Postnatal Liver Development Research Articles

Energy restriction affect liver development in Hu sheep ram lambs through Hippo signaling pathway.

This study aimed to evaluate the effects of dietary energy restriction on postnatal liver development in Hu sheep ram lambs. A total of 16 ram lambs were randomly divided into two groups: 100% energy requirements diet and 55% energy requirements diet, which were fed for 75 d. Results showed that the final body and liver weights decreased with energy restriction (p <0.05). Energy restriction caused a significant decrease in the levels of circulating insulin-like growth factor 1 (IGF1) and an increase in growth hormone secretion (p <0.05), which can be explained by the decreased mRNA expression levels of the growth hormone receptor (GHR) and IGF1 (p <0.05). The proliferating cell nuclear antigen (PCNA), Ki-67 and apoptosis-related proteins (BAX and BCL2) were mainly located in the nucleus and the cytoplasm of hepatocytes, respectively. The transcription and protein levels of PCNA and BAX were significantly decreased and increased by energy restriction, respectively (p <0.05). The caspase9 and caspase3 mRNA and activity were increased in energy restriction group (p <0.05). Moreover, Hippo signaling pathway proteins [mammalian sterile 20-like protein kinase 1 (MST1), large tumor suppressor kinase 1 (LATS1), and yes-associated protein 1 (YAP1)] were mainly observed around the hepatic portal area, and the expression levels of their mRNA and proteins were significantly decreased in energy restriction group (p <0.05). In summary, energy restriction in ram lambs impairs liver development by increasing apoptosis, which may occur via the Hippo signaling pathway.

Expression profiling of adhesion proteins during prenatal and postnatal liver development in rats.

Culturing of primary hepatocytes and stem cell-derived hepatocytes faces a major issue of dedifferentiation due to absence of cell–cell adhesion and 3D structures. One of the possible ways to eliminate the problem of dedifferentiation is mimicking the expression pattern of adhesion proteins during the normal developmental process of liver cells. The purpose of this study was to evaluate the expression pattern of some key adhesion proteins, namely, E-cadherin, N-cadherin, epithelial CAM (EpCAM), intracellular CAM (ICAM), collagen 1α1, α-actinin, β-catenin and vimentin, in the liver tissue during prenatal and postnatal stages. Furthermore, differences in their expression between prenatal, early postnatal and adult stages were highlighted. Wistar rats were used to isolate livers at prenatal Day 14 and 17 as well as on postnatal Day 1, 3, 7 and 14. The liver from adult rats was used as control. Both conventional and real-time quantitative polymerase chain reactions (PCRs) were performed. For most of the adhesion proteins such as E-cadherin, N-cadherin, EpCAM, ICAM, collagen 1α1 and α-actinin, low expression was observed around prenatal Day 14 and an increasing expression was observed in the postnatal period. Moreover, β-catenin and vimentin showed higher expression in the early prenatal period, which decreased gradually in the postnatal period, but still this low expression was considerably higher than that in the adult control rats. This basic knowledge of the regulation of expression of adhesion proteins during different developmental stages indicates their vital role in liver development. This pattern can be further studied and imitated under in vitro conditions to achieve better cell–cell interactions.

MRNA N6-methyladenosine methylation of postnatal liver development in pig.

N6-methyladenosine (m6A) is a ubiquitous reversible epigenetic RNA modification that plays an important role in the regulation of post-transcriptional protein coding gene expression. Liver is a vital organ and plays a major role in metabolism with numerous functions. Information concerning the dynamic patterns of mRNA m6A methylation during postnatal development of liver has been long overdue and elucidation of this information will benefit for further deciphering a multitude of functional outcomes of mRNA m6A methylation. Here, we profile transcriptome-wide m6A in porcine liver at three developmental stages: newborn (0 day), suckling (21 days) and adult (2 years). About 33% of transcribed genes were modified by m6A, with 1.33 to 1.42 m6A peaks per modified gene. m6A was distributed predominantly around stop codons. The consensus motif sequence RRm6ACH was observed in 78.90% of m6A peaks. A negative correlation (average Pearson’s r = -0.45, P < 10−16) was found between levels of m6A methylation and gene expression. Functional enrichment analysis of genes consistently modified by m6A methylation at all three stages showed genes relevant to important functions, including regulation of growth and development, regulation of metabolic processes and protein catabolic processes. Genes with higher m6A methylation and lower expression levels at any particular stage were associated with the biological processes required for or unique to that stage. We suggest that differential m6A methylation may be important for the regulation of nutrient metabolism in porcine liver.

Role of Farnesoid X Receptor in the Determination of Liver Transcriptome during Postnatal Maturation in Mice.

The liver is a vital organ with critical functions in metabolism of various biologically useful materials, synthesis of several vital proteins, detoxification of toxic substances, and immune defense. Most liver functions are not mature at birth and many changes happen during postnatal liver development, which lead to differential vulnerabilities of the liver at different developmental stages. However, the details of what changes occur in liver after birth, at what developmental stages they occur, and molecular mechanisms in the regulation of the developmental process are not clearly known. The nuclear receptor Farnesoid X receptor (FXR) is an important transcriptional regulator in liver. Here, we used RNA-Sequencing to analyze the transcriptome of mouse liver from perinatal to adult ages in both C57BL/6 and Fxr−/− mice. We have defined a clear timeline of functional transition from prenatal through neonatal and adolescent to adult in C57BL/6 mice. Without FXR, activation of neonatal-specific pathways was prolonged and maturation of multiple metabolic pathways was delayed. The loss of FXR also led to increased expression of 27 other transcription regulators. Our data support a conclusion that developmental transcriptome revealed significant functional transition during postnatal liver development and FXR plays an important role in control of postnatal liver maturation.

Abstract IA16: Breaking the balance between E2F activators and atypical repressors: Consequences to development and cancer

Abstract E2F transcriptional activators and repressors regulate cell cycle-dependent gene expression. Using a series of loss- and gain-of-function alleles to dial E2F transcriptional output we show that mouse development is relatively insensitive to alterations in E2F levels and composition. However, modest increases in E2F output resulted in spontaneous hepatocellular carcinoma (HCC) without additional organ involvement, whereas decreases protected against HCC. Cell type- and temporal-specific gene ablation strategies defined a cell-autonomous oncogenic role for E2F1/3B and a tumor suppressor role for E2F7/8 in hepatocytes. Genetic, biochemical and profiling studies suggest a mechanism involving E2F1/3B-mediated activation and E2F7/8-mediated repression of a common set of targets during early post-natal liver development, which are associated with HCC progression. Thus, while overall development is sufficiently robust, the liver is uniquely sensitive to perturbations in E2Fs. In summary, we identified the critical network components in immature hepatocytes that control E2F output and foster a cancer-free life span. Citation Format: Gustavo W. Leone. Breaking the balance between E2F activators and atypical repressors: Consequences to development and cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr IA16.

Hepatic Loss of Borealin Impairs Postnatal Liver Development, Regeneration, and Hepatocarcinogenesis

Borealin, a member of the chromosomal passenger complex, plays a key regulatory role at centromeres and the central spindle during mitosis. Loss of Borealin leads to defective cell proliferation and early embryonic lethality. The in vivo functions of Borealin in mammalian postnatal development, tissue homeostasis, and tumorigenesis remain elusive. We specifically analyzed the role of Borealin in regulating postnatal liver development, damage-induced liver regeneration, and liver carcinogenesis using mice carrying conditional Borealin alleles. Perinatal loss of Borealin caused increased genome ploidy and enlarged cell size in hepatocytes, likely due to the impaired function of the chromosomal passenger complex in mitosis. Borealin deletion also showed attenuated expansion of Sox9+HNF4α+ progenitor-like cells in liver regeneration during 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet-induced liver injury. Moreover, ΔN90-β-Catenin and c-Met-induced hepatocarcinogenesis development was largely impeded by Borealin deletion. These findings indicate that Borealin plays a key role in liver development, regeneration, and tumorigenesis and suggests that Borealin could be a potential target for related liver diseases.

E2f8 mediates tumor suppression in postnatal liver development.

E2F-mediated transcriptional repression of cell cycle-dependent gene expression is critical for the control of cellular proliferation, survival, and development. E2F signaling also interacts with transcriptional programs that are downstream of genetic predictors for cancer development, including hepatocellular carcinoma (HCC). Here, we evaluated the function of the atypical repressor genes E2f7 and E2f8 in adult liver physiology. Using several loss-of-function alleles in mice, we determined that combined deletion of E2f7 and E2f8 in hepatocytes leads to HCC. Temporal-specific ablation strategies revealed that E2f8's tumor suppressor role is critical during the first 2 weeks of life, which correspond to a highly proliferative stage of postnatal liver development. Disruption of E2F8's DNA binding activity phenocopied the effects of an E2f8 null allele and led to HCC. Finally, a profile of chromatin occupancy and gene expression in young and tumor-bearing mice identified a set of shared targets for E2F7 and E2F8 whose increased expression during early postnatal liver development is associated with HCC progression in mice. Increased expression of E2F8-specific target genes was also observed in human liver biopsies from HCC patients compared to healthy patients. In summary, these studies suggest that E2F8-mediated transcriptional repression is a critical tumor suppressor mechanism during postnatal liver development.

The Temporal Expression and Regulation of an Imprinted Gene Network during Postnatal Liver Development in Mice

Postnatal liver maturation involves a unique process after birth, in which hepatocytes proliferate and differentiate simultaneously until the organ gains a mature gene expression profile and adult hepatic functions. During this developmental period, most liver functions, including drug metabolism, are not fully mature as the genes involved are not expressed at adult levels. It is known that the neonatal liver undergoes extensive changes in its transcriptome during early life as it matures from a hematopoietic to a metabolic organ, however genetic mechanisms regulating postnatal liver maturation are largely unknown. The goal of our study is to understand how an imprinted gene network controls organ growth and hepatocyte proliferation and promotes the expression of mature hepatic genes during postnatal liver maturation. Mouse is selected as a model. RNA‐Seq was performed on mouse livers collected at 6 ages of postnatal development: days ‐2 (day 17 of gestation), 1, 5, 20, 25, and 60 (adult). A total of 88 genes that were previously proven to be imprinted in mice, meaning they have monoallelic expression, were selected for further analysis. A total of 46 of these genes were found to be expressed in the liver during at least one of the ages. The majority of expressed genes (27) had decreasing expression with age, showing high expression levels closer to birth, then little to no expression in adult ages, with a coordinated notable decrease in expression at day 20. Imprinted genes with the highest expression at neonatal ages included the long non‐coding RNA (lncRNA) H19 and its neighborhood genes, along with another lncRNA maternally expressed 3 (Meg3) and the genes in its cluster. Generally, the majority of imprinted genes are found in clusters on the chromosomes. After examining the expression of imprinted genes in each cluster, it was found that groups of genes with decreasing expression with age all contained an imprinted lncRNA, suggesting that lncRNAs may be critical for controlling the coordinate decline of many imprinted genes levels during postnatal development. To investigate a possible factor responsible for the decline of many imprinted genes with age, we also performed RNA‐Seq on farnesoid X receptor (Fxr) null mice at matching ages to wild type mice. FXR is a nuclear receptor involved in bile acid homeostasis that increases in expression postnatally and is expressed at high levels in adults. We found that Fxr null mice have significantly higher expression of 28 imprinted genes in liver during neonatal ages. H19 and Meg3 showed the greatest increase in expression, suggesting FXR may play a role, possibly through the activation of small heterodimer partner (SHP), in repressing the expression of many imprinted genes. The expression of imprinted genes in the liver seems to be highly coordinated with both the age of mice and their inclusion in certain clusters on chromosomes. Imprinted genes are known to be critical for mammalian development, and we suggest they may control the maturation of the postnatal liver. Our study also suggests that lncRNAs and the nuclear receptor FXR play a role in coordinating the expression patterns of imprinted genes with age and therefore can have a significant effect on liver development and function.Support or Funding InformationNIH/NIEHS R01ES019487

Developmental programming of long non-coding RNAs during postnatal liver maturation in mice.

The liver is a vital organ with critical functions in metabolism, protein synthesis, and immune defense. Most of the liver functions are not mature at birth and many changes happen during postnatal liver development. However, it is unclear what changes occur in liver after birth, at what developmental stages they occur, and how the developmental processes are regulated. Long non-coding RNAs (lncRNAs) are involved in organ development and cell differentiation. Here, we analyzed the transcriptome of lncRNAs in mouse liver from perinatal (day −2) to adult (day 60) by RNA-Sequencing, with an attempt to understand the role of lncRNAs in liver maturation. We found around 15,000 genes expressed, including about 2,000 lncRNAs. Most lncRNAs were expressed at a lower level than coding RNAs. Both coding RNAs and lncRNAs displayed three major ontogenic patterns: enriched at neonatal, adolescent, or adult stages. Neighboring coding and non-coding RNAs showed the trend to exhibit highly correlated ontogenic expression patterns. Gene ontology (GO) analysis revealed that some lncRNAs enriched at neonatal ages have their neighbor protein coding genes also enriched at neonatal ages and associated with cell proliferation, immune activation related processes, tissue organization pathways, and hematopoiesis; other lncRNAs enriched at adolescent ages have their neighbor protein coding genes associated with different metabolic processes. These data reveal significant functional transition during postnatal liver development and imply the potential importance of lncRNAs in liver maturation.

Downregulation of Sox9 Expression Associates with Hepatogenic Differentiation of Human Liver Mesenchymal Stem/Progenitor Cells

Understanding the mechanisms triggering hepatogenic differentiation of stem/progenitor cells would be useful for studying postnatal liver regeneration and development of liver cell therapies. Many evidences support the involvement of Sox9 transcription factor in liver development. Here, we investigate the possibility of liver mesenchymal stem/progenitor cells to constitutively express Sox9 by using reverse transcription-quantitative polymerase chain reaction, immunocytochemistry, and western blotting. The involvement of Sox9 in hepatogenic differentiation was assessed by following its expression at different steps of the process, evaluating the impact of its altered expression, and analyzing its expression in human liver disease specimen. Liver mesenchymal stem/progenitor cells constitutively express Sox9 at both the mRNA and protein levels. Upon hepatogenic differentiation, Sox9 expression is downregulated mainly in the maturation step after oncostatin M treatment. Induction of Sox9 expression using transforming growth factor beta is accompanied with a decrease of the quality of hepatogenic differentiation. Blunting Sox9 expression using specific ShRNA clearly alters the levels of several hepatic markers, an effect confirmed in HepG2 cells. In human liver disease specimen, Sox9 expression is enhanced at both the mRNA and protein levels compared with healthy donors. The current data demonstrate that Sox9 may play a pivotal role in hepatocyte lineage development, including adult liver mesenchymal stem/progenitor cells. Further studies on the identification of pathways regulated by or regulating Sox9 will certainly gain insight into the molecular networks controlling hepatogenic differentiation.

Hepatic loss of survivin impairs postnatal liver development and promotes expansion of hepatic progenitor cells in mice

Hepatocytes possess a remarkable capacity to regenerate and reconstitute the parenchyma after liver damage. However, in the case of chronic injury, their proliferative potential is impaired and hepatic progenitor cells (HPCs) are activated, resulting in a ductular reaction known as oval cell response. Proapoptotic and survival signals maintain a precise balance to spare hepatocytes and progenitors from hyperplasia and cell death during regeneration. Survivin, a member of the family of inhibitor of apoptosis proteins (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 liver development and homeostasis using mice carrying conditional Survivin alleles. Hepatic perinatal loss of Survivin causes impaired mitosis, increased genome ploidy, and enlarged cell size in postnatal livers, which eventually leads to hepatocyte apoptosis and triggers tissue damage and inflammation. Subsequently, HPCs that retain genomic Survivin alleles are activated, which finally differentiate into hepatocytes and reconstitute the whole liver. By contrast, inducible ablation of Survivin in adult hepatocytes does not affect HPC activation and liver homeostasis during a long-life period. Perinatal Survivin deletion impairs hepatic mitosis in postnatal liver development, which induces HPC activation and reconstitution in the liver, therefore providing a novel HPC induction model.

The time point of β-catenin knockout in hepatocytes determines their response to xenobiotic activation of the constitutive androstane receptor

The constitutive androstane receptor (CAR) controls the expression of drug-metabolizing enzymes and regulates hepatocyte proliferation. Studies with transgenic mice with an early postnatal conditional hepatocyte-specific knockout of the β-catenin gene Ctnnb1 revealed that β-catenin deficiency decreases the magnitude of induction of drug-metabolizing enzymes by CAR activators, abrogates zonal differences in the hepatocytes’ susceptibility to these compounds, and impacts on hepatocyte proliferation. These data, however, do not allow distinguishing between effects caused by β-catenin deficiency during postnatal liver development and acute effects of β-catenin deficiency in the adult animal at the time point of CAR activation. Therefore, CAR activation was now studied in a different mouse model allowing for the hepatocyte-specific knockout of β-catenin in adult mice. Treatment of these mice with 3mg/kg body weight of the model CAR activator 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) confirmed previous findings related to the coordinate regulation of drug metabolism by β-catenin and CAR. More importantly, the present study clarified that the impact of β-catenin signaling on CAR-mediated enzyme induction in the liver is not merely due to developmental defects caused by a postnatal lack of β-catenin, but depends on the presence of β-catenin at the time point of xenobiotic treatment. The study also revealed interesting differences between the two mouse models: hepatic zonation of TCPOBOP-dependent induction of drug-metabolizing enzymes was restored in mice with late knockout of β-catenin, and the strong proliferative response of female mice was exclusively abolished when using animals with a late β-catenin knockout. This suggests a β-catenin-dependent postnatal priming of hepatocytes during postnatal liver development, later affecting the proliferative response of adult animals to CAR-activating xenobiotics.

RNA-Sequencing Quantification of Hepatic Ontogeny and Tissue Distribution of mRNAs of Phase II Enzymes in Mice

Phase II conjugating enzymes play key roles in the metabolism of xenobiotics. In the present study, RNA sequencing was used to elucidate hepatic ontogeny and tissue distribution of mRNA expression of all major known Phase II enzymes, including enzymes involved in glucuronidation, sulfation, glutathione conjugation, acetylation, methylation, and amino acid conjugation, as well as enzymes for the synthesis of Phase II cosubstrates, in male C57BL/6J mice. Livers from male C57BL/6J mice were collected at 12 ages from prenatal to adulthood. Many of these Phase II enzymes were expressed at much higher levels in adult livers than in perinatal livers, such as Ugt1a6b, -2a3, -2b1, -2b5, -2b36, -3a1, and -3a2; Gsta1, -m1, -p1, -p2, and -z1; mGst1; Nat8; Comt; Nnmt; Baat; Ugdh; and Gclc. In contrast, hepatic mRNA expression of a few Phase II enzymes decreased during postnatal liver development, such as mGst2, mGst3, Gclm, and Mat2a. Hepatic expression of certain Phase II enzymes peaked during the adolescent stage, such as Ugt1a1, Sult1a1, Sult1c2, Sult1d1, Sult2as, Sult5a1, Tpmt, Glyat, Ugp2, and Mat1a. In adult mice, the total transcripts for Phase II enzymes were comparable in liver, kidney, and small intestine; however, individual Phase II enzymes displayed marked tissue specificity among the three organs. In conclusion, this study unveils for the first time developmental changes in mRNA abundance of all major known Phase II enzymes in mouse liver, as well as their tissue-specific expression in key drug-metabolizing organs. The age- and tissue-specific expression of Phase II enzymes indicate that the detoxification of xenobiotics is highly regulated by age and cell type.

Hepatic ontogeny and tissue distribution of mRNAs of epigenetic modifiers in mice using RNA-sequencing

Developmental regulation of gene expression is controlled by distinct epigenetic signatures catalyzed by various epigenetic modifiers. Little is known about the ontogeny and tissue distribution of these epigenetic modifiers. In the present study, we used a novel approach of RNA-sequencing to elucidate hepatic ontogeny and tissue distribution of mRNA expression of 142 epigenetic modifiers, including enzymes involved in DNA methylation/demethylation, histone acetylation/deacetylation, histone methylation/demethylation, histone phosphorylation and chromosome remodeling factors in male C57BL/6 mice. Livers from male C57BL/6 mice were collected at 12 ages from prenatal to adulthood. Many of these epigenetic modifiers were expressed at much higher levels in perinatal livers than adult livers, such as Dnmt1, Dnmt3a, Dnmt3b, Apobec3, Kat1, Ncoa4, Setd8, Ash2l, Dot1l, Cbx1, Cbx3, Cbx5, Cbx6, Ezh2, Suz12, Eed, Suv39h1, Suv420h2, Dek, Hdac1, Hdac2, Hdac7, Kdm2b, Kdm5c, Kdm7, Prmt1–5, Prmt7, Smarca4, Smarcb1, Chd4 and Ino80e. In contrast, hepatic mRNA expression of a few epigenetic modifiers increased during postnatal liver development, such as Smarca2, Kdm1b, Cbx7 and Chd3. In adult mice (60 d of age), most epigenetic modifiers were expressed at moderately (1–3-fold) higher levels in kidney and/or small intestine than liver. In conclusion, this study, for the first time, unveils developmental changes in mRNA abundance of all major known epigenetic modifiers in mouse liver. These data suggest that ontogenic changes in mRNA expression of epigenetic modifiers may play important roles in determining the addition and/or removal of corresponding epigenetic signatures during liver development.

Neighbor of Punc E 11: Expression Pattern of the New Hepatic Stem/Progenitor Cell Marker During Murine Liver Development

We have previously identified Neighbor of Punc E 11 (Nope) as a specific cell surface marker of stem/progenitor cells in the murine fetal liver that is also expressed in hepatocellular carcinoma. Here, we focus on the differential expression pattern of Nope during murine fetal and postnatal liver development as well as in a normal and regenerating adult liver including oval cell activation. In the fetal liver, Nope shows a constantly high expression level and is a useful surface marker for the identification of Dlk, E-cadherin, and CD133-positive hepatoblasts by flow cytometry. Postnatally, Nope expression declines rapidly and remains barely detectable in the adult liver as shown by quantitative real-time reverse-transcriptase polymerase chain reaction and western blot analyses. Immunohistochemically, costainings for Nope- and epithelial-specific markers (E-cadherin), markers of early hepatoblasts (alpha-fetoprotein), and biliary marker proteins (CK19) demonstrate that Nope is initially expressed on bipotent hepatoblasts and persists thereafter on commited hepatocytic as well as cholangiocytic progenitor cells during late fetal liver development. Postnatally, Nope loses its circular expression pattern and is specifically directed to the sinusoidal membrane of early hepatocytes. While Nope is only weakly expressed on cholangiocytes in the normal adult liver, activated stem/progenitor (oval) cells clearly coexpress Nope together with the common markers A6, EpCAM, and CD24 in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse model. In conclusion, Nope should be most useful in future research to define the differentiation stage of hepatic-specified cells of various sources and is a promising candidate to identify and isolate hepatic stem cells from the adult liver.

Sex-specific mouse liver gene expression: genome-wide analysis of developmental changes from pre-pubertal period to young adulthood

BackgroundEarly liver development and the transcriptional transitions during hepatogenesis are well characterized. However, gene expression changes during the late postnatal/pre-pubertal to young adulthood period are less well understood, especially with regards to sex-specific gene expression.MethodsMicroarray analysis of male and female mouse liver was carried out at 3, 4, and 8 wk of age to elucidate developmental changes in gene expression from the late postnatal/pre-pubertal period to young adulthood.ResultsA large number of sex-biased and sex-independent genes showed significant changes during this developmental period. Notably, sex-independent genes involved in cell cycle, chromosome condensation, and DNA replication were down regulated from 3 wk to 8 wk, while genes associated with metal ion binding, ion transport and kinase activity were up regulated. A majority of genes showing sex differential expression in adult liver did not display sex differences prior to puberty, at which time extensive changes in sex-specific gene expression were seen, primarily in males. Thus, in male liver, 76% of male-specific genes were up regulated and 47% of female-specific genes were down regulated from 3 to 8 wk of age, whereas in female liver 67% of sex-specific genes showed no significant change in expression. In both sexes, genes up regulated from 3 to 8 wk were significantly enriched (p < E-76) in the set of genes positively regulated by the liver transcription factor HNF4α, as determined in a liver-specific HNF4α knockout mouse model, while genes down regulated during this developmental period showed significant enrichment (p < E-65) for negative regulation by HNF4α. Significant enrichment of the developmentally regulated genes in the set of genes subject to positive and negative regulation by pituitary hormone was also observed. Five sex-specific transcriptional regulators showed sex-specific expression at 4 wk (male-specific Ihh; female-specific Cdx4, Cux2, Tox, and Trim24) and may contribute to the developmental changes that lead to global acquisition of liver sex-specificity by 8 wk of age.ConclusionsOverall, the observed changes in gene expression during postnatal liver development reflect the deceleration of liver growth and the induction of specialized liver functions, with widespread changes in sex-specific gene expression primarily occurring in male liver.

Yes-associated protein is involved in proliferation and differentiation during postnatal liver development.

It is known that the liver undergoes size increase and differentiation simultaneously during the postnatal period. Cells in the liver undergo a period of well-controlled proliferation to achieve the adult liver-to-body weight ratio. The postnatal liver growth is also accompanied by simultaneous hepatic differentiation. However, the mechanisms of liver size regulation and differentiation are not completely clear. Herein we report that yes-associated protein (Yap), the downstream effector of the Hippo Kinase signaling pathway, plays a role in liver size regulation and differentiation during the postnatal liver growth period. Postnatal liver growth was studied in C57BL/6 mice over a time course of postnatal days (PND) 0-30. Analysis of nuclear Yap by Western blot indicated peak Yap activation between PND15-20, which coincided with increased cyclin D1 expression and liver cell proliferation. Analysis of postnatal liver development in Yap(+/-) mice revealed a significant decrease in the liver-to-body weight ratio compared with Yap(+/+) mice at PND15 and -30. Yap(+/-) mice exhibited a significant decrease in postnatal liver cell proliferation, but no change in apoptosis was observed. Furthermore, global gene expression analysis of Yap(+/-) livers revealed a role of Yap in regulation of genes involved in bile acid metabolism, retinoic acid metabolism, ion transport, and extracellular matrix proteins. Taken together, these data indicate that Yap plays a role in both cell proliferation and possibly in hepatic differentiation during postnatal liver development.

In utero exposure to di-(2-ethylhexyl) phthalate affects liver morphology and metabolism in post-natal CD-1 mice

The plasticizer di-(2-ethylhexyl)phthalate (DEHP) affects reproductive development, glycogen and lipid metabolism. Whereas liver is a main DEHP target in adult rodents, the potential impact on metabolic programming is unknown. Effects of in utero DEHP exposure on liver development were investigated upon treatment of pregnant CD-1 mice on gestational days (GD)11–19. F1 mice were examined at post-natal days 21 (weaning) and 35 (start of puberty): parameters included liver histopathological, immunocytochemical and α-fetoprotein (AFP) gene expression analyses. In utero DEHP exposure altered post-natal liver development in weanling mice causing significant, dose-related (i) increased hepatosteatosis, (ii) decreased glycogen storage, (iii) increased β-catenin intracytoplasmic localization (females only). At puberty, significantly decreased glycogen storage was still present in males. A treatment-induced phenotype was identified with lack of glycogen accumulation and intracytoplasmic localization of β-catenin which was associated with increased AFP gene expression. Our findings suggested that DEHP alters post-natal liver development delaying the programming of glycogen metabolism.

Enrichment and Clonal Culture of Progenitor Cells During Mouse Postnatal Liver Development in Mice

Stem and progenitor cells exist in normal postnatal livers. However, it has not been possible to clonally isolate or analyze postnatal liver stem/progenitor-like cells (PLSCs) derived from noninjured livers because of a lack of specific surface markers. This study aimed to establish a primary culture system for clone-sorted PLSCs. To investigate proliferation and differentiation of PLSCs, subpopulations of nonparenchymal cells derived from noninjured livers were purified and cultured using a single-cell culture system. Cells were grown in fetal liver cell-derived conditioned medium in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632. We identified CD13 and CD133 as markers expressed on the PLSC-containing population in noninjured livers and established an efficient single-cell culture system to clonally analyze PLSCs. Culture of PLSCs is difficult, even using conditioned medium, but the addition of Y-27632 increased PLSC cell proliferation. The proportion of progenitor cells among nonparenchymal cells decreased during postnatal liver development; however, a PLSC population was still preserved in 3-month-old mice. Long-term cultivated cells derived from clone-sorted cells in normal livers were established and were called normal-liver-derived stem-like cells (NLS cells). NLS cells could differentiate into hepatocyte-like and cholangiocyte-like cells under appropriate culture conditions and underwent self-renewal-like activity in serial reclone-sorted culture. CD13 and CD133 were expressed on progenitor cells derived from fetal and postnatal liver, whereas CD49f (integrin alpha6 subunit) was strongly expressed only on PLSCs. These results demonstrate the presence of progenitor cells in the CD13(+)CD49f(+)CD133(+) subpopulation of nonhematopoietic cells derived from noninjured postnatal livers.

Ontogenic expression of hepatic Ahr mRNA is associated with histone H3K4 di-methylation during mouse liver development

The aryl hydrocarbon receptor (Ahr) is a xenobiotic sensor that regulates the expression of a battery of drug-metabolizing genes. However, Ahr is also important for normal liver development. The purpose of the present study was to examine the ontogeny of Ahr mRNA in mouse liver, and determine the epigenetic mechanisms regulating Ahr gene transcription during postnatal liver development. There was a 224% increase in hepatic Ahr mRNA from 2 days before birth to 45 days after birth. ChIP-on-chip analysis demonstrated that DNA methylation and histone H3K27 tri-methylation (H3K27Me3), two epigenetic marks for suppression of gene transcription, were consistently low around the Ahr gene locus. In contrast, enrichment of histone H3K4 di-methylation (H3K4Me2), a hallmark for gene activation, increased 182% from prenatal to young adult period around the Ahr gene locus. Regression analysis revealed a strong correlation between enrichment of H3K4Me2 and Ahr mRNA (r=0.91). In conclusion, postnatal H3K4Me2 enrichment positively associates with Ahr mRNA in developing mouse liver, providing a permissive chromatin state allowing Ahr gene transactivation in postnatal liver development.