hepatic loss of survivin impairs postnatal liver development and promotes expansion of hepatic...

Hepatic Loss of Survivin Impairs Postnatal LiverDevelopment and Promotes Expansion of Hepatic

Progenitor Cells in MiceDan Li,1,2 Jin Cen,2 Xiaotao Chen,2 Edward M. Conway,3 Yuan Ji,4 and Lijian Hui2

Hepatocytes possess a remarkable capacity to regenerate and reconstitute the paren-chyma after liver damage. However, in the case of chronic injury, their proliferativepotential is impaired and hepatic progenitor cells (HPCs) are activated, resulting in aductular reaction known as oval cell response. Proapoptotic and survival signals main-tain a precise balance to spare hepatocytes and progenitors from hyperplasia and celldeath during regeneration. Survivin, a member of the family of inhibitor of apoptosisproteins (IAPs), plays key roles in the proliferation and apoptosis of various cell types.Here, we characterized the in vivo function of Survivin in regulating postnatal liverdevelopment and homeostasis using mice carrying conditional Survivin alleles. Hepaticperinatal loss of Survivin causes impaired mitosis, increased genome ploidy, andenlarged cell size in postnatal livers, which eventually leads to hepatocyte apoptosis andtriggers tissue damage and inflammation. Subsequently, HPCs that retain genomic Sur-vivin alleles are activated, which finally differentiate into hepatocytes and reconstitutethe whole liver. By contrast, inducible ablation of Survivin in adult hepatocytes doesnot affect HPC activation and liver homeostasis during a long-life period. Conclusion:Perinatal Survivin deletion impairs hepatic mitosis in postnatal liver development, whichinduces HPC activation and reconstitution in the liver, therefore providing a novelHPC induction model. (HEPATOLOGY 2013;58:2109-2121)

The liver possesses the remarkable capacity torestore damaged hepatic tissues after injury.1-4

During normal hepatocyte turnover or partialhepatectomy-regeneration, this is achieved by replica-tion of highly differentiated parenchymal hepatocytesin response to loss of liver mass. When the prolifera-tive capacity of mature liver cells is blocked or com-promised, a population of small portal zone cells witha high nuclear cytoplasmic ratio and an ovoid nucleus,

known as “oval cells,” proliferate extensively and differ-entiate into hepatocytes and bile duct cholangiocytesto restore damaged liver tissue.5-7 Oval cells areregarded as the facultative hepatic progenitor cells(HPCs) in adult livers.1-4

Chemically injured animal models are commonlyused to trigger oval cell activation. For example, feed-ing mice with a diet supplemented with 3,5-diethoxy-carbonyl-1,4-dihydro-collidine (DDC) is a protocol

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK19, cytokeratin 19; CPC, chromosomal passenger complex; DDC, 3,5-dieth-oxycarbonyl-1,4-dihydro-collidine; EpCAM, epithelial cell adhesion molecule; F2, fraction 2; FGF7, fibroblast growth factor 7; HGF, hepatocyte growth factor;HPC, hepatic progenitor cells; IAP, inhibitor of apoptosis proteins; IF, immunofluorescent; IHC, immunohistochemical; IP, intraperitoneally; mRNA, messengerRNA; p-H3S10, phosphorylated H3S10; PI, propidium iodide; qPCR, quantitative PCR; Sox9, SRY (sex determining region Y)-box 9; TGF-a, transforminggrowth factor alpha; TUNEL, TdT-mediated dUTP nick end labeling; TWEAK, tumor necrosis factor–related weak inducer of apoptosis.

From the 1Graduate University of Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China;2State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,Shanghai, China; 3Center for Blood Research, Division of Hematology-Oncology, Department of Medicine, University of British Columbia, Vancouver, BritishColumbia, Canada; and 4Department of Pathology, Zhongshan Hospital, Fudan University, Shanghai, China.

Received February 16, 2013; accepted June 14, 2013.L.H.’s laboratory is funded by the National Science Foundation of China (31071238), the Ministry of Science and Technology of China (2012CB945001 and

2011ZX09307-302-01), and the Chinese Academy of Sciences (the Hundred Talents Program). E.M.C. is supported by the Canadian Institutes for HealthResearch, the Canada Foundations for Innovation, and the Natural Sciences and Engineering Research Council of Canada. E.M.C. holds a CSL-Behring ResearchChair and a Canada Research Chair in Endothelial Cell Biology and is an adjunct scientist with the Canadian Blood Services (CBS). The authors thank Dr. V.Factor (National Cancer Institute, National Institutes of Health, Bethesda, MD) for A6 antibody and Dr. X. Wang (Inner Mongolia University, Inner Mongolia,China) for antibodies against CK19.

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for oval cell induction.8-10 However, chemicals causesevere, unpredictable toxicity to both hepatocytes andnonparenchymal cells. Chemical injury fails to distin-guish damaged cells from nondamaged cells during tis-sue repair, specifically newly derived hepatocytes fromactivated HPCs. Recent progresses have developedgenetic mouse models for HPC activation. Liver-specific deletion of b-catenin11 or DDB112,13 causedHPCs induction and reconstitution during hepatocyteturnover in aged mice. However, the ductular reactioneventually engenders liver tumors in b-catenin orDDB1 models. Other studies showed that liver-specificoverexpression of tumor necrosis factor–related weakinducer of apoptosis (TWEAK)14 and fibroblastgrowth factor 7 (FGF7)15 stimulated HPC prolifera-tion. Nevertheless, mature hepatocytes in these modelsretain proliferation capacity, which makes it difficult toanalyze the hepatic differentiation and reconstitutionof HPCs. Animal models that selectively target hepato-cytes for cell death and proliferation arrest, but spareprogenitor cells, would bring additional insight incharacterizing HPCs.

Survivin is a unique member of the family of inhib-itor of apoptosis proteins (IAPs), which contains a sin-gle N-terminal baculovirus IAP repeat domain.16 Asan essential regulator of mitosis, Survivin localizes tokinetochores by associating with Aurora B, INCENP,and Borealin to constitute the chromosomal passengercomplex (CPC).17,18 It was thus proposed that Survi-vin contributes to mitosis by facilitating CPC assem-bling in kinetochores and microtubule stabilization. Inline with this, Survivin depletion causes mitotic arrestand the formation of polyploid cells.19 Survivin is alsoa key player in antagonizing cell death through directlyinhibiting active caspases.16 The capacity of Survivinto inhibit active caspases likely involves cooperationwith hepatitis B X-interacting protein and X-linkedIAP.20,21

Consistent with its prominent role in cell-cycle pro-gression and cell survival, Survivin is expressed inhighly proliferating cells, such as embryonic cells andadult stem cells.22 Survivin is indispensable for onto-genesis, because Survivin-deficient mice died in uteroas a result of dys-regulated microtubule organization

and cellular degeneration.23 Using conditional alleles,it has been demonstrated that tissue-specific depletionof Survivin impaired the proliferation and maturationof hematopoietic cells, neuronal cells, endothelial cells,cardiomyocytes, and pancreatic beta-cells.24-29 In addi-tion, Survivin is considered to play a key role in tumoronset and recurrence.16 A recent study showed thatSurvivin promotes liver carcinogenesis through enhanc-ing the survival of initiated cancer cells.30 Interestingly,the in vivo function of Survivin in postnatal liverdevelopment and liver homeostasis remains largelyundefined.

Using mouse lines carrying conditional alleles, wefound that in neonatal livers, but not in adult livers,hepatocytes lacking Survivin are blocked in mitosisand display increased genome ploidy and enlarged cellsize. Moreover, loss of Survivin-deficient hepatocytesdramatically activates HPCs that retain Survivin allelesduring postnatal liver development. HPCs differentiateinto hepatocytes and eventually reconstitute the liver.Our study demonstrates that Survivin is essential forhepatocyte proliferation during postnatal liver develop-ment. Furthermore, mice with liver-specific Survivindeletion provide a novel model to study HPC induc-tion and differentiation.

Materials and MethodsMice. Alb-cre; Survivinf/f (svvDli) and Mx-cre; Survi-

vinf/f (svvDli*) were generated by crossing svvf/f mice toAlb-Cre mice31 and Mx-Cre mice,32 respectively. Liversamples were collected at indicated time points for var-ious assays.

Mouse Hepatocyte Isolation. Mouse hepatocyteswere isolated after a modified two-step perfusion andcollagenase digestion. Cell viability was determinedusing Trypan blue exclusion assay. HPCs were purifiedby Nycodenz density-gradient centrifugation, as previ-ously described.8

Histological Analysis. Liver tissue samples werefixed by paraformaldehyde and embedded in paraffin.Sections were subjected to hematoxylin and eosin(H&E), immunohistochemistry (IHC), Sirius Red,and TdT-mediated dUTP nick end labeling (TUNEL)staining.

Address reprint requests to: Lijian Hui, Ph.D., State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes forBiological Sciences, Chinese Academy of Sciences, 320 Yue-yang Road, 200031 Shanghai, China. E-mail: [email protected]; fax: 186-21-5492132.

Copyright VC 2013 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.26601Potential conflict of interest: Nothing to report.Additional Supporting Information may be found in the online version of this article.

2110 LI ET AL. HEPATOLOGY, December 2013

Cryosections and Immunofluorescence Staining.Liver tissue samples were snap-frozen in liquid nitro-gen and embedded in optimal cutting temperaturetissue-freezing media. Frozen sections were subjectedto immunofluorescent (IF) staining.

Statistical Analysis. Data were subjected to theStudent t test. P < 0.05 was considered statisticallysignificant. Data are presented as mean 6 standarddeviation.

Further detailed description of the materials andmethods used are provided in the SupportingInformation.

ResultsEfficient Deletion of Survivin in Hepatocytes of

svvDli Mice. Mice homozygous for floxed Survivin(svv) alleles26 were crossed with mice that carry Crerecombinase under the control of the rat albumin pro-moter (Alb-Cre)31 to specifically delete Survivin inhepatocytes perinatally (Fig. 1A). Alb-Cre, svvf/f (svvDli)mice were born indistinguishable from their littermates(svvf/f ) with the expected Mendelian frequency. SvvDli

mice had normal body weight (Fig. 1B). The floxedsvv alleles were only weakly deleted in neonatal livers,but efficiently deleted in adult livers, as shown by gen-otyping polymerase chain reaction (PCR; Fig. 1C). Tomonitor deletion efficiency during postnatal liverdevelopment, total liver RNA was isolated from svvDli

livers. Consistent with previous reports,22 quantitativePCR (qPCR) revealed that messenger RNA (mRNA)levels of Survivin progressively decreased in control liv-ers. Notably, expression of Survivin was almostunchanged in postnatal day 1 svvDli livers (Fig. 1D).Survivin mRNA levels were reduced by 70% at theage of 1 week, and were further decreased by 80%-90% at the age of 2 and 3 weeks, indicating a progres-sive and efficient deletion of Survivin in livers (Fig.1D). Moreover, IHC staining showed that Survivinprotein was undetectable in svvDli livers at 2 weeks ofage (Fig. 1E).

Survivin Is Required for Mitotic Transition Dur-ing Postnatal Liver Development. Although svvDli

mice did not appear ill, their livers were markedlyenlarged, with 30% increase of liver/body weight ratios(Fig. 2A). Histological inspection revealed that svvDli

hepatocytes underwent progressive hypertrophy with3-fold increase in diameter, compared to controls (Fig.2B). Single hepatocytes prepared by collagenase perfu-sion were highly polyploid (Fig. 2C,D), suggestingimpairments in their cell cycle. Interestingly, the devel-opment of bile ducts and biliary epithelial cells seemednot to be affected in svvDli livers (Supporting Fig. 1).

Previous studies have shown that Survivin is highlyexpressed in the G2/M phase and is required formitotic transition through enhancing histone H3 phos-phorylation.17,18 We analyzed hepatocyte mitosis byIHC staining of phosphorylated H3S10 (p-H3S10).The numbers of p-H3S10-positive hepatocytes weredramatically decreased in 2-week-old svvDli mice (Fig.2E). Additionally, expression levels of mitotic phasecyclins, such as cyclin A1 and cyclin B2, were signifi-cantly decreased in svvDli livers (Fig. 2F). Collectively,these data demonstrate that during postnatal liverdevelopment, svvDli hepatocytes are blocked in mitosis,leading to genome polyploidy and cell hypertrophy.Almost all hepatocytes showed hypertrophy in svvDli

livers, because each hepatocyte was proliferating duringpostnatal development.

Progressive Death of Survivin-Deficient Hepato-cytes in Young Mice. We characterized the mitoticallyarrested svvDli hepatocytes over an extended period oftime. At 2-3 months of age, hepatocytes containingapoptotic bodies were evident in svvDli livers, as shownby morphological changes and TUNEL (Fig. 3A,B).Quantification of TUNEL-positive cells showed thatcell death was initiated at the age of 8 weeks and con-tinued for several weeks (Fig. 3B). Moreover, levels ofcleaved caspase-3 were significantly increased in svvDli

livers (Fig. 3C). Expression levels of proapoptotic geneBak1 were also increased, whereas antiapoptotic genes,such as XIAP, Bruce, and ML-IAP, were reduced insvvDli livers (Fig. 3D).

Dead hepatocytes triggered infiltration of inflamma-tory cells in parenchyma of svvDli livers (Fig. 3A),resulting in elevated levels of several inflammatorycytokines (Fig. 3E). Consistent with increased hepato-cyte death and inflammation in svvDli livers, serum lev-els of alanine aminotransferase (ALT), aspartateaminotransferase (AST), total bilirubin, and total bileacid were significantly elevated in svvDli mice (Fig. 3F).These data indicate that mitotically arrested svvDli

hepatocytes undergo cell death, which consequentlytriggers liver damage and dysfunction.

Activation of HPCs in svvDli Livers. Previousstudies showed that HPCs are expanded in number torepopulate the injured liver parenchyma when the pro-liferative capacity of hepatocytes is impaired.1-4

Remarkably, in 8- to 12-week-old svvDli livers, expan-sion of small ductular cells with a high ratio ofnuclear/cytoplasm was observed (Fig. 4A). The expan-sion of small epithelial cells in svvDli livers generatedcell cords that bridged the portal tracts and delineatedthe polygonal architecture of hepatic lobules (Fig. 4A).Morphologically, extensive hyperplasia of small

HEPATOLOGY, Vol. 58, No. 6, 2013 LI ET AL. 2111

epithelial cells fits the description of facultative mouseHPCs.8

Consistent with classic characteristics of HPCs,8,9 thesmall epithelial cells stained positively with anti–cytokeratin 19 (CK19), anti-Sox9 (SRY [sex determin-ing region Y]-box 9), and A6 antibodies (Fig. 4B,C). Incontrast, expression of the CK19, Sox9, and A6 anti-gens were either restricted to bile duct epithelial cells orundetectable in control livers (Fig. 4B,C). Furthermore,genes that are enriched in HPCs were significantlyincreased in svvDli livers (Fig. 4D). Double staining ofA6 antigens and p-H3S10 demonstrated that a portion

of HPCs underwent proliferation (Fig. 4E). Further-more, quantification of proliferating cells by Ki67 stain-ing showed that all proliferating cells were HPCs in 8-week-old livers, whereas Ki67-positive hepatocytes wereundetectable (Supporting Fig. 2). In line with HPCactivation, expression of growth factors essential toHPC proliferation, such as hepatocyte growth factor(HGF), transforming growth factor alpha (TGF-a), andFGF7,15,33 were increased in svvDli livers (Fig. 4F).Taken together, these data suggest that deletion of Sur-vivin in hepatocytes causes cell death and liver damage,which triggers extensive HPC expansion.

Fig. 1. Efficient deletion of Survivin in hepatocytes of svvDli mice. (A) Schematic diagram of the svv gene locus and related alleles. The svvfloxed allele has two loxP sites (triangles) flanking the four exons (boxes). Mice with svv floxed alleles were crossed with a Cre line to generatethe deleted svv allele. K, KpnI; S, SacI; E, EcoRV restriction enzyme sites. (B) Body weights of svvDli and control svvf/f mice were measured. (C)Genotypes were determined by PCR using liver genomic DNA. (D) Survivin mRNA levels were measured by qPCR in livers at indicated ages. (E)Survivin protein levels were characterized by Survivin IHC staining in livers. Arrowheads indicate Survivin positive cells, which have brown-stainednuclei. *P < 0.05; t test. Scale bars, 50 lm.


Fig. 2. Survivin is required for hepatocyte mitosis and maintains cell size and genome ploidy. (A) Eight-week old svvDli mice showed largerlivers. (B) Hematoxylin and eosin staining of liver sections of 8-week-old mice. Hepatocyte diameters were quantified. (C) Primary hepatocytesharvested by liver perfusion. (D) The genome ploidy of hepatocytes was characterized by PI staining, followed by fluorescence-activated cell sort-ing. (E) Hepatocyte mitosis was determined by p-H3S10 IHC staining. Arrowheads indicate p-H3S10-positive cells. Ratio of p-H3S10-positivehepatocytes was quantified. (F) Cyclin A1 and cyclin B2 mRNA levels in livers of 2-week-old mice were measured by qPCR. *P < 0.05. Scalebars, 50 lm.


Fig. 3. Survivin deficiency causes hepatocyte death and inflammation. (A) Hematoxylin and eosin staining of liver sections from 12-week-old svvDli

mice. Apoptotic hepatocytes are indicated. (B) Apoptosis of hepatocytes is confirmed by TUNEL staining. Positive cells were stained green in nuclei. Theratio of TUNEL positive hepatocytes in svvDli livers was quantified. (C) Cleaved caspase-3 was detected by western blotting. (D) Expression levels ofcell-death–related genes and IAP family genes were measured by qPCR. (E) mRNA and serum levels of TNFa, IFN-c, and IL-6 in svvDli livers were deter-mined by qPCR and ELISA. (F) Serum ALT, AST, total bilirubin, and bile acid levels were measured in svvf/f and svvDli mice at indicated ages. *P <0.05. Scale bars, 50 lm. TNFa, tumor necrosis factor alpha; IFN-c, interferon-gamma; IL-6, interleukin-6; ELISA, enzyme-linked immunosorbent assay.


Fig. 4. HPC activation in postnatal svvDli livers. (A) Hematoxylin and eosin staining of liver sections from 12-week-old svvDli mice. Anincreased number of oval cells, defined as small cells with an oval nucleus and scant cytoplasm, spreading from the periportal region wasobserved. (B and C) Co-IF staining of HPC markers A6 antigen, CK19, and Sox9 on frozen liver sections from 12-week-old svvDli mice. Cells dou-ble positive for either A6 antigen and CK19 (B) or A6 antigen and Sox9 (C) were identified. (D) mRNA levels of HPC marker genes were meas-ured by qPCR. (E) Co-IF staining of proliferation marker p-H3S10 and HPC marker A6 antigen in livers of 12-week-old svvDli mice. (F) mRNAlevels of growth factors were quantified by qPCR. *P < 0.05. Scale bars, 50 lm.


Activated HPCs in svvDli Livers Are Comparableto Those in the DDC Model. We compared HPCsinduced in svvDli mice at the age of 8-12 weeks tothose in DDC-treated svvf/f livers. HPCs positive forboth A6 antigens and CK19 were induced in peripor-tal areas 3 weeks after DDC treatment (Fig. 5A).CK19-positive HPCs induced in svvDli and DDC-treated livers had similarly scant cytoplasm, ovoidnuclei (Fig. 5B), and increased collagen deposition inthe extracellular matrix (Fig. 5C). HPCs in bothmodels were organized in duct-like structures in theperiportal region and extended into the liver lobule.The duct-like structures were usually irregularlyshaped with poorly defined lumen. Intriguingly,expression levels of genes enriched in HPCs werecomparable in both svvDli livers and DDC-treated liv-ers (Fig. 5D).

HPCs were purified by Nycodenz density gradientcentrifugation, as previously described.8 Three fractionsof nonparenchymal cell populations were obtained,and HPCs were enriched in fraction 2 (F2)-containingsmall cells of 7-10 lm in diameter. Cell numbers ofF2 were significantly increased in both svvDli livers andDDC-treated livers, with highly elevated expression ofepithelial cell adhesion molecule (EpCAM), CD133,and CD44 (Fig. 5E). Importantly, the majority of F2cells were positive for both A6 antigens and CK19(Fig. 5F). Collectively, these data suggest that in svvDli

livers, HPCs are activated and form pathological struc-tures comparable to HPCs in DDC-treated livers.

HPCs Reconstitute the Whole Liver Without Car-cinogenesis in Aged svvDli Mice. The expansion anddifferentiation of HPCs were progressive, yieldingregenerative hepatic nodules containing hepatocytes ofnormal size at periportal regions in livers of 12- to 14-week-old svvDli mice (Supporting Fig. 3A). Strikingly,hepatocytes in these regenerative nodules were prolifer-ating, as determined by Ki67 staining (Supporting Fig.3B). In line with reports that floxed alleles were ineffi-ciently deleted by Cre recombinase in HPC-derivedregenerative livers,11,12 IHC staining revealed that Sur-vivin protein was expressed in the nodules (SupportingFig. 3C). We microdissected tissues containing hyper-trophic hepatocytes and regenerative nodules contain-ing normal-sized hepatocytes from svvDli liver sections.Though floxed Survivin alleles were completely deletedin liver tissues containing hypertrophic hepatocytes,genotyping by PCR revealed that Survivin was mostlyundeleted in regenerative nodules (Supporting Fig.3D).

The long-term effects of HPC expansion andreconstitution were assessed in aged svvDli mice. At

the age of 10 months, svvDli mice showed normalliver size (Fig. 6A) with poorly deleted Survivin (Fig.6B). ALT, AST, and total bilirubin in svvDli micereverted to control levels (Fig. 6C), indicating thatliver function recovered in aged svvDli mice. Impor-tantly, histological analysis showed that hepatocytesize in 10-month-old svvDli mice was the same as thatobserved in svvf/f livers (Fig. 6D). Propidium iodide(PI) staining also showed a comparable spectrum ofhepatocyte ploidy in aged svvDli and control mice(Fig. 6E). Intriguingly, liver function and hepatocytesize were already recovered in 4-month-old svvDli

mice (Supporting Fig. 4A-C), suggesting that HPC-derived hepatocytes could reconstitute the liver effi-ciently within 1-2 months.

HPC expansion has been reported to cause tumorformation in aged mice.11,12 However, despite the liverdamage observed at the age of 8-12 weeks, svvDli micesurvived as long as control mice (Supporting Fig. 5A).Moreover, the liver morphology and function in 18-month-old svvDli mice remained the same as that incontrols (Supporting Fig. 5B,C). Importantly, notumors have been found in livers of svvDli mice at theage of 18 months.

Survivin Is Dispensable in Adult Livers. Giventhat Survivin-expressing HPCs repopulated liver insvvDli mice, it was not possible to address whetherSurvivin is required in adult livers. To that end, wegenerated an inducible mouse line, Mx-Cre; svvf/f.Survivin can be deleted in liver and hematopoieticcells in adult mice through intraperitoneal (IP) injec-tion of poly(IC).29 To avoid the mortality induced bySurvivin deficiency in hematopoietic cells,29 Mx-Cre;svvf/f mice were gamma-irradiated and transplantedwith littermate svvf/f bone marrows (Fig. 7A). Afterreconstitution of the hematopoietic system, Mx-Cre;svvf/f mice were injected with poly(IC) to induceliver-specific Survivin deletion (referred to as svvDli*;Fig. 7A). All svvDli* mice survived poly(IC) treatmentand appeared healthy. Six months after poly(IC) treat-ment, the floxed Survivin gene was efficiently deletedin svvDli* livers (Fig. 7B). SvvDli* mice displayedunchanged liver size and serum levels of ALT andAST (Fig. 7C,D). Also, hepatocyte size was notincreased, and no ductular reaction was detected insvvDli* livers (Fig. 7E). Although a slight, but not sig-nificant, increase in the number of tetraploid hepato-cytes was detected, the spectrum of hepatocyte ploidywas similar in svvDli* and control mice (Fig. 7F).These results suggested that Survivin is dispensablefor the normal function of mature hepatocytes inadult mice.


Fig. 5. HPCs in svvDli livers are similar to those induced in DDC model. (A) Co-IF staining of HPC markers A6 antigen and CK19 in DDC-treated svvf/f livers. (B) CK19 IHC staining in 12-week-old svvDli livers and DDC-treated svvf/f livers. (C) Sirius Red staining in 12-week-old svvDli

livers and DDC-treated svvf/f livers. (D) mRNA levels of HPC marker genes were measured by qPCR in 12-week-old mice. n 5 5 for each group.(E) HPCs were harvested by Nycodenz density-gradient centrifugation. The cell numbers of three fractions (F1, F2, and F3) were counted. mRNAlevels of epithelial cell adhesion molecule (EpCAM), CD133, and CD44 in F2 cells were quantified by qPCR. (F) Expression of HPC markers A6antigen and CK19 in F2 cells was assessed by co-IF staining. *P < 0.05. Scale bars, 50 lm.


Fig. 6. HPCs reconstitute aged svvDli livers. (A) Liver size of 10-month-old svvDli mice was normal. (B) Deletion efficiency of Survivin in liverswas determined by genotyping PCR using liver genomic DNA extracted from 10-month-old mice. (C) Serum ALT, AST, and total bilirubin levelswere measured. (D) Hematoxylin and eosin staining shows normal liver morphology of 10-month-old svvDli mice. Hepatocyte diameters werequantified. (E) The genome ploidy of hepatocytes in 10-month-old mice was assessed by PI staining, followed with fluorescence-activated cellsorting sorting. Scale bars, 50 lm.


Discussion

In this study, we demonstrated that Survivin isessential for postnatal liver development. Survivin defi-

ciency results in hepatocyte mitotic arrest, hypertrophy,and genome polyploidy. Furthermore, mitoticallyarrested Survivin-deficient hepatocytes undergo cell

Fig. 7. Survivin is dispensable for adult liver homeostasis. (A) Mx-Cre; Survivinf/f mice were treated by gamma-irradiation, followed by trans-plantation with svvf/f bone marrows. Mx-Cre; Survivinf/f mice were then injected with poly(IC) IP to induce liver-specific Survivin gene deletion(svvDli*). (B) Deletion efficiency of Survivin 6 months after poly(IC) treatment was determined by genotyping PCR using liver genomic DNA. (C)Aged svvDli* mice showed normal liver size. (D) Serum ALT and AST levels in aged svvDli* mice were measured. (E) Hematoxylin and eosin stain-ing shows normal liver morphology in aged svvDli* mice. (F) Genome ploidy of hepatocytes in aged svvDli* mice was analyzed by fluorescence-activated cell sorting sorting. Scale bars, 50 lm.


death, which subsequently stimulates Survivin-positiveHPCs to proliferate and reconstitute svvDli livers. Inaged svvDli mice, the liver is completely repopulated byHPC-derived hepatocytes retaining Survivin andappears normal, in comparison to the control. Deple-tion of Survivin in adult livers has no obvious effecton liver homeostasis and HPC activation. These datasupport the notion that liver-specific deletion of Survi-vin could serve as a novel progenitor induction modelduring postnatal liver development.

During the course of hepatocyte polyploidization,we detected extensive apoptosis of hepatocytes withincreased DNA content in 8- to 12-week-old svvDli

mice. Intriguingly, Survivin was already efficientlydeleted in hepatocytes of svvDli mice by approximately2 weeks. Therefore, apoptosis in svvDli livers was notdirectly caused by Survivin deficiency. It is likely thatmitotic defects and polyploidy pose a stress on prolif-erating hepatocytes and eventually lead to cell death.In support of this hypothesis, depletion of Survivin inadult livers did not cause cell death in svvDli* livers.This finding would be of particular importance in tar-geting Survivin for human cancer treatment. Becauseof its essential role in normal cell survival and division,Survivin targeting has been always viewed with cau-tion.34,35 Our results unambiguously demonstrate thatSurvivin is dispensable for adult liver homeostasis,therefore suggesting that local inactivation of Survivinis safe for liver cancer treatment. We recognize thatalthough Survivin deletion did not affect adult liverfunction, it is possible that Survivin deficiency mightresult in impaired proliferation in pathologies, such aspartial hepatectomy and tumorigenesis.

Extensive hepatocyte apoptosis in svvDli livers causedloss of liver mass and inflammation. The cell deathlasted for more than 1 month, and this level of liverdamage should trigger remarkable liver regeneration.Survivin-deficient hepatocytes lost their proliferativecapacity and could not restore the liver mass. Instead,HPCs were activated to proliferate and expand innumber. Onset of HPC induction occurred at approxi-mately 8 weeks. Interestingly, cell death was still unde-tectable at the age of 6 weeks, suggesting that HPCinduction and cell death are closely coupled eventsduring onset of tissue regeneration. Proliferative HPCswere found to express Survivin, suggesting that theyescaped Cre recombinase-mediated excision of floxedalleles, as previously reported.11,12 The Alb-Cre trans-gene uses the promoter and upstream enhancer of therat albumin gene.31 It is likely that the rat albuminpromoter does not respond identically to that of themouse.

Notably, the liver was completely repopulated byHPC-derived hepatocytes in aged svvDli mice. In theDDC model, liver parenchyma was only partiallyreplaced by HPC-derived hepatocytes,36 which made itdifficult to examine the differentiation capacity of theHPCs and their contribution to the restoration of liverparenchyma. Unlike chemicals, genetic ablation of Sur-vivin is restricted to hepatocytes and leaves nonparen-chymal cells, including HPCs, intact. Therefore, thismodel is ideal for analyzing the extracellular signalsthat mediate cross-talk between the dying hepatocytesand activated HPCs. Indeed, our study indicate thatliver growth factors, which play important roles inHPC induction, are increased in svvDli livers, includingHGF, TGF-a, and FGF7.15,33

Ductular reaction by wild-type HPCs has beendocumented in genetically modified mouse lines.Liver-specific overexpression of TWEAK14 andFGF715 stimulated HPC activation. However, unlikeSurvivin-deficient hepatocytes that are mitoticallyarrested, mature hepatocytes in these models possessproliferation capacity. Liver-specific DDB1 deletionabrogates the proliferative capacity of hepatocytes,resulting in proliferation of DDB1-expressing HPCs.Intriguingly, these HPCs were activated in the micro-environment with minimal tissue damage.12,13 Anotherstudy showed that hepatocyte turnover was compro-mised in livers lacking b-catenin, leading to b-catenin-positive HPC proliferation and liver mass reconstitu-tion at the age of 11-18 months.11 Because of the longperiod of ductular reaction, it was difficult to definewhen b-catenin-proficient HPCs were activated toreconstitute the liver. By contrast, in Survivin-deficientlivers, hepatocytes undergo cell death within 8-12weeks of age, thereby providing a clear onset of pro-genitor activation. Moreover, the repopulation ofHPC-derived hepatocytes is fast and complete inSurvivin-deficient livers. By the age of 4 months, thewhole liver was almost reconstituted by newly gener-ated hepatocytes.

Remarkably, in DDB1-deficient mice, ductular reac-tion appeared at the age of 4 weeks and lasted for along time. Liver tumors were eventually found at theage of 16-20 months. In b-catenin-deficient mice,ductular reaction was evident at approximately 11months of age, and this led to the development ofliver tumors at 18-20 months. In both models, pro-longed ductular reaction may cause the accumulationof mutations in HPCs in aged livers. However, in ourstudy, no tumors were found in aged svvDli livers. Weobserved that HPC-derived hepatocytes reconstitutedyoung svvDli livers within 1-2 months, and liver


function was well maintained thereafter. The differentductular reactions in these models might explain whysvvDli mice did not develop liver tumors. Together, ourstudy provides a new model with a defined process ofactivation, differentiation, and repopulation of HPCs.

Acknowledgment: The authors thank P. Huang fortechnical support. The authors also thank the AnimalCore Facility and Cell Biology Core Facility at Shang-hai institute of Biochemistry and Cell Biology forexperimental support.

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