Figure 2. Scanned images of original autoradiographs showing Western Blot analysis for β-Catenin,... more Figure 2. Scanned images of original autoradiographs showing Western Blot analysis for β-Catenin, GAPDH and LaminB1 in embryonic liver samples taken at the time points indicated (see Figure 1B).
Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and patho... more 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/b-catenin pathway, we challenged the allele combinations by genetically restricting intracellular b-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/b-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/b-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 genotypephenotype analysis suggests tissue-specific response levels for the Wnt/b-catenin pathway that regulate both physiological and pathophysiological conditions.
Pancreatic cells can be converted to hepatocytes by overexpression of C/EBPβ (Shen, C-N, Slack, J... more Pancreatic cells can be converted to hepatocytes by overexpression of C/EBPβ (Shen, C-N, Slack, J.M.W. and Tosh, D., 2000. Molecular basis of transdifferentiation of pancreas to liver. Nature Cell Biology 2: 879-887). This suggested that expression of one or more C/EBP factors may distinguish liver and pancreas in early development. We have now studied the early expression of C/EBPα and C/EBPβ in the mouse embryo and show that both are expressed exclusively in the early liver bud and not in the pancreatic buds. Their expression is identical to that of hepatocyte nuclear factor 4 (HNF4), another key hepatic transcription factor and alpha-fetoprotein (AFP), a differentiation product characteristic of immature hepatocytes. Both are complementary to the early expression of Pdx1, a key pancreatic transcription factor. These results are consistent with the idea that C/EBP factors are master regulators for liver development.
The canonical Wnt (Wnt/β-catenin) signalling pathway is highly conserved and plays a critical rol... more The canonical Wnt (Wnt/β-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/β-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/β-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 crossta...
Breast cancer is one of the most prevalent types of cancers worldwide and yet, its pathophysiolog... more 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 mm 2 , 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 Ca 2+ 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.
After publication of this article [1], concerns were raised about irregularities in Fig 4B and ve... more After publication of this article [1], concerns were raised about irregularities in Fig 4B and vertical lines regarding C/EBPβ. The authors would like to make the following clarification regarding the western blotting data for C/EBPβ in Fig 4B. The scanned image of the CEBPβ western blot was used and this data is presented with westerns for a set of liver markers (Albumin, Transferrin and alpha fetoprotein) and alpha tubulin. However, the samples for these western blots (Albumin, Transferrin, alpha fetoprotein and alpha-tubulin) were loaded in a different order to the samples in the CEBPβ western (omitting the 14day data). Therefore, the scanned image for the CEBPβ western was cropped and rearranged so that the order of the samples corresponded across all western blots included in Fig 4B. All images were adjusted to have a similar size in Photoshop (so that the lanes lined up and all panels were of a similar width and height) and then compiled into a single annotated figure. Please view the updated Fig 4 and updated legend here.
While the Wnt/β-catenin pathway plays a critical role in the maintenance of the zonation of ammon... more While the Wnt/β-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/β-catenin pathway in the development of zonation in embryonic mouse liver by conditional deletion of Apc and β-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 β-catenin coincides with the restriction of GS expression to the terminal perivenous hepatocytes. Conditional loss of Apc resulted in the expressio...
Differentiation; research in biological diversity, Jan 19, 2016
Barrett's metaplasia is the only known morphological precursor to oesophageal adenocarcinoma ... more 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, K...
Proceedings of the National Academy of Sciences of the United States of America, Jan 10, 2015
SUuL2Y3LTqgMf2SrMKk5mK%2FkMM2t7VccfGaeF6E% 2BQvmfVmEjQWBk6Lj1fM3gGaUbSC0b8G7pBrNd%0D% 0Ais0nWNkkD... more SUuL2Y3LTqgMf2SrMKk5mK%2FkMM2t7VccfGaeF6E% 2BQvmfVmEjQWBk6Lj1fM3gGaUbSC0b8G7pBrNd%0D% 0Ais0nWNkkDw%3D%3D)." should instead appear as "We have deposited the array data in NCBI's Gene Expression Omnibus, which is accessible through accession number GSE65476 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE65476)."
Transdifferentiation is defined as the conversion of one cell type to another. One well-documente... more 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.
Glucocorticoids are known to exert multiple effects upon neuronal systems and neuronal gene expre... more Glucocorticoids are known to exert multiple effects upon neuronal systems and neuronal gene expression but the molecular mechanisms through which these effects are mediated are largely undefined. In this study, a transgenic mouse model that expresses a bovine vasopressin transgene was used to investigate the mechanisms by which this neuropeptide gene is repressed by glucocorticoids. Using both northern analysis and a reverse transcriptase polymerase chain reaction assay, depletion of glucocorticoids with the 11,beta-hydroxylase inhibitor metyrapone was shown to result in a dexamethasone-reversed increase in ectopic adrenal transgene messenger RNA levels. This result shows that sequences within the confines of the 3.5 kb transgene are sufficient to mediate repression by glucocorticoids, and indicates the involvement of a type II glucocorticoid receptor mechanism which is independent of cellular context. Evidence for the involvement of cis-acting repressive elements in the proximal 5' flanking sequence was obtained in further studies in which bovine transgene constructs were shown to be negatively regulated by dexamethasone in 293 cells. The further demonstration that recombinant glucocorticoid receptor binds to a vasopressin promoter fragment in an in vitro electrophoretic mobility shift assay provided additional evidence of a direct mechanism of repression. Both in vitro studies were consistent with the presence of a glucocorticoid regulatory element within the region -300 to 155 of the transcription start site. The use of an in vivo transgenic system combined with in vitro analyses of gene promoter fragments enabled the characterization of the molecular mechanisms which effect physiological changes in vasopressin gene expression, and provided evidence of a direct mechanism of repression mediated by sequences within the vasopressin gene promoter.
Results and Problems in Cell Differentiation, 2012
States of terminal cell differentiation are often considered to be fixed. There are examples, how... more 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.
The bacterial gene chloramphenicol acetyltransferase (CAT) is a widely used reporter in both in-v... more The bacterial gene chloramphenicol acetyltransferase (CAT) is a widely used reporter in both in-vitro and in-vivo studies of genetic regulation. We have recently generated novel rat transgenic lines carrying an arylalkylamine N-acetyltransferase (AA-NAT) promoter-reporter construct in which CAT (with associated SV40 small-t antigen sequence) is the reporter. In addition to the predicted transgene transcript (1.9kb), we identified an abundant 1.5kb transcript which derives from an alternative splicing event that utilises a cryptic splice donor site located within the CAT gene. The native CAT open reading frame (ORF) is lost in the 1.5kb transcript, and a western analysis has shown that protein deriving from an aberrant open reading frame is not expressed at detectable levels.
The International Journal of Developmental Biology, 2006
Pancreatic cells can be converted to hepatocytes by overexpression of C/EBPβ (Shen, C-N, Slack, J... more Pancreatic cells can be converted to hepatocytes by overexpression of C/EBPβ (Shen, C-N, Slack, J.M.W. and Tosh, D., 2000. Molecular basis of transdifferentiation of pancreas to liver. Nature Cell Biology 2: 879-887). This suggested that expression of one or more C/EBP factors may distinguish liver and pancreas in early development. We have now studied the early expression of C/EBPα and C/EBPβ in the mouse embryo and show that both are expressed exclusively in the early liver bud and not in the pancreatic buds. Their expression is identical to that of hepatocyte nuclear factor 4 (HNF4), another key hepatic transcription factor and alpha-fetoprotein (AFP), a differentiation product characteristic of immature hepatocytes. Both are complementary to the early expression of Pdx1, a key pancreatic transcription factor. These results are consistent with the idea that C/EBP factors are master regulators for liver development.
Background: The pancreatic exocrine cell line AR42J-B13 can be reprogrammed to hepatocytes follow... more 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/EBPb (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/ EBPb 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/EBPb, 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/EBPb.
... anterior pituitary. LJ Chew a , ZD Burke b , H. Morgan b , I. Gozes c , D. Murphy a and DA Ca... more ... anterior pituitary. LJ Chew a , ZD Burke b , H. Morgan b , I. Gozes c , D. Murphy a and DA Carter b , *. a Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge, Singapore 0511, Singapore. b School ...
Figure 2. Scanned images of original autoradiographs showing Western Blot analysis for β-Catenin,... more Figure 2. Scanned images of original autoradiographs showing Western Blot analysis for β-Catenin, GAPDH and LaminB1 in embryonic liver samples taken at the time points indicated (see Figure 1B).
Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and patho... more 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/b-catenin pathway, we challenged the allele combinations by genetically restricting intracellular b-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/b-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/b-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 genotypephenotype analysis suggests tissue-specific response levels for the Wnt/b-catenin pathway that regulate both physiological and pathophysiological conditions.
Pancreatic cells can be converted to hepatocytes by overexpression of C/EBPβ (Shen, C-N, Slack, J... more Pancreatic cells can be converted to hepatocytes by overexpression of C/EBPβ (Shen, C-N, Slack, J.M.W. and Tosh, D., 2000. Molecular basis of transdifferentiation of pancreas to liver. Nature Cell Biology 2: 879-887). This suggested that expression of one or more C/EBP factors may distinguish liver and pancreas in early development. We have now studied the early expression of C/EBPα and C/EBPβ in the mouse embryo and show that both are expressed exclusively in the early liver bud and not in the pancreatic buds. Their expression is identical to that of hepatocyte nuclear factor 4 (HNF4), another key hepatic transcription factor and alpha-fetoprotein (AFP), a differentiation product characteristic of immature hepatocytes. Both are complementary to the early expression of Pdx1, a key pancreatic transcription factor. These results are consistent with the idea that C/EBP factors are master regulators for liver development.
The canonical Wnt (Wnt/β-catenin) signalling pathway is highly conserved and plays a critical rol... more The canonical Wnt (Wnt/β-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/β-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/β-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 crossta...
Breast cancer is one of the most prevalent types of cancers worldwide and yet, its pathophysiolog... more 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 mm 2 , 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 Ca 2+ 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.
After publication of this article [1], concerns were raised about irregularities in Fig 4B and ve... more After publication of this article [1], concerns were raised about irregularities in Fig 4B and vertical lines regarding C/EBPβ. The authors would like to make the following clarification regarding the western blotting data for C/EBPβ in Fig 4B. The scanned image of the CEBPβ western blot was used and this data is presented with westerns for a set of liver markers (Albumin, Transferrin and alpha fetoprotein) and alpha tubulin. However, the samples for these western blots (Albumin, Transferrin, alpha fetoprotein and alpha-tubulin) were loaded in a different order to the samples in the CEBPβ western (omitting the 14day data). Therefore, the scanned image for the CEBPβ western was cropped and rearranged so that the order of the samples corresponded across all western blots included in Fig 4B. All images were adjusted to have a similar size in Photoshop (so that the lanes lined up and all panels were of a similar width and height) and then compiled into a single annotated figure. Please view the updated Fig 4 and updated legend here.
While the Wnt/β-catenin pathway plays a critical role in the maintenance of the zonation of ammon... more While the Wnt/β-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/β-catenin pathway in the development of zonation in embryonic mouse liver by conditional deletion of Apc and β-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 β-catenin coincides with the restriction of GS expression to the terminal perivenous hepatocytes. Conditional loss of Apc resulted in the expressio...
Differentiation; research in biological diversity, Jan 19, 2016
Barrett's metaplasia is the only known morphological precursor to oesophageal adenocarcinoma ... more 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, K...
Proceedings of the National Academy of Sciences of the United States of America, Jan 10, 2015
SUuL2Y3LTqgMf2SrMKk5mK%2FkMM2t7VccfGaeF6E% 2BQvmfVmEjQWBk6Lj1fM3gGaUbSC0b8G7pBrNd%0D% 0Ais0nWNkkD... more SUuL2Y3LTqgMf2SrMKk5mK%2FkMM2t7VccfGaeF6E% 2BQvmfVmEjQWBk6Lj1fM3gGaUbSC0b8G7pBrNd%0D% 0Ais0nWNkkDw%3D%3D)." should instead appear as "We have deposited the array data in NCBI's Gene Expression Omnibus, which is accessible through accession number GSE65476 (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE65476)."
Transdifferentiation is defined as the conversion of one cell type to another. One well-documente... more 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.
Glucocorticoids are known to exert multiple effects upon neuronal systems and neuronal gene expre... more Glucocorticoids are known to exert multiple effects upon neuronal systems and neuronal gene expression but the molecular mechanisms through which these effects are mediated are largely undefined. In this study, a transgenic mouse model that expresses a bovine vasopressin transgene was used to investigate the mechanisms by which this neuropeptide gene is repressed by glucocorticoids. Using both northern analysis and a reverse transcriptase polymerase chain reaction assay, depletion of glucocorticoids with the 11,beta-hydroxylase inhibitor metyrapone was shown to result in a dexamethasone-reversed increase in ectopic adrenal transgene messenger RNA levels. This result shows that sequences within the confines of the 3.5 kb transgene are sufficient to mediate repression by glucocorticoids, and indicates the involvement of a type II glucocorticoid receptor mechanism which is independent of cellular context. Evidence for the involvement of cis-acting repressive elements in the proximal 5' flanking sequence was obtained in further studies in which bovine transgene constructs were shown to be negatively regulated by dexamethasone in 293 cells. The further demonstration that recombinant glucocorticoid receptor binds to a vasopressin promoter fragment in an in vitro electrophoretic mobility shift assay provided additional evidence of a direct mechanism of repression. Both in vitro studies were consistent with the presence of a glucocorticoid regulatory element within the region -300 to 155 of the transcription start site. The use of an in vivo transgenic system combined with in vitro analyses of gene promoter fragments enabled the characterization of the molecular mechanisms which effect physiological changes in vasopressin gene expression, and provided evidence of a direct mechanism of repression mediated by sequences within the vasopressin gene promoter.
Results and Problems in Cell Differentiation, 2012
States of terminal cell differentiation are often considered to be fixed. There are examples, how... more 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.
The bacterial gene chloramphenicol acetyltransferase (CAT) is a widely used reporter in both in-v... more The bacterial gene chloramphenicol acetyltransferase (CAT) is a widely used reporter in both in-vitro and in-vivo studies of genetic regulation. We have recently generated novel rat transgenic lines carrying an arylalkylamine N-acetyltransferase (AA-NAT) promoter-reporter construct in which CAT (with associated SV40 small-t antigen sequence) is the reporter. In addition to the predicted transgene transcript (1.9kb), we identified an abundant 1.5kb transcript which derives from an alternative splicing event that utilises a cryptic splice donor site located within the CAT gene. The native CAT open reading frame (ORF) is lost in the 1.5kb transcript, and a western analysis has shown that protein deriving from an aberrant open reading frame is not expressed at detectable levels.
The International Journal of Developmental Biology, 2006
Pancreatic cells can be converted to hepatocytes by overexpression of C/EBPβ (Shen, C-N, Slack, J... more Pancreatic cells can be converted to hepatocytes by overexpression of C/EBPβ (Shen, C-N, Slack, J.M.W. and Tosh, D., 2000. Molecular basis of transdifferentiation of pancreas to liver. Nature Cell Biology 2: 879-887). This suggested that expression of one or more C/EBP factors may distinguish liver and pancreas in early development. We have now studied the early expression of C/EBPα and C/EBPβ in the mouse embryo and show that both are expressed exclusively in the early liver bud and not in the pancreatic buds. Their expression is identical to that of hepatocyte nuclear factor 4 (HNF4), another key hepatic transcription factor and alpha-fetoprotein (AFP), a differentiation product characteristic of immature hepatocytes. Both are complementary to the early expression of Pdx1, a key pancreatic transcription factor. These results are consistent with the idea that C/EBP factors are master regulators for liver development.
Background: The pancreatic exocrine cell line AR42J-B13 can be reprogrammed to hepatocytes follow... more 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/EBPb (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/ EBPb 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/EBPb, 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/EBPb.
... anterior pituitary. LJ Chew a , ZD Burke b , H. Morgan b , I. Gozes c , D. Murphy a and DA Ca... more ... anterior pituitary. LJ Chew a , ZD Burke b , H. Morgan b , I. Gozes c , D. Murphy a and DA Carter b , *. a Institute of Molecular and Cell Biology, National University of Singapore, Kent Ridge, Singapore 0511, Singapore. b School ...
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