The Canonical Wnt Pathway as a Key Regulator in Liver Development, Differentiation and Homeostatic Renewal.

The canonical Wnt (Wnt/ß-catenin) signalling pathway is highly conserved and plays a critical role in regulating cellular processes both during development and in adult tissue homeostasis. The Wnt/ß-catenin signalling pathway is vital for correct body patterning and is involved in fate specification of the gut tube, the primitive precursor of liver. In adults, the Wnt/ß-catenin pathway is increasingly recognised as an important regulator of metabolic zonation, homeostatic renewal and regeneration in response to injury throughout the liver. Herein, we review recent developments relating to the key role of the pathway in the patterning and fate specification of the liver, in the directed differentiation of pluripotent stem cells into hepatocytes and in governing proliferation and zonation in the adult liver. We pay particular attention to recent contributions to the controversy surrounding homeostatic renewal and proliferation in response to injury. Furthermore, we discuss how crosstalk between the Wnt/ß-catenin and Hedgehog (Hh) and hypoxia inducible factor (HIF) pathways works to maintain liver homeostasis. Advancing our understanding of this pathway will benefit our ability to model disease, screen drugs and generate tissue and organ replacements for regenerative medicine.

Author Correction: Spatiotemporal regulation of liver development by the Wnt/ß-catenin pathway.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human Breast Cancer Cells Demonstrate Electrical Excitability.

Breast cancer is one of the most prevalent types of cancers worldwide and yet, its pathophysiology is poorly understood. Single-cell electrophysiological studies have provided evidence that membrane depolarization is implicated in the proliferation and metastasis of breast cancer. However, metastatic breast cancer cells are highly dynamic microscopic systems with complexities beyond a single-cell level. There is an urgent need for electrophysiological studies and technologies capable of decoding the intercellular signaling pathways and networks that control proliferation and metastasis, particularly at a population level. Hence, we present for the first time non-invasive in vitro electrical recordings of strongly metastatic MDA-MB-231 and weakly/non-metastatic MCF-7 breast cancer cell lines. To accomplish this, we fabricated an ultra-low noise sensor that exploits large-area electrodes, of 2 mm2, which maximizes the double-layer capacitance and concomitant detection sensitivity. We show that the current recorded after adherence of the cells is dominated by the opening of voltage-gated sodium channels (VGSCs), confirmed by application of the highly specific inhibitor, tetrodotoxin (TTX). The electrical activity of MDA-MB-231 cells surpasses that of the MCF-7 cells, suggesting a link between the cells' bioelectricity and invasiveness. We also recorded an activity pattern with characteristics similar to that of Random Telegraph Signal (RTS) noise. RTS patterns were less frequent than the asynchronous VGSC signals. The RTS noise power spectral density showed a Lorentzian shape, which revealed the presence of a low-frequency signal across MDA-MB-231 cell populations with propagation speeds of the same order as those reported for intercellular Ca2+ waves. Our recording platform paves the way for real-time investigations of the bioelectricity of cancer cells, their ionic/pharmacological properties and relationship to metastatic potential.

Correction: Dexamethasone Treatment Induces the Reprogramming of Pancreatic Acinar Cells to Hepatocytes and Ductal Cells.

[This corrects the article DOI: 10.1371/journal.pone.0013650.].

Spatiotemporal regulation of liver development by the Wnt/ß-catenin pathway.

While the Wnt/ß-catenin pathway plays a critical role in the maintenance of the zonation of ammonia metabolizing enzymes in the adult liver, the mechanisms responsible for inducing zonation in the embryo are not well understood. Herein we address the spatiotemporal role of the Wnt/ß-catenin pathway in the development of zonation in embryonic mouse liver by conditional deletion of Apc and ß-catenin at different stages of mouse liver development. In normal development, the ammonia metabolising enzymes carbamoylphosphate synthetase I (CPSI) and Glutamine synthetase (GS) begin to be expressed in separate hepatoblasts from E13.5 and E15.5 respectively and gradually increase in number thereafter. Restriction of GS expression occurs at E18 and becomes increasingly limited to the terminal perivenous hepatocytes postnatally. Expression of nuclear ß-catenin coincides with the restriction of GS expression to the terminal perivenous hepatocytes. Conditional loss of Apc resulted in the expression of nuclear ß-catenin throughout the developing liver and increased number of cells expressing GS. Conversely, conditional loss of ß-catenin resulted in loss of GS expression. These data suggest that the Wnt pathway is critical to the development of zonation as well as maintaining the zonation in the adult liver.

Hnf4α is a key gene that can generate columnar metaplasia in oesophageal epithelium.

Barrett's metaplasia is the only known morphological precursor to oesophageal adenocarcinoma and is characterized by replacement of stratified squamous epithelium by columnar epithelium. The cell of origin is uncertain and the molecular mechanisms responsible for the change in cellular phenotype are poorly understood. We therefore explored the role of two transcription factors, Cdx2 and HNF4α in the conversion using primary organ cultures. Biopsy samples from cases of human Barrett's metaplasia were analysed for the presence of CDX2 and HNF4α. A new organ culture system for adult murine oesophagus is described. Using this, Cdx2 and HNF4α were ectopically expressed by adenoviral infection. The phenotype following infection was determined by a combination of PCR, immunohistochemical and morphological analyses. We demonstrate the expression of CDX2 and HNF4α in human biopsy samples. Our oesophageal organ culture system expressed markers characteristic of the normal SSQE: p63, K14, K4 and loricrin. Ectopic expression of HNF4α, but not of Cdx2 induced expression of Tff3, villin, K8 and E-cadherin. HNF4α is sufficient to induce a columnar-like phenotype in adult mouse oesophageal epithelium and is present in the human condition. These data suggest that induction of HNF4α is a key early step in the formation of Barrett's metaplasia and are consistent with an origin of Barrett's metaplasia from the oesophageal epithelium.

MicroRNA-122: a novel hepatocyte-enriched in vitro marker of drug-induced cellular toxicity.

Emerging hepatic models for the study of drug-induced toxicity include pluripotent stem cell-derived hepatocyte-like cells (HLCs) and complex hepatocyte-non-parenchymal cellular coculture to mimic the complex multicellular interactions that recapitulate the niche environment in the human liver. However, a specific marker of hepatocyte perturbation, required to discriminate hepatocyte damage from non-specific cellular toxicity contributed by non-hepatocyte cell types or immature differentiated cells is currently lacking, as the cytotoxicity assays routinely used in in vitro toxicology research depend on intracellular molecules which are ubiquitously present in all eukaryotic cell types. In this study, we demonstrate that microRNA-122 (miR-122) detection in cell culture media can be used as a hepatocyte-enriched in vitro marker of drug-induced toxicity in homogeneous cultures of hepatic cells, and a cell-specific marker of toxicity of hepatic cells in heterogeneous cultures such as HLCs generated from various differentiation protocols and pluripotent stem cell lines, where conventional cytotoxicity assays using generic cellular markers may not be appropriate. We show that the sensitivity of the miR-122 cytotoxicity assay is similar to conventional assays that measure lactate dehydrogenase activity and intracellular adenosine triphosphate when applied in hepatic models with high levels of intracellular miR-122, and can be multiplexed with other assays. MiR-122 as a biomarker also has the potential to bridge results in in vitro experiments to in vivo animal models and human samples using the same assay, and to link findings from clinical studies in determining the relevance of in vitro models being developed for the study of drug-induced liver injury.

Barrett's metaplasia as a paradigm for understanding the development of cancer.

The conversion of one cell type to another is defined as metaplasia (or sometimes it is referred to as transdifferentiation or cellular reprogramming). Metaplasia is important clinically and may predispose to the development of cancer. Barrett's metaplasia is one such example and is the focus of the present review. Barrett's is a pathological condition in which the normal oesophageal stratified squamous epithelium is replaced by intestinal-type columnar epithelium and is associated with gastro-oesophageal reflux disease. The appearance of columnar epithelium in the oesophagus predisposes to the development of adenocarcinoma. Herein we review the latest evidence on the cellular origin of Barrett's metaplasia. Until recently it was thought that the cellular origin of the columnar epithelium was from a pre-existing cell within the oesophagus. However, recent evidence suggests that this may not be the case. Instead two recent publications indicate that the columnar cells may migrate from a site distal to the oesophagus. These new data contravene our current understanding of metaplasia and raise important questions about the cellular origin of cancer.

Cellular reprogramming during mouse development.

States of terminal cell differentiation are often considered to be fixed. There are examples, however, in which cells of one type can be converted to a completely different cell type. The process whereby one cell type can be converted to another is referred to as cellular reprogramming. Cellular reprogramming is also referred to in the literature as transdifferentiation (or the direct conversion of one cell type to another without dedifferentiation to an intermediate cell type). Where the conversion between cell types occurs in the developing embryo, the process is referred to as transdetermination. Herein we examine some well-defined examples of transdetermination. Defining the molecular and cellular basis of transdetermination will help us to understand the normal developmental biology of the cells that interconvert, as well as identifying key regulatory transcription factors (master switch genes) that may be important for the reprogramming of stem cells. Harnessing the therapeutic potential of reprogramming and master genes is an important goal in regenerative medicine.

Ontogenesis of hepatic and pancreatic stem cells.

In the embryo, the liver and pancreas exhibit a close developmental relationship. Both tissues arise from neighbouring regions of the developing endoderm. As well as this close developmental relationship, the liver and pancreas can, under certain circumstances, regenerate functional components. Understanding the normal development of the two tissue types and the underlying cellular and molecular mechanisms governing normal development and regeneration is critical to the production of novel therapies for treating liver disease and pancreatic disorders such as diabetes and pancreatitis. Herein we discuss the development of the liver and pancreas from progenitor cells in the embryo and the existence of potential stem cells in the adult tissues.

Molecular aspects of esophageal development.

The following on molecular aspects of esophageal development contains commentaries on esophageal striated myogenesis and transdifferentiation; conversion from columnar into stratified squamous epithelium in the mouse esophagus; the roles for BMP signaling in the developing esophagus and forestomach; and evidence of a direct conversion from columnar to stratified squamous cells in the developing esophagus.

Dexamethasone treatment induces the reprogramming of pancreatic acinar cells to hepatocytes and ductal cells.

BACKGROUND: The pancreatic exocrine cell line AR42J-B13 can be reprogrammed to hepatocytes following treatment with dexamethasone. The question arises whether dexamethasone also has the capacity to induce ductal cells as well as hepatocytes. METHODOLOGY/PRINCIPAL FINDINGS: AR42J-B13 cells were treated with and without dexamethasone and analyzed for the expression of pancreatic exocrine, hepatocyte and ductal markers. Addition of dexamethasone inhibited pancreatic amylase expression, induced expression of the hepatocyte marker transferrin as well as markers typical of ductal cells: cytokeratin 7 and 19 and the lectin peanut agglutinin. However, the number of ductal cells was low compared to hepatocytes. The proportion of ductal cells was enhanced by culture with dexamethasone and epidermal growth factor (EGF). We established several features of the mechanism underlying the transdifferentiation of pancreatic exocrine cells to ductal cells. Using a CK19 promoter reporter, we show that a proportion of the ductal cells arise from differentiated pancreatic exocrine-like cells. We also examined whether C/EBPß (a transcription factor important in the conversion of pancreatic cells to hepatocytes) could alter the conversion from acinar cells to a ductal phenotype. Overexpression of an activated form of C/EBPß in dexamethasone/EGF-treated cells provoked the expression of hepatocyte markers and inhibited the expression of ductal markers. Conversely, ectopic expression of a dominant-negative form of C/EBPß, liver inhibitory protein, inhibited hepatocyte formation in dexamethasone-treated cultures and enhanced the ductal phenotype. CONCLUSIONS/SIGNIFICANCE: These results indicate that hepatocytes and ductal cells may be induced from pancreatic exocrine AR42J-B13 cells following treatment with dexamethasone. The conversion from pancreatic to hepatocyte or ductal cells is dependent upon the expression of C/EBPß.

Isolation and culture of embryonic pancreas and liver.

Culturing embryonic tissue in an in vitro setting offers the unique ability to manipulate the external medium and therefore to investigate the pathways involved in regulating normal organogenesis as well as providing models for developmental disorders. Here we describe a system for the in vitro culture of the dorsal pancreatic buds and liver buds from mouse embryos. The tissues are dissected from day 9.0 or 11.5 mouse embryos. The tissues are placed on fibronectin-coated coverslips in serum-containing medium and allowed to attach. Over the next few days, the buds grow as flattened structures which are thin enough to allow the use of wholemount immunostaining methods.

In vitro reprogramming of pancreatic cells to hepatocytes.

Transdifferentiation is defined as the conversion of one cell type to another. One well-documented example of transdifferentiation is the conversion of pancreatic cells to hepatocytes. Here we describe a robust in vitro model to study pancreas to liver transdifferentiation. It is based on the addition of the synthetic glucocorticoid dexamethasone to the rat pancreatic exocrine cell line AR42J. Following glucocorticoid treatment, cells resembling hepatocytes are induced. Transdifferentiated hepatocytes express many of the properties of bona fide hepatocytes, e.g. production of albumin and ability to respond to xenobiotics. These hepatocytes can be used for studying liver function in vitro as well as studying the molecular basis of transdifferentiation.

Genetic dissection of differential signaling threshold requirements for the Wnt/beta-catenin pathway in vivo.

Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/beta-catenin pathway, we challenged the allele combinations by genetically restricting intracellular beta-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/beta-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/beta-catenin signaling capacity similar to that in the germline of the Apc(min) mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apc(min) mice arise independently of intestinal tumorigenesis. Together, the present genotype-phenotype analysis suggests tissue-specific response levels for the Wnt/beta-catenin pathway that regulate both physiological and pathophysiological conditions.

Liver zonation occurs through a beta-catenin-dependent, c-Myc-independent mechanism.

BACKGROUND AND AIMS: The Wnt pathway has previously been shown to play a role in hepatic zonation. Herein, we have explored the role of 3 key components (Apc, beta-catenin, and c-Myc) of the Wnt pathway in the zonation of ammonia metabolizing enzymes. METHODS: Conditional deletion of Apc, beta-catenin, and c-Myc was induced in the livers of mice and the expression of periportal and perivenous hepatocyte markers was determined by polymerase chain reaction, Western blotting, and immunohistochemical techniques. RESULTS: Under normal circumstances, the urea cycle enzyme carbamoylphosphate synthetase I (CPS I) is present in the periportal, intermediate, and the first few layers of the perivenous zone. In contrast, glutamine synthetase (GS)--and nuclear beta-catenin--is expressed in a complementary fashion in the last 1-2 cell layers of the perivenous zone. Conditional loss of Apc resulted in the expression of nuclear beta-catenin and GS in most hepatocytes irrespective of zone. Induction of GS in hepatocytes outside the normal perivenous zone was accompanied by a reduction in the expression of CPS I. Deletion of beta-catenin induces a loss of GS and a complementary increase in expression of CPS I irrespective of whether Apc is present. Remarkably, deletion of c-Myc did not perturb the pattern of zonation. CONCLUSIONS: It has been shown that the Wnt pathway is key to imposing the pattern of zonation within the liver. Herein we have addressed the relevance of 3 major Wnt pathway components and show critically that the zonation is c-Myc independent but beta-catenin dependent.

B-catenin deficiency, but not Myc deletion, suppresses the immediate phenotypes of APC loss in the liver.

Dysregulated Wnt signaling is seen in approximately 30% of hepatocellular carcinomas; thus, finding pathways downstream of the activation of Wnt signaling is key. Here, using cre-lox technology, we deleted the Apc gene in the adult mouse liver and observed a rapid increase in nuclear beta-catenin and c-Myc, which is associated with an induction of proliferation that led to hepatomegaly within 4 days of gene deletion. To investigate the downstream pathways responsible for these phenotypes, we analyzed the impact of inactivating APC in the context of deficiency of the potentially key effectors beta-catenin and c-Myc. beta-catenin loss rescues both the proliferation and hepatomegaly phenotypes after APC loss. However, c-Myc deletion, which rescues the phenotypes of APC loss in the intestine, had no effect on the phenotypes of APC loss in the liver. The consequences of the deregulation of the Wnt pathway within the liver are therefore strikingly different from those observed within the intestine, with the vast majority of Wnt targets being beta-catenin-dependent but c-Myc-independent in the liver.

Stem cells in the adult pancreas and liver.

Stem cells are undifferentiated cells that can self-renew and generate specialized (functional) cell types. The remarkable ability of stem cells to differentiate towards functional cells makes them suitable modalities in cellular therapy (which means treating diseases with the body's own cells). Potential targets for cellular therapy include diabetes and liver failure. However, in order for stem cells to be clinically useful, we must learn to identify them and to regulate their differentiation. We will use the intestine as a classical example of a stem cell compartment, and then examine the evidence for the existence of adult stem cells in two endodermally derived organs: pancreas and liver. We will review the characteristics of the putative stem cells in these tissues and the transcription factors controlling their differentiation towards functional cell types.

The Wnt/beta-catenin pathway: master regulator of liver zonation?

The liver contains two systems for the removal of ammonia - the urea cycle and the enzyme glutamine synthetase. These systems are expressed in a complementary fashion in two distinct populations of hepatocytes, referred to as periportal and perivenous cells. One of the unresolved problems in hepatology has been to elucidate the molecular mechanisms responsible for induction and maintenance of the cellular heterogeneity for ammonia detoxification. There is now a potential molecular explanation for the zonation of the urea cycle and glutamine synthetase based on the Wnt/beta-catenin pathway.