Michele A. Battle

Highly efficient generation of human hepatocyte–like cells from induced pluripotent stem cells

There exists a worldwide shortage of donor livers available for orthotropic liver transplantation and hepatocyte transplantation therapies. In addition to their therapeutic potential, primary human hepatocytes facilitate the study of molecular and genetic aspects of human hepatic disease and development and provide a platform for drug toxicity screens and identification of novel pharmaceuticals with potential to treat a wide array of metabolic diseases. The demand for human hepatocytes, therefore, heavily outweighs their availability. As an alternative to using donor livers as a source of primary hepatocytes, we explored the possibility of generating patient‐specific human hepatocytes from induced pluripotent stem (iPS) cells. Conclusion: We demonstrate that mouse iPS cells retain full potential for fetal liver development and describe a procedure that facilitates the efficient generation of highly differentiated human hepatocyte‐like cells from iPS cells that display key liver functions and can integrate into the hepatic parenchyma in vivo. (HEPATOLOGY 2010.)

Crosstalk between B lymphocytes, microbiota and the intestinal epithelium governs immunity versus metabolism in the gut

Using a systems biology approach, we discovered and dissected a three-way interaction between the immune system, the intestinal epithelium and the microbiota. We found that, in the absence of B cells, or of IgA, and in the presence of the microbiota, the intestinal epithelium launches its own protective mechanisms, upregulating interferon-inducible immune response pathways and simultaneously repressing Gata4-related metabolic functions. This shift in intestinal function leads to lipid malabsorption and decreased deposition of body fat. Network analysis revealed the presence of two interconnected epithelial-cell gene networks, one governing lipid metabolism and another regulating immunity, that were inversely expressed. Gene expression patterns in gut biopsies from individuals with common variable immunodeficiency or with HIV infection and intestinal malabsorption were very similar to those of the B cell–deficient mice, providing a possible explanation for a longstanding enigmatic association between immunodeficiency and defective lipid absorption in humans.

Loss of both GATA4 and GATA6 blocks cardiac myocyte differentiation and results in acardia in mice.

Despite significant advances in identifying signaling molecules that induce cardiogenesis in mammals, the transcription factors that control the onset of cardiac myocyte gene expression have remained elusive. Candidates include the zinc finger transcription factors GATA binding proteins 4 and 6 (GATA4, GATA6). The individual loss of either protein in mice results in lethality prior to the onset of heart development due to defects in the extra-embryonic endoderm; however, when this extra-embryonic deficiency is circumvented using tetraploid embryo complementation, cardiac myocyte differentiation initiates normally. Here we show that these factors have redundant roles in controlling the onset of cardiac myocyte differentiation. As a consequence, Gata4(-/-)Gata6(-/-) embryos completely lack hearts, although second heart field progenitor cells are still generated. Our data support a model whereby GATA4 or GATA6 are essential for expression of the network of transcription factors that regulate the onset of cardiac myocyte gene expression during mammalian development.

Pancreas-specific deletion of mouse Gata4 and Gata6 causes pancreatic agenesis

Pancreatic agenesis is a human disorder caused by defects in pancreas development. To date, only a few genes have been linked to pancreatic agenesis in humans, with mutations in pancreatic and duodenal homeobox 1 (PDX1) and pancreas-specific transcription factor 1a (PTF1A) reported in only 5 families with described cases. Recently, mutations in GATA6 have been identified in a large percentage of human cases, and a GATA4 mutant allele has been implicated in a single case. In the mouse, Gata4 and Gata6 are expressed in several endoderm-derived tissues, including the pancreas. To analyze the functions of GATA4 and/or GATA6 during mouse pancreatic development, we generated pancreas-specific deletions of Gata4 and Gata6. Surprisingly, loss of either Gata4 or Gata6 in the pancreas resulted in only mild pancreatic defects, which resolved postnatally. However, simultaneous deletion of both Gata4 and Gata6 in the pancreas caused severe pancreatic agenesis due to disruption of pancreatic progenitor cell proliferation, defects in branching morphogenesis, and a subsequent failure to induce the differentiation of progenitor cells expressing carboxypeptidase A1 (CPA1) and neurogenin 3 (NEUROG3). These studies address the conserved and nonconserved mechanisms underlying GATA4 and GATA6 function during pancreas development and provide a new mouse model to characterize the underlying developmental defects associated with pancreatic agenesis.

Hepatocyte nuclear factor 4α is implicated in endoplasmic reticulum stress-induced acute phase response by regulating expression of cyclic adenosine monophosphate responsive element binding protein H

Loss of the nuclear hormone receptor hepatocyte nuclear factor 4α (HNF4α) in hepatocytes results in a complex pleiotropic phenotype that includes a block in hepatocyte differentiation and a severe disruption to liver function. Recent analyses have shown that hepatic gene expression is severely affected by the absence of HNF4α, with expression of 567 genes reduced by ≥2.5‐fold (P ≤ 0.05) in Hnf4α−/− fetal livers. Although many of these genes are direct targets, HNF4α has also been shown to regulate expression of other liver transcription factors, and this raises the possibility that the dependence on HNF4α for normal expression of some genes may be indirect. We postulated that the identification of transcription factors whose expression is regulated by HNF4α might reveal roles for HNF4α in controlling hepatic functions that were not previously appreciated. Here we identify cyclic adenosine monophosphate responsive element binding protein H (CrebH) as a transcription factor whose messenger RNA can be identified in both the embryonic mouse liver and adult mouse liver and whose expression is dependent on HNF4α. Analyses of genomic DNA revealed an HNF4α binding site upstream of the CrebH coding sequence that was occupied by HNF4α in fetal livers and facilitated transcriptional activation of a reporter gene in transient transfection analyses. Although CrebH is highly expressed during hepatogenesis, CrebH−/− mice were viable and healthy and displayed no overt defects in liver formation. However, upon treatment with tunicamycin, which induces an endoplasmic reticulum (ER)–stress response, CrebH−/− mice displayed reduced expression of acute phase response proteins. Conclusion: These data implicate HNF4α in having a role in controlling the acute phase response of the liver induced by ER stress by regulating expression of CrebH. (HEPATOLOGY 2008.)

GATA4 is essential for jejunal function in mice

BACKGROUND & AIMS Although the zinc-finger transcription factor GATA4 has been implicated in regulating jejunal gene expression, the contribution of GATA4 in controlling jejunal physiology has not been addressed. METHODS We generated mice in which the Gata4 gene was specifically deleted in the small intestinal epithelium. Measurements of plasma cholesterol and phospholipids, intestinal absorption of dietary fat and cholesterol, and gene expression were performed on these animals. RESULTS Mice lacking GATA4 in the intestine displayed a dramatic block in their ability to absorb cholesterol and dietary fat. Comparison of the global gene expression profiles of control jejunum, control ileum, and GATA4 null jejunum by gene array analysis revealed that GATA4 null jejunum lost expression of 53% of the jejunal-specific gene set and gained expression of 47% of the set of genes unique to the ileum. These alterations in gene expression included a decrease in messenger RNAs (mRNAs) encoding lipid and cholesterol transporters as well as an increase in mRNAs encoding proteins involved in bile acid absorption. CONCLUSIONS Our data demonstrate that GATA4 is essential for jejunal function including fat and cholesterol absorption and confirm that GATA4 plays a pivotal role in determining jejunal vs ileal identity.

Hepatocyte nuclear factor 4Α is implicated in endoplasmic reticulum stress–induced acute phase response by regulating expression of cyclic adenosine monophosphate responsive element binding protein H Potential conflict of interest: Nothing to report.

Loss of the nuclear hormone receptor hepatocyte nuclear factor 4α (HNF4α) in hepatocytes results in a complex pleiotropic phenotype that includes a block in hepatocyte differentiation and a severe disruption to liver function. Recent analyses have shown that hepatic gene expression is severely affected by the absence of HNF4α, with expression of 567 genes reduced by ≥2.5‐fold (P ≤ 0.05) in Hnf4α−/− fetal livers. Although many of these genes are direct targets, HNF4α has also been shown to regulate expression of other liver transcription factors, and this raises the possibility that the dependence on HNF4α for normal expression of some genes may be indirect. We postulated that the identification of transcription factors whose expression is regulated by HNF4α might reveal roles for HNF4α in controlling hepatic functions that were not previously appreciated. Here we identify cyclic adenosine monophosphate responsive element binding protein H (CrebH) as a transcription factor whose messenger RNA can be identified in both the embryonic mouse liver and adult mouse liver and whose expression is dependent on HNF4α. Analyses of genomic DNA revealed an HNF4α binding site upstream of the CrebH coding sequence that was occupied by HNF4α in fetal livers and facilitated transcriptional activation of a reporter gene in transient transfection analyses. Although CrebH is highly expressed during hepatogenesis, CrebH−/− mice were viable and healthy and displayed no overt defects in liver formation. However, upon treatment with tunicamycin, which induces an endoplasmic reticulum (ER)–stress response, CrebH−/− mice displayed reduced expression of acute phase response proteins. Conclusion: These data implicate HNF4α in having a role in controlling the acute phase response of the liver induced by ER stress by regulating expression of CrebH. (HEPATOLOGY 2008.)

E-cadherin is required for intestinal morphogenesis in the mouse.

E-cadherin, the primary epithelial adherens junction protein, has been implicated as playing a critical role in nucleating formation of adherens junctions, tight junctions, and desmosomes. In addition to its role in maintaining structural tissue integrity, E-cadherin has also been suggested as an important modulator of cell signaling via interactions with its cytoplasmic binding partners, catenins, as well as with growth factor receptors. Therefore, we proposed that loss of E-cadherin from the developing mouse intestinal epithelium would disrupt intestinal epithelial morphogenesis and function. To test this hypothesis, we used a conditional knockout approach to eliminate E-cadherin specifically in the intestinal epithelium during embryonic development. We found that E-cadherin conditional knockout mice failed to survive, dying within the first 24 hours of birth. Examination of intestinal architecture at E18.5 demonstrated severe disruption to intestinal morphogenesis in animals lacking E-cadherin in the epithelium of the small intestine. We observed changes in epithelial cell shape as well as in the morphology of villi. Although junctional complexes were evident, junctions were abnormal, and barrier function was compromised in E-cadherin mutant intestine. We also identified changes in the epithelial cell populations present in E-cadherin conditional knockout animals. The number of proliferating cells was increased, whereas the number of enterocytes was decreased. Although Wnt/β-catenin target mRNAs were more abundant in mutants compared with controls, the amount of nuclear activated β-catenin protein was dramatically lower in mutants compared with controls. In summary, our data demonstrate that E-cadherin is essential for intestinal epithelial morphogenesis and homeostasis during embryonic development.

Transcriptional regulation of the human hepatic lipase (LIPC) gene promoter

Hepatic lipase (HL) plays a key role in the metabolism of plasma lipoproteins, and its level of activity requires tight regulation, given the association of both low and high levels with atherosclerosis and coronary artery disease. However, little is known about the factors responsible for HL expression. Here, we report that the human hepatic lipase gene (LIPC) promoter is regulated by hepatocyte nuclear factor 4α (HNF4α), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), apolipoprotein A-I regulatory protein-1 (ARP-1), and hepatocyte nuclear factor 1α (HNF1α). Reporter analysis showed that HNF4α directly regulates the LIPC promoter via two newly identified direct repeat elements, DR1 and DR4. PGC-1α is capable of stimulating the HNF4α-dependent transactivation of the LIPC promoter. ARP-1 displaces HNF4α from the DR1 site and blocks its ability to activate the LIPC promoter. Induction by HNF1α requires the HNF1 binding site and upon cotransfection with HNF4α leads to an additive effect. In addition, the in vivo relevance of HNF4α in LIPC expression is shown by the ability of the HNF4α antagonist Medica 16 to repress endogenous LIPC mRNA expression. Furthermore, disruption of Hnf4α in mice prevents the expression of HL mRNA in liver. The overall effect these transcription factors have on HL expression will ultimately depend on the interplay between these various factors and their relative intracellular concentrations.

GATA4 and GATA6 regulate intestinal epithelial cytodifferentiation during development

The intestinal epithelium performs vital roles in organ function by absorbing nutrients and providing a protective barrier. The zinc-finger containing transcription factors GATA4 and GATA6 regulate enterocyte gene expression and control regional epithelial cell identity in the adult intestinal epithelium. Although GATA4 and GATA6 are expressed in the developing intestine, loss of either factor alone during the period of epithelial morphogenesis and cytodifferentiation fails to disrupt these processes. Therefore, we tested the hypothesis that GATA4 and GATA6 function redundantly to control these aspects of intestinal development. We used Villin-Cre, which deletes specifically in the intestinal epithelium during the period of villus development and epithelial cytodifferentiation, to generate Gata4Gata6 double conditional knockout embryos. Mice lacking GATA4 and GATA6 in the intestinal epithelium died within 24h of birth. At E18.5, intestinal villus architecture and epithelial cell populations were altered. Enterocytes were lost, and goblet cells were increased. Proliferation was also increased in GATA4-GATA6 deficient intestinal epithelium. Although villus morphology appeared normal at E16.5, the first time at which both Gata4 and Gata6 were efficiently reduced, changes in expression of markers of enterocytes, goblet cells, and proliferative cells were detected. Moreover, goblet cell number was increased at E16.5. Expression of the Notch ligand Dll1 and the Notch target Olfm4 were reduced in mutant tissue indicating decreased Notch signaling. Finally, we found that GATA4 occupies chromatin near the Dll1 transcription start site suggesting direct regulation of Dll1 by GATA4. We demonstrate that GATA4 and GATA6 play an essential role in maintaining proper intestinal epithelial structure and in regulating intestinal epithelial cytodifferentiation. Our data highlight a novel role for GATA factors in fine tuning Notch signaling during intestinal epithelial development to repress goblet cell differentiation.