1000, Cell Signaling), Akt (1:1000, Cell Signaling),phospho-Atm (1:600, Rockland), Atm (1:1000, Sigma).

We performed the StressXpress Ras Activation kit (Stressgen,Bioreagents) on protein extracts from larvae and juvenile fishaccording to the manufacturer’s instructions. Band intensities werequantified using ImageJ.

β-gal histochemistryAcidic β-gal staining was performed on formalin-fixed zebrafishcryosections, as described previously (Dimri et al., 1995).

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BrdU treatment, immunofluorescence and antiseraEmbryos and adults were incubated in fish water containing 10mM of BrdU for 2 hours (embryos and larvae) or overnight(juveniles and adults). Embryos and larvae were fixed in 4%paraformaldehyde, adult and juveniles were embedded unfixed inOCT, and sections were fixed in 2% paraformaldehyde for 2minutes. For BrdU-phospho H3 (PH3) immunostaining, sectionsand larvae were treated with 2 N HCl for 20 minutes, followed bytwo rinses in 0.1 M sodium tetraborate. For γH2AX and pAtmimmunostaining, unfixed cryostat sections were fixed in a 50:50methanol:acetone mix for 2 minutes and then rinsed in PBS. Inaddition to the antisera used in the western blots, mousemonoclonal anti-BrdU (Sigma) and rabbit polyclonal anti-PH3(Upstate) antisera were used. We used secondary antibodiesconjugated with Alexa 488 or 568 (Molecular Probes).

Chromosome preparation and visualizationAt 24 hpf, tg(Hsp70:GFP-H-RASV12)io3 embryos were incubatedfor 30 minutes at 39°C. At 32 hpf, 100 transgenic embryos, 100irradiated embryos (20 Gy) and 100 untreated embryos, wereincubated in colchicine (4 mg/ml) overnight and dechorionated bypronase treatment. Chromosome preparations were dropped ontoslides and allowed to dry overnight at 37°C. Samples were mounted

in Vectashield (Vector Laboratories) with DAPI. The images ofchromosome spreads were acquired and elaborated with the BandView software system (Applied Spectral Imaging).

Counts of BrdU+ and TUNEL+ cellsThe numbers of BrdU- or TUNEL-positive cells in whole-mountlarvae were counted with a confocal microscope (Leica SP2 TCS).Counts were performed in the whole tail region (from thebeginning of the yolk extension) or in the forebrain, where thecount refers to a 100 μm thick section from the area between theolfactory bulb and the diencephalon. Section counts refer to anarea of 2.4 mm2, from a serial section with a thickness of 20 μm,from the whole forebrain or whole heart. At least 5 sections,including the areas of interest (see Results), from 6 differentspecimens were analyzed.

Gene, constructs and morpholinosThe tbx20 (hRT) plasmid (Szeto et al., 2002) and the cdkn1a/p21plasmid were used to transcribe antisense probes. We injected 10ng of tp53 mo4 (www.zfin.org) into Tg (hsp70I: GFP-H-RASV12)io3embryos at the one-cell stage. Larvae were heat shocked at 24 hpffor 30 minutes and sorted by fluorescence 4 hours later.ACKNOWLEDGEMENTSWe especially thank Keith Cheng, Giuseppe Testa, Giorgio Scita and Bruno Amatifor critically reading the manuscript and for the constructive comments. We thankO. Malazzi for help with the chromosomal spread and analysis, M. Fumagalli and R.di Micco for sharing reagents and protocols, A. Renna and C. Buratti for fishmaintenance, P. M. Essomba for photography of adult fish and T. Schilling forconsultation in bone staining. C.S. was supported by a 1-year fellowship from theItalian Association for Cancer Research (AIRC).

COMPETING INTERESTSThe authors declare no competing financial interests.

AUTHOR CONTRIBUTIONSM.M. and C.S. conceived and designed the experiments; C.S. and M.M., with somecontribution from F.P., G.D. and L.L., performed all of the experiments; F.d.d.F.helped with data analysis and interpretation; K.K. provided the Tol2 plasmid andadvice; M.M. and C.S. wrote the manuscript.

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/content/2/1-2/56/suppl/DC1

Received 24 June 2008; Accepted 16 October 2008.

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TRANSLATIONAL IMPACT

Clinical issueSomatic gain-of-function mutations in the Ras genes are commonly found inthe majority of human cancers; whereas, germline gain-of-function mutationsin genes of the Ras-Raf-Mek and ERK pathways cause a class of developmentaldisorders including Noonan, Costello and cardio-facio-cutaneous (CFC)syndromes. It is still unclear how the same activating mutations of the Rasgenes can induce both oncogenic transformation, as seen in somaticmutations in cancer patients, as well as developmental abnormalities, as aresult of germline mutations, in Noonan, Costello and CFC patients. Onepossible explanation is that, during development, Ras activates target genes ina limited subtype of cells or tissues, thus curbing the oncogenic activity ofmutant Ras and avoiding a lethal phenotype.

ResultsIn this paper, the zebrafish is used to model Costello syndrome and elucidateoncogenic H-RAS function during development. The authors generatedtransgenic zebrafish lines expressing oncogenic H-RAS through the germline.These fish show several developmental defects similar to those found inCostello patients. Similar to recent reports in mouse models, no overtactivation of the Mek-ERK or PI3K-Akt pathway could be detected in Costello-like fish, suggesting that the molecular mechanism underlying Costellosyndrome does not involve activation of these common Ras targets. Amongseveral cellular responses commonly induced by oncogenic Ras, such asproliferation, invasiveness and apoptosis, the authors also observed cellularsenescence in adult progenitor cells of the brain and heart – two of the mostaffected organs in Costello patients.

Implications and future directionsIn this zebrafish model of Costello syndrome, some defects are caused by anoncogene-induced senescence program in adult proliferating cells. Thisfinding encourages further investigation of cellular senescence in Costellopatients, who show an age-dependent worsening phenotype that iscompatible with premature exhaustion of adult progenitor cells. Additionally,understanding how a developing organism restricts oncogenic signalactivation might lead to novel therapeutic strategies in cancer.

doi:10.1242/dmm.002238

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expression of h-rasv12 in a zebrafish model of costello ... · that fish carrying the integrated...

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