FIGURE SUMMARY
Title

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.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Dev. Dyn.