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. 2009 Jan-Feb;2(1-2):56-67.
doi: 10.1242/dmm.001016. Epub 2008 Dec 22.

Expression of H-RASV12 in a zebrafish model of Costello syndrome causes cellular senescence in adult proliferating cells

Cristina Santoriello  1 , Gianluca Deflorian, Federica Pezzimenti, Koichi Kawakami, Luisa Lanfrancone, Fabrizio d'Adda di Fagagna, Marina Mione
Affiliations

Expression of H-RASV12 in a zebrafish model of Costello syndrome causes cellular senescence in adult proliferating cells

Cristina Santoriello et al. Dis Model Mech. 2009 Jan-Feb.

Abstract

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 have generated transgenic zebrafish that constitutively express low levels, or can be induced to express high levels, of oncogenic H-RAS. We observed that fish carrying the integrated transgene in their germline display several hallmarks of Costello syndrome, a rare genetic disease caused by activating mutations 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 associated with both reduced proliferation and an increase in senescence markers in adult progenitor cell compartments in the brain and heart, together with activated DNA damage responses. Overexpression of H-RAS through a heat-shock-inducible promoter in larvae led to hyperproliferation, activation of the DNA damage response and tp53-dependent cell cycle arrest. Thus, oncogene-induced senescence of adult proliferating cells contributes to the development of Costello syndrome and provides an alternative pathway to transformation in the presence of widespread constitutively active H-RAS expression.

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Figures

Fig. 1
Fig. 1
Embryos and adults with germline integration and constitutive expression of GFP-H-RASV12 are healthy, whereas somatic integrations generate tumors. (A) Diagram of the gene trap T2KSAG:GFP-H-RASV12 construct used in this study, containing Tol2 sequences, a splice acceptor (SA) and the sequence coding 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 not affect 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 a cryostat section of the tumor (lower right panel). Bars, 40 μm. (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, 10 μm. (E) Southern blot analysis using genomic DNA extracted from 1-month-old wild-type (WT) zebrafish. Analysis of 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.
Fig. 2
Fig. 2
Homozygous P1 fish develop a Costello (CS)-like phenotype. Developmental defects in a 6-week-old CS-like fish. (A) CS-like fish have reduced body size compared with normal sibling fish. They also have a smaller heart (B), an enlarged gill area (C) and craniofacial dysmorphogenesis with increased ossification (Alizarin Red staining) of the Weberian complex (arrow) (D). (E) Alizarin Red staining also reveals fusion of vertebrae (arrowheads). An increase in cancer development is observed in transgenic fish. (F) Trunk rhabdomyosarcoma in a heterozygous P1 5-month-old fish. (G,H) H&E staining of a paraffin section of the tumor, showing the overall appearance (G) and the mature striated muscle component (H). Bars, 40 μm.
Fig. 3
Fig. 3
Ras targets are not activated in embryos and adult P1/CS-like zebrafish. (A) Western blot analysis shows that, in heterozygous transgenic (+/–) fish, the expression level of GFP-H-RASV12 from the transgene (T) is slightly higher than the expression of endogenous (E) Ras proteins. The antibodies used are indicated on the right of each blot and molecular weights are indicated on the left. (B,C) Protein extracts from 4 dpf P1 larvae (B) and adult CS-like fish (C) show active Ras (immunoprecipitated with Raf1), but there is no increase in ERK or Akt phosphorylation.
Fig. 4
Fig. 4
DDR and cellular senescence in proliferating cells from the adult brain and heart of CS-like fish. CS-like fish show signs of cellular senescence in areas of adult neurogenesis as indicated by reduced BrdU incorporation (A) and increased SA-βgal staining (B) in the cerebellum. The area of the valvula cerebelli analyzed here is shown in the line diagram (left panel). In contrast, no changes in BrdU incorporation (C) and SA-βgal staining (D) were observed in the gills of CS-like fish. The immunostaining and SA-βgal staining were performed on cryosections from 6-week-old CS-like fish and their sibling controls. Bars, 50 μm (B); 20 μm (D). (E) Quantification of proliferating cells (BrdU+ cells) in the brain, heart, gills, skin, gut, liver and kidney. Black bars indicate wild-type adult control fish and gray bars represent CS-like fish (n=5, mean±s.d.; analysis of variance).
Fig. 5
Fig. 5
DDR markers in tissues of CS-like zebrafish. BrdU staining on cryosections 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 following tissues 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.
Fig. 6
Fig. 6
Heat-shock-induced H-RASV12 expression causes a CS-like phenotype within 5 days and ERK/Akt phosphorylation. Embryos were subject to heat shock at 24 hpf. At 5 days, fluorescent transgenic larvae (inset) display abnormal phenotypes, characterized by (A) strong pericardial edema (arrow) and (B) craniofacial defects as revealed by Alcian Blue-stained splanchnocranium (right) and neurocranium (left). Bar, 50 μm. (C) Western blot analysis revealed that, in 4 dpf larvae, the inducible and high expression of H-RASV12 causes an increase in active Ras and the phosphorylation of ERK/Akt at 2 and 6 hours following heat shock, respectively. ‘WT’ or ‘ctrl’ indicates heat-shocked non-fluorescent sibling control fish and ‘hs-fluo’ indicates heat-shocked fluorescent transgenic fish.
Fig. 7
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 initial increase 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 in both skin (middle panels) and brain (right panels). The percentage of mitotic, PH3-positive, cells (red) is unchanged. (B) Quantification of BrdU+ cells shows a reduced number in 5-day-old transgenic larvae (n=5; mean±s.d.) following heat shock. Quantification of apoptotic cells revealed an increase in TUNEL-positive cells 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 dpf larvae (C), and in cells dissociated from transgenic larvae and heat shocked in vitro (D). GFP-H-RASV12 expression induces aneuploidy, a sign of genomic instability, in mitotic spreads from 32 hpf embryos (E). Bars, 40 μm (A,C,D); 10 μm (E).
Fig. 8
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. Persistence of 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 of BrdU+ 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-type larvae (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-fluo larva 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 an enlargement of the boxed area stained for BrdU (green) and PH3 (red). Bars, 40 μm.
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