CCAAT/enhancer-binding protein α is required for hepatic outgrowth via the p53 pathway in zebrafish

CCAAT/enhancer-binding protein α (C/ebpα) is a transcription factor that plays important roles in the regulation of hepatogenesis, adipogenesis and hematopoiesis. Disruption of the C/EBPα gene in mice leads to disturbed liver architecture and neonatal death due to hypoglycemia. However, the precise stages of liver development affected by C/ebpα loss are poorly studied. Using the zebrafish embryo as a model organism, we show that inactivation of the cebpa gene by TALENs results in a small liver phenotype. Further studies reveal that C/ebpα is distinctively required for hepatic outgrowth but not for hepatoblast specification. Lack of C/ebpα leads to enhanced hepatic cell proliferation and subsequent increased cell apoptosis. Additional loss of p53 can largely rescue the hepatic defect in cebpa mutants, suggesting that C/ebpα plays a role in liver growth regulation via the p53 pathway. Thus, our findings for the first time demonstrate a stage-specific role for C/ebpα during liver organogenesis.

The cebpa mutant exhibits a small liver phenotype. To study the function of C/ebpα in embryonic liver development, we first analyzed the expression pattern of cebpa by whole mount in situ hybridization. The results showed that cebpa expression was enriched in the developing liver (supplemental Fig. S1), consistent with the previous observation 13 . We then examined the expression of liver fatty acid binding protein (lfabp), a liver-specific marker 14 . The data revealed that the liver size of the cebpa mutant was strikingly reduced, compared to that of sibling controls at 72 hpf and 5 dpf, respectively ( Fig. 2A and supplemental Fig. S2). In contrast, the development of other endoderm-derived tissues such as exocrine pancreas, endocrine pancreas and intestine was not obviously affected as determined by assessing the expression of trypsin, insulin and fatty acid binding protein 2 (fabp2), respectively ( Fig. 2B-D). Together, these results suggest that cebpa is essential for liver development in zebrafish. C/ebpα is required for hepatic outgrowth but not for hepatoblast specification. The failure of liver development in the cebpa mutant could be attributable to defects in hepatoblast specification from endodermal cells or budding prior to hepatic outgrowth. To test which stage had been affected, we examined the expression of hhex and prox1, representing the earliest markers for hepatoblasts 15,16 . Data Luciferase activity assays were performed in 293T cells using C/ebpα constructs indicated. Renilla was used as an internal control. Data shown are the mean ± SD of three independent experiments, **P < 0.005 by student's t-test. NS, not significant. Note that the mutant C/ebpα ( CA), with two nucleotide deletions, loses its transcriptional activity.
showed that these genes displayed similar expression patterns in the liver primordia of both sibling and mutant embryos at 30 hpf (completion of specification) (Fig. 3A,B) and 48 hpf (completion of budding) ( Fig. 3D-E). Moreover, the expression of foxa3, a pan-endodermal marker, was also unaffected in the mutant embryos (Fig. 3C,F). Therefore, these results, together with that of lfabp, indicate that C/ebpα is not required for liver specification but for liver expansion growth during hepatogenesis. cebpa deficiency results in enhanced hepatic cell proliferation and subsequent increased apoptosis. The small liver phenotype in the cebpa mutant could potentially result from abnormal cell proliferation and/or apoptotic cell death. We next examined the terminal fate of the cebpa-deficient hepatic cells using immunostaining. Firstly, using an antibody against phospho-histone H3 (pH3), a marker of cell proliferation, we found that the pH3-positive hepatic cells exhibited a 2.5-fold increase in the cebpa mutant sectioned embryos at 60 and 72 hpf, respectively ( Fig. 4A-D,I). Next, we examined apoptotic events using the TUNEL assay. The mutant hepatic cells underwent significant activation of cell apoptosis at 72 hpf, whereas almost no apoptotic cells were detected in the developing liver of sibling controls or at earlier developmental stage ( Fig. 4E-H,J). Collectively, these results suggest that loss of C/ ebpα results in enhanced hepatic cell proliferation and subsequent increased cell apoptosis which may account for the small liver phenotype. p53 pathway activation in the cebpa mutant. To study the molecular mechanisms that may underlie the small liver phenotype in the cebpa mutant, we examined the expression levels of genes which are involved in cell proliferation and apoptosis. It was previously shown that the expression of c-myc and c-jun were induced in the liver of C/EBPα knockout mice 10 . In agreement with this, we also detected elevated expression of mycb and jun in the cebpa mutant zebrafish embryos (Fig. 5A), underscoring the evolutionary conserved role of these genes in the regulation of normal liver development. Importantly, we also observed that the expression of bcl2 and bcl2l were significantly increased in these mutant embryos (Fig. 5B), supporting the notion that an increased portion of the cebpa-deficient hepatic cells were in an apoptotic state. It is well known that the p53 signaling pathway plays a key role in controlling cell proliferation and apoptosis 17 . The aberrant cell proliferation and apoptosis observed in the cebpa mutant suggests that p53 pathway is likely activated. In order to test this, we knocked down p53 using morpholino (MO) in the cebpa mutant embryos. As expected, the hepatic defect of the cebpa mutant embryos could be efficiently rescued by p53 knockdown (Fig. 5C,D). To further confirm the role of p53 in this process, we next generated cebpa and p53 double mutant zebrafish. The data showed that additional loss of p53 could restore normal liver development in cebpa-deficient embryos (Fig. 5 E,F). Moreover, the defects of cell proliferation and apoptosis could be also largely recovered in the cebpa and p53 double mutant (supplemental Fig. S3). Thus, these results demonstrate that the p53 pathway is indeed involved in C/ebpα -dependent hepatic outgrowth.

Discussion
Targeted disruption of the C/EBPα gene in mice leads to disturbed liver architecture and results in neonatal death due to hypoglycemia 9,10 . However, the exact stages affected by C/EBPα deficiency during liver development have not been analyzed in detail. Furthermore, the fetal liver of mammals is also a hematopoietic organ, and hepatogenesis and hematopoiesis are intertwined. Therefore, dysfunction of one process may prevent the proper development of the other. C/EBPα not only plays important roles in hepatogenesis, but also is required for hematopoiesis 18 , which obfuscates its role in these two processes. To assess the independent function of C/EBPα in liver development, we used the popular model organism, zebrafish, due to the fact that embryonic hematopoiesis does not take place in the liver. Here, we found that the expression of cebpa was enriched in the developing liver of zebrafish. Inactivation of the cebpa gene by TALENs led to a small liver phenotype. Detailed analysis revealed that C/ebpα was required for hepatic outgrowth but not for hepatoblast specification during liver development.
An increasing number of reports have implicated C/EBPα as a suppressor of cell proliferation 8 . In support of this idea, we found that loss-of-function of C/ebpα induced hepatic proliferation in the developing liver of zebrafish. Interestingly, we also detected increased hepatic cell apoptosis in the cebpa mutant embryos at later developmental stage. These ambivalent results suggest that the cebpa-deficient hepatic cells seemed to be in an inappropriate proliferative state and then underwent apoptosis, eventually resulting in the small liver phenotype. It is not a rare phenomenon in the liver development, since disruption of Apc (adenomatous polyposis coli) in the liver of mice also leads to increased hepatocyte proliferation and apoptosis, which may be caused by elevated DNA damage, accumulation of p53 and increased levels of anaphase bridges 19 . Additionally, in the partial hepatectomy (PH)-induced mouse liver regeneration study, Nur77 knock-out livers exhibited enhanced hepatocyte proliferation coincided with hepatocyte apoptosis 20 . Further studies are required to investigate the switch between cell proliferation and apoptosis regulated by C/ebpα , such as the role of C/ebpα in controlling chromosome segregation and genomic stability.
The p53 pathway is composed of a network of genes responding to a variety of intrinsic and extrinsic stress signals. Activation of the p53 protein induces cell cycle arrest, cellular senescence or apoptosis 17,21 . p53, as a well-known tumor suppressor, also plays a critical role during organogenesis, including hepatogenesis. In zebrafish def hi429 (digestive-organ expansion factor) mutant, the expression of ∆113p53, a newly identified isoform of p53, was selectively up-regulated within the mutant digestive organs, and then triggered the arrest of cell proliferation, resulting in compromised organ growth. Furthermore, knock-down of p53 and  113p53 levels could rescue the developmental defects of the mutants 22,23 .
Moreover, the Def-p53 pathway was also involved in scar formation at the amputation site after PH in zebrafish 24 . Here, we showed that the p53 pathway was activated in the cebpa-deficient embryos, which may be triggered by the aberrant cell proliferation, and additional loss of p53 could largely rescue the hepatic defects in the cebpa mutants. However, p53 might not be a direct target gene of C/ebpα in the liver organogenesis, since the transcriptional level of p53 has no obvious changes in the cebpa mutants compared with sibling controls (supplemental Fig. S4). It will be of interest in future studies to determine how C/ebpα regulates p53 activities in the developing liver.
Taken together, we hereby provide novel evidence that C/ebpα is specifically required for hepatic outgrowth via the p53 pathway, and accordingly have expanded our understanding of liver development. Moreover, these new findings may help to identify new targets for therapeutic manipulation in the treatment of liver failure and liver cancer.

Methods
Zebrafish. Zebrafish maintenance and staging were performed as described previously 25 . The zebrafish facility and study were approved by the Institutional Review Board of the Institute of Health Sciences, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and the methods were carried out in accordance with the approved guidelines. The cebpa rj31/+ and p53 zdf1/zdf1 mutants were used in this study 12,26 . Generation of constructs. The zebrafish cebpa∆CA was generated by genomic PCR with the following primers: Forward 5' CCGGAATTCATGGAGCAAGCAAACCTCTACGAGG 3' , Reverse 5' CCGCTCGAGTTAAGCGCAGTTGCCCATGGCTTTGAC 3' , then cloned into the pCS2+ vector.
Luciferase reporter assay. 293T cells were transfected with the indicated plasmids using Effectene Transfection Reagent (QIAGEN). Tetramer of the CEBP site of human GCSFR was inserted into the promoterless luciferase vector pTK81-luc and used as a reporter plasmid 27 . Cells were harvested 36 hours after transfection and luciferase activities were analyzed using the Dual Luciferase Reporter Assay Kit (Promega), according to the manufacturer's protocols. Luciferase activity was normalized to Renilla activity.
Morpholino. The morpholino oligonucleotide (MO) of p53 (TCTTGGCTGTCGTTTTGCGCCATTG) was used as previously described 28 . (D,F) The relative liver area measured in (C,E) respectively. The result shown is fold difference compared with the level (set to 100) detected in control embryos (mean ± SD, n ≥ 3, **P < 0.005 by student's t-test).
Phospho-histone H3 (pH3) immunostaining and TUNEL assay. Embryos were fixed in 4% paraformaldehyde at 4 °C overnight. For sectioning, the embryos were embedded in OCT compound (SAKURA) and cryosectioned into 14μ m slices. After blocking with 10% FBS for 1 hr at room temperature, the sections were incubated with rabbit anti-pH3 antibody (1:100 dilution, Santa Cruz) at 4 °C overnight. Secondary antibody of Alexa Fluor 488 conjugated anti-rabbit IgG (Invitrogen) was then incubated for 1 hr at room temperature. The sections were counterstained with DAPI (Vector Labs) to label cell nuclei.
Terminal transferase UTP nick end labeling (TUNEL) was carried out on cryosections using the In Situ Cell Death Detection Kit, TMR red (Roche) according to the manufacturer's recommendations.
Quantitative PCR. Total RNA was extracted from the head region containing liver dissected from embryos at 72 hpf, and the trunks of the embryos were used for genotyping. Quantitative PCR was performed using a LightCycler 1.5 (Roche) following the manufacturer's protocol. Primers are listed in supplemental Table S1.