- Title
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Liver-specific expression of p53-negative regulator mdm2 leads to growth retardation and fragile liver in zebrafish
- Authors
- Chen, L.J., Hsu, C.C., Hong, J.R., Jou, L.K., Tseng, H.C., Wu, J.L., Liou, Y.C., and Her, G.M.
- Source
- Full text @ Dev. Dyn.
Generation and characterization of zebrafish transgenic Gmdm2-liver lines. A: Transgenic construct (LF2.8-GFP::Mdm2) of the engineering of transgene GFP::Mdm2, which is GFP fused with zebrafish Mdm2 (GeneBank accession no: NM_131364) into the zebrafish genome. The Not I/Sfi I fragment consists of the zebrafish L-FABP gene 2.8-kb promoter (LF2.8-pro) (Her et al.,[2003a]), transgene GFP::Mdm2, and the SV40 poly(A) signal (SV40 pA). B: Genomic PCR analysis of the production of transgenic zebrafish possessing the transgene GFP::Mdm2. The PCR primer pair P1-P2 was used to identify transgenic zebrafish during breeding. Three transgenic F0 founder lines (Gmdm2-liver#1, Gmdm2-liver#2, and Gmdm2-liver#3) were generated. pLF2.8-GFP::Mdm2 (C+) and WT/G-liver genomic DNA were used as positive and negative controls, respectively. C: Three different cycles of RT-PCR showing the amount of total GFP::Mdm2 and Mdm2 transcripts in hepatic tissues (hepatocytes) of stable transgenic Gmdm2-liver#1, Gmdm2-liver#2, G-liver lines (Her et al.,[2003a]), and wild-type zebrafish. β-actin transcripts are the internal control. D: Immunoblot analysis of GFP:mdm2 expression in the livers of Gmdm2-liver#1 and #2 transgenic zebrafish. An ectopic 60-kDa GFP:mdm2 fusion protein was expressed exclusively in the hepatic tissues of Gmdm2-liver#1 and #2 transgenic zebrafish; higher levels of this protein were expressed exclusively in hepatic tissues of Gmdm-liver#2. Hepatic lysates were isolated from 4-month-old G-liver, WT (wild-type), and Gmdm2-liver#1, and 2 transgenic zebrafish. Hepatic lysates from the G-liver and WT were used as positive and negative controls, respectively. A monoclonal anti-GFP antibody was used for detecting both GFP::Mdm2 and GFP. The same filter was probed with a monoclonal anti-β-actin antibody to calibrate the amount of protein extracts loaded in each lane. |
The chronic effects of GFP::Mdm2 expression in Gmdm2-liver larvae and adult fish resulting in developmental delay and growth retardation. A-F: G-liver and Gmdm2 larvae and their growth were assessed at 14, 50, and 100 days post-fertilization (dpf). Representative pictures of the fish are shown. Gmdm2-liver fish are significantly shorter and thinner than G-liver fish until 100 dpf. G: Genetic statistical analysis of developmental delay and growth retardation in two Gmdm2-liver lines compared with G-liver and wild-type fish. H: Severe growth retardation of two 10-month-old Gmdm2-liver fish lines compared with G-liver and wild-type fish. Insets show higher magnification images of the cross-sections of zebrafish liver at various stages. Scale bars = 200 μm (A,D); 10 mm (B,E); 1 cm (C,F,H). |
Liver degeneration in Gmdm2-liver fish. A: Whole-body image of normal, atrophic, contractive, and hypoplastic livers in a G-liver and three Gmdm2-liver#1 fish with various levels of growth retardation or illness at 5 months of age. Normal liver (1-3); atrophic liver (4-6); contractive liver (7-9); hypoplastic liver (10-12). Bright field image of the livers (1, 4, 7, and 10); GFP fluorescent image of the same livers (2, 5, 8, and 11); merge image of the same livers (3, 6, 9, and 12). B: Hepatic lesions in Gmdm2-liver transgenic zebrafish. GFP::Mdm2 over-expression leads to progression from normal liver (i) to liver atrophy (ii), contraction (iii), and hyperplasia (iv). Coalescence of hepatic foci as depicted by the arrow. Hematoxylin and eosin (H&E)-stained slides of paraffin-embedded fixed livers from G-liver#1 (1) and from Gmdm2-liver#1 transgenic zebrafish (4, 7, and 10). Magnifications at 100x. |
Morphological analysis of chronic disease in Gmdm2-liver#1 and 2 transgenic zebrafish with different levels of eating disorders and illness. A,B: Gross appearance of 10-month-old male Gmdm2-liver#1 with small liver polyps (A) and liver cysts (B). C-E: Liver agenesis in a Gmdm2-liver#2 transgenic zebrafish with severe emaciation (C). Macroscopic appearance of the stomach region (abdominal chamber) in the dissected whole body of 4-month-old male G-liver#1 and Gmdm2-liver #2 fish. Whole liver lobes were absent in the abdominal chamber of the Gmdm2-liver#2 fish compared with the liver of G-liver fish (D, E). Bright field image of the livers (C); GFP fluorescent image of the same livers (D); merge image of the same livers (E). F,G: Histological cross-sections from G-liver in C (F) and Gmdm2-liver#2 in C (G). Scale bars = 20 mm. |
Immunohistochemical staining of active caspase 3 and proliferating cell nuclear antigen (PCNA) on the liver sections of 4-month Gmdm2-liver and G-liver transgenic zebrafish.A-C: Detection of apoptotic cells in normal liver of 4-month G-liver (A) and hypoplastic liver of 4-month Gmdm2-liver #1 (B) and Gmdm2-liver #2 (B) by immunohistochemical staining of active caspase 3 and haematoxylin counterstain. The active caspase 3 immunopositive (brown reaction product) cells were considered to be apoptotic cells. Comparison of the intensity of nuclear staining and the number of active caspase 3 immunopositive cells in the normal (A) and hypoplastic (B and C) livers revealed a significantly increased level of cell death in the hypoplastic livers of 4-month Gmdm2-liver #1 and Gmdm2-liver #2 where GFP::Mdm2 is over-expressed. D-F: Detection of the proliferative activity of cells in normal liver of 4-month G-liver (D) and hypoplastic liver of 4-month Gmdm2-liver #1 (E) and Gmdm2-liver #2 (F) by immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and haematoxylin counterstain. The PCNA immunopositive (brown reactth G-liver (D) and hypoplastic liver of 4-month Gmdm2-liver #1 (E) and Gmdm2-liver #2 (F) by immunohistochemical staining of proliferion product) cells were considered to be proliferative cells. Comparison of the normal (D) and hyperplastic (E and F) livers revealed a cell-cycle arrest or decreased number of immunopositive cells in the hypoplastic livers of 4-month Gmdm2-liver #1 and Gmdm2-liver #2 where GFP::Mdm2 is over-expressed. Arrows indicate examples of immunopositive (brown reaction product) cells. Scale bar = 50 μm. |
Detection of p53 protein and gene expression changes associated with altered cell death and growth in the degenerated liver of Gmdm2-liver lines. A: Expression of zebrafish p53 in the liver of G-liver, Gmdm2-liver#1, and Gmdm2-liver#2 using a p53 polyclonal antibody (Santa Cruz Biotechnology). B: Quantification of p53 regulatory genes of p21, 14-3-3-σ, and Gadd45-α were slightly down-regulated in the liver of Gmdm2-liver#1 and Gmdm2-liver#2 fish compared with G-liver by quantitative PCR. Expression analyses of the p53 regulatory genes using cDNA prepared from G-liver (n = 4-6) and Gmdm2-liver#1 (n = 4-7) and Gmdm2-liver#2 (n = 4-8). Data represent mean values ± SEM. Values were normalized against β-actin as a housekeeping gene, and represent relative fold change of mRNA expression to G-liver. The asterisks represent a statistically significant difference when compared with the G-liver: *P < 0.05 and **P < 0.01 levels. C: Quantification of cell apoptotic genes of zBid, zBik, zBax1, zBok1, and zPuma were highly up-regulated and the cell survival (anti-apoptotic) related-genes of zBlp1, zMcl-1a, and zNR13were slightly down-regulated in the liver of Gmdm2-liver#1 and Gmdm2-liver#2 fish compared with G-liver by quantitative PCR. Expression analyses of the apoptotic genes using cDNA prepared from G-liver (n = 4-8), Gmdm2-liver#1 (n = 4-6). and Gmdm2-liver#2 (n = 4-7). Data represent mean values ± SEM. Values were normalized against β-actin as a housekeeping gene, and represent relative fold change of mRNA expression to G-liver. The asterisks represent a statistically significant difference when compared with the G-liver: *P < 0.05 and **P < 0.01 levels. D: Quantification of cell cycle-related genes of cyclin A2, B1, D1, E, H, and G1 where there was no significant difference in the liver of Gmdm2-liver#1 and Gmdm2-liver#2 fish compared with G-liver by quantitative PCR. The β-actin primer was used as an internal control for real time PCR reaction. Relative fold-changes were determined by calculating the ratio of the mean expression values from the G-liver and Gmdm2-liver lines. PCR reactions were carried out in triplicate. Expression analyses of the cell cycle-related genes using cDNA prepared from G-liver (n = 4-6), Gmdm2-liver#1 (n = 4-8), and Gmdm2-liver#2 (n = 4-8). Data represent mean values ± SEM. Values were normalized against β-actin as a housekeeping gene, and represent relative fold change of mRNA expression to G-liver. The asterisk represents a statistically significant difference when compared with the G-liver: *P < 0.05 and **P < 0.01 levels. |