Extracellular matrix microarrays to study inductive signaling for endoderm specification.

During tissue development, stem and progenitor cells are faced with fate decisions coordinated by microenvironmental cues. Although insights have been gained from in vitro and in vivo studies, the role of the microenvironment remains poorly understood due to the inability to systematically explore combinations of stimuli at a large scale. To overcome such restrictions, we implemented an extracellular matrix (ECM) array platform that facilitates the study of 741 distinct combinations of 38 different ECM components in a systematic, unbiased and high-throughput manner. Using embryonic stem cells as a model system, we derived definitive endoderm progenitors and applied them to the array platform to study the influence of ECM, including the interactions of ECM with growth factor signaling, on the specification of definitive endoderm cells towards the liver and pancreas fates. We identified ECM combinations that influence endoderm fate decisions towards these lineages, and demonstrated the utility of this platform for studying ECM-mediated modifications to signal activation during liver specification. In particular, defined combinations of fibronectin and laminin isoforms, as well as combinations of distinct collagen subtypes, were shown to influence SMAD pathway activation and the degree of hepatic differentiation. Overall, our systematic high-throughput approach suggests that ECM components of the microenvironment have modulatory effects on endoderm differentiation, including effects on lineage fate choice and cell adhesion and survival during the differentiation process. This platform represents a robust tool for analyzing effects of ECM composition towards the continued improvement of stem cell differentiation protocols and further elucidation of tissue development processes. STATEMENT OF SIGNIFICANCE: Cellular microarrays can provide the capability to perform high-throughput investigations into the role of microenvironmental signals in a variety of cell functions. This study demonstrates the utility of a high-throughput cellular microarray approach for analyzing the effects of extracellular matrix (ECM) in liver and pancreas differentiation of endoderm progenitor cells. Despite an appreciation that ECM is likely involved in these processes, the influence of ECM, particularly combinations of matrix proteins, had not been systematically explored. In addition to the identification of relevant ECM compositions, this study illustrates the capability of the cellular microarray platform to be integrated with a diverse range of cell fate measurements, which could be broadly applied towards the investigation of cell fate regulation in other tissue development and disease contexts.

Nuclear mobility and mitotic chromosome binding: similarities between pioneer transcription factor FoxA and linker histone H1.

There exists a hierarchy by which transcription factors can engage their target sites in chromatin, in that a subset of factors can bind transcriptionally silent, nucleosomal DNA, whereas most factors cannot, and this hierarchy is reflected, at least in part, in the developmental function of the factors. For example, transcription factors possessing the Forkhead box (Fox) DNA-binding domain contain an overall fold resembling that of linker histone and thus are structured to bind DNA, site specifically, in a nucleosomal context. Where tested, Fox factors bind early in the developmental or physiological activation of target genes, thereby enabling the binding of other factors that cannot engage chromatin on their own. To investigate the basis for early chromatin binding, we have used fluorescence recovery after photobleaching (FRAP) to analyze the mobility, in the live cell nucleus, of FoxA factors in comparison to linker histone and other transcription factors. We have further analyzed the factors for their ability to bind to chromatin in mitosis and thereby serve as epigenetic marks. The results indicate that the "pioneer" features of FoxA factors involve various chromatin-binding parameters seen in linker histones and that distinguish the factors with respect to their regulatory and mechanistic functions.

Pioneer factors, genetic competence, and inductive signaling: programming liver and pancreas progenitors from the endoderm.

The endoderm is a multipotent progenitor cell population in the embryo that gives rise to the liver, pancreas, and other cell types and provides paradigms for understanding cell-type specification. Studies of isolated embryo tissue cells and genetic approaches in vivo have defined fibroblast growth factor/mitogen-activated protein kinase (FGF/MAPK) and bone morphogenetic protein (BMP) signaling pathways that induce liver and pancreatic fates in the endoderm. In undifferentiated endoderm cells, the FoxA and GATA transcription factors are among the first to engage silent genes, helping to endow competence for cell-type specification. FoxA proteins can bind their target sites in highly compacted chromatin and open up the local region for other factors to bind; hence, they have been termed "pioneer factors." We recently found that FoxA proteins remain bound to chromatin in mitosis, as an epigenetic mark. In embryonic stem cells, which lack FoxA, FoxA target sites can be occupied by FoxD3, which in turn helps to maintain a local demethylation of chromatin. By these means, a cascade of Fox factors helps to endow progenitor cells with the competence to activate genes in response to tissue-inductive signals. Understanding such epigenetic mechanisms for transcriptional competence coupled with knowledge of the relevant signals for cell-type specification should greatly facilitate efforts to predictably differentiate stem cells to liver and pancreatic fates.

Transcription factor FoxA (HNF3) on a nucleosome at an enhancer complex in liver chromatin.

Nucleosome-like particles and acetylated histones occur near active promoters and enhancers, and certain transcription factors can recognize their target sites on the surface of a nucleosome in vitro; yet it has been unclear whether transcription factors can occupy target sites on nucleosomes in native chromatin. We developed a method for sequential chromatin immunoprecipitation of distinct nuclear proteins that are simultaneously cross-linked to nucleosome-sized genomic DNA segments. We find that core histone H2A co-occupies, along with the FoxA (hepatocyte nuclear factor-3) transcription factor, DNA for the albumin transcriptional enhancer in native liver chromatin, where the enhancer is active. Because histone H2A on nuclear DNA is only known to exist in nucleosomes, we conclude that transcription factors can form a stable complex on nucleosomes at an active enhancer element in vivo.

Liver organogenesis promoted by endothelial cells prior to vascular function.

The embryonic role of endothelial cells and nascent vessels in promoting organogenesis, prior to vascular function, is unclear. We find that early endothelial cells in mouse embryos surround newly specified hepatic endoderm and delimit the mesenchymal domain into which the liver bud grows. In flk-1 mutant embryos, which lack endothelial cells, hepatic specification occurs, but liver morphogenesis fails prior to mesenchyme invasion. We developed an embryo tissue explant system that permits liver bud vasculogenesis and show that in the absence of endothelial cells, or when the latter are inhibited, there is a selective defect in hepatic outgrowth. We conclude that vasculogenic endothelial cells and nascent vessels are critical for the earliest stages of organogenesis, prior to blood vessel function.

Hepatocyte differentiation: from the endoderm and beyond.

Hepatocytes differentiate from the endoderm during embryonic development. Recent studies show, however, that hepatocytes can also be derived from rare cells that reside in the pancreas, bone marrow, and brain. Indeed, the latest discoveries indicate that embryonic hepatocytes normally arise by diversion of an endodermal cell population that would otherwise default to a pancreatic fate. Convergent FGF and BMP signals from distinct mesodermal cell types control this transition. Molecular signals that govern the differentiation of hepatocytes from non-endodermal cells and the role of such cells in normal liver physiology remain to be discovered.

Distinct mesodermal signals, including BMPs from the septum transversum mesenchyme, are required in combination for hepatogenesis from the endoderm.

Mesodermal signaling is critical for patterning the embryonic endoderm into different tissue domains. Classical tissue transplant experiments in the chick and recent studies in the mouse indicated that interactions with the cardiogenic mesoderm are necessary and sufficient to induce the liver in the ventral foregut endoderm. Using molecular markers and functional assays, we now show that septum transversum mesenchyme cells, a distinct mesoderm cell type, are closely apposed to the ventral endoderm and contribute to hepatic induction. Specifically, using a mouse Bmp4 null mutation and an inhibitor of BMPs, we find that BMP signaling from the septum transversum mesenchyme is necessary to induce liver genes in the endoderm and to exclude a pancreatic fate. BMPs apparently function, in part, by affecting the levels of the GATA4 transcription factor, and work in parallel to FGF signaling from the cardiac mesoderm. BMP signaling also appears critical for morphogenetic growth of the hepatic endoderm into a liver bud. Thus, the endodermal domain for the liver is specified by simultaneous signaling from distinct mesodermal sources.

A bipotential precursor population for pancreas and liver within the embryonic endoderm.

The pancreas emerges independently from dorsal and ventral domains of embryonic gut endoderm. Gene inactivation experiments in mice have identified factors required for dorsal pancreas development, but factors that initiate the ventral pancreas have remained elusive. In this study, we investigated the hypothesis that the emergence of the ventral pancreas is related to the emergence of the liver. We find that the liver and ventral pancreas are specified at the same time and in the same general domain of cells. Using embryo tissue explantation experiments, we find that the default fate of the ventral foregut endoderm is to activate the pancreas gene program. FGF signalling from the cardiac mesoderm diverts this endoderm to express genes for liver instead of those for pancreas. No evidence was found to indicate that the cell type choice for pancreas or liver involves a selection for growth or viability. Cardiac mesoderm or FGF induces the local expression of sonic hedgehog, which in turn is inhibitory to pancreas but not to liver. The bipotential precursor cell population for pancreas and liver in embryonic development and its fate selection by FGF has features that appear to be recapitulated in the adult pancreas and are reflected in the evolution of these organs.

Repressive and restrictive mesodermal interactions with gut endoderm: possible relation to Meckel's Diverticulum.

The midgut and hindgut endoderm of the mouse embryo give rise to the intestinal epithelium, yet it is not known how the intestinal program is chosen in contrast to other endoderm-derived cell types. Previous tissue explant studies with embryos at 8.5 to 11.5 days gestation (d) showed that when the gut mesoderm is removed from the prospective intestinal endoderm, the endoderm activates the expression of liver-specific genes such as serum albumin, demonstrating the endoderm's pluripotence. This reversible repression of liver genes does not affect the expression of the endodermal transcription factors HNF3 and GATA4, nor these factors' ability to engage target sites in chromatin. We have now found that at 13.5 d, the mesoderm gains a second inhibitory activity, resulting in the irreversible loss of expression of HNF3 (Foxa2) and GATA factors in the endoderm and the absence of factors binding to their target sites in chromatin. The second inhibitory activity causes the endoderm to lose the potential to activate a liver gene, and this restriction precedes the normal cytodifferentiation of the intestinal epithelium. In summary, two inhibitory interactions with mesoderm successively restrict the developmental potential of the gut endoderm, leading to intestinal differentiation. We also observed rare gut bud structures in midgestation embryos that appear to represent murine examples of Meckel's Diverticulum, a congenital abnormality in human development. The absence of restrictive mesodermal interactions could explain how Meckel's diverticula express diverse non-intestinal, endoderm-derived cell types.

Creating temperature-sensitive winged helix transcription factors. Amino acids that stabilize the DNA binding domain of HNF3.

Winged helix transcription factors contain two polypeptide loops, or "wings," that make minor groove contacts with DNA from either side of a three-helix bundle that binds the DNA major groove. While wing 1 is stabilized by a beta-sheet, parameters that stabilize wing 2 are unknown. Herein we identify two bulky aromatic residues in wing 2 that stabilize the loop structure and, thereby, the entire protein's DNA binding and transcriptional stimulatory activity by interacting with other residues in the three-helix bundle. Mutations of these wing 2 residues create proteins that are temperature-sensitive for transcriptional activity. Aromatic and/or hydrophobic residues are highly conserved among the 150 known winged helix proteins, suggesting conserved function. We suggest that the winged helix structure evolved by the acquisition of aromatic and/or hydrophobic residues in distal polypeptide sequences that helped stabilize the association of a protein loop (wing 2) with the three-helix bundle, thereby enhancing DNA binding.

Liver specification and early morphogenesis.

The classically defined induction of the liver from the endoderm, elicited by the cardiac mesoderm, has recently been discovered to involve signaling by fibroblast growth factors (FGFs). Multiple FGFs induce hepatic gene expression independent of an effect on growth. A subset of these FGFs cooperates with other factors to promote morphogenesis of the newly specified hepatocytes. Subsequent to the formation of the liver bud, distinct mesenchymal signals and hepatic response pathways stimulate further growth and differentiation of the hepatic parenchymal cells and prevent apoptosis. The initial stages of hepatogenesis are therefore beginning to be understood, and serve as a paradigm for the development of other tissues from the endoderm.

Initiation of mammalian liver development from endoderm by fibroblast growth factors.

The signaling molecules that elicit embryonic induction of the liver from the mammalian gut endoderm or induction of other gut-derived organs are unknown. Close proximity of cardiac mesoderm, which expresses fibroblast growth factors (FGFs) 1, 2, and 8, causes the foregut endoderm to develop into the liver. Treatment of isolated foregut endoderm from mouse embryos with FGF1 or FGF2, but not FGF8, was sufficient to replace cardiac mesoderm as an inducer of the liver gene expression program, the latter being the first step of hepatogenesis. The hepatogenic response was restricted to endoderm tissue, which selectively coexpresses FGF receptors 1 and 4. Further studies with FGFs and their specific inhibitors showed that FGF8 contributes to the morphogenetic outgrowth of the hepatic endoderm. Thus, different FGF signals appear to initiate distinct phases of liver development during mammalian organogenesis.

An early developmental transcription factor complex that is more stable on nucleosome core particles than on free DNA.

In vivo footprinting studies have shown that transcription factor binding sites for HNF3 and GATA-4 are occupied on the albumin gene enhancer in embryonic endoderm, prior to the developmental activation of liver gene transcription. We have investigated how these factors can stably occupy silent chromatin. Remarkably, we find that HNF3, but not GATA-4 or a GAL4 control protein, binds far more stably to nucleosome core particles than to free DNA. In the presence of HNF3, GATA-4 binds stably to an HNF3-positioned nucleosome. Histone acetylation does not affect HNF3 binding. This is evidence for stable nucleosome binding by a transcription factor and shows that a winged helix protein is sufficient to initiate the assembly of an enhancer complex on nonacetylated nucleosomes.

GATA transcription factors as potentiators of gut endoderm differentiation.

Gene inactivation studies have shown that members of the GATA family of transcription factors are critical for endoderm differentiation in mice, flies and worms, yet how these proteins function in such a conserved developmental context has not been understood. We use in vivo footprinting of mouse embryonic endoderm cells to show that a DNA-binding site for GATA factors is occupied on a liver-specific, transcriptional enhancer of the serum albumin gene. GATA site occupancy occurs in gut endoderm cells at their pluripotent stage: the cells have the potential to initiate tissue development but they have not yet been committed to express albumin or other tissue-specific genes. The GATA-4 isoform accounts for about half of the nuclear GATA-factor-binding activity in the endoderm. GATA site occupancy persists during hepatic development and is necessary for the activity of albumin gene enhancer. Thus, GATA factors in the endoderm are among the first to bind essential regulatory sites in chromatin. Binding occurs prior to activation of gene expression, changes in cell morphology or functional commitment that would indicate differentiation. We suggest that GATA factors at target sites in chromatin may generally help potentiate gene expression and tissue specification in metazoan endoderm development.

Alpha3beta1-integrin as a critical mediator of the hepatic differentiation response to the extracellular matrix.

The extracellular matrix (ECM) promotes the differentiation of many cell types, and ECM remodeling in the liver has been implicated in embryonic development, tissue injury, and oncogenesis. Integrins are heterodimeric ECM receptors that play critical roles in transducing the composition of the ECM in the cell environment. We previously showed that mouse H2.35 cells, a conditionally transformed, liver-derived cell line, assume a more differentiated hepatocyte morphology and enhanced liver-specific gene expression when the cells are cultured on gelatinous ECM substrata. Here we show that H2. 35 cells express relatively high levels of alpha3beta1-integrins, similar to that previously shown for immature hepatocytes, transformed hepatocytes, and biliary cells. However, the cell morphological responses that depend on alpha3beta1-integrin have not been defined. We found that transfecting H2.35 cells with antisense RNA construct directed to alpha3-subunit messenger RNA perturbs the initial cell attachment to laminin and collagen, and strongly inhibits cell morphological, proliferative, and gene expression responses to a collagen gel substratum. In situ hybridization to mouse embryo tissues demonstrates the presence of alpha3-subunit messenger RNAs in newly formed hepatocytes. We suggest that alpha3beta1-integrins are important for immature and transformed hepatocytes to respond morphologically to the extracellular matrix.

Chromatin structure and functional analysis of the mouse HNF3alpha gene.

The transcription factor HNF3alpha is a member of the winged-helix family of regulatory proteins. It is expressed in the definitive endoderm, notochord, and neural tube in embryos, but in the adult is expressed primarily in endoderm-derived tissues such as liver, lung, and pancreas. We present here the cloning of the mouse HNF3alpha gene and a characterization of its chromatin structure and regulatory sequences. The HNF3alpha gene is encoded by two exons and its transcription initiates at multiple start sites at a TATA-less promoter that is highly conserved between mouse and rat. We found different patterns of DNaseI hypersensitive sites in HNF3alpha gene chromatin in different adult tissues in which HNF3alpha is expressed, suggesting distinct regulatory mechanisms occurring within different tissue derivatives of the endoderm germ layer. Cell transfection data indicate that sequences spanning certain upstream hypersensitive sites can enhance transcription from the HNF3alpha promoter, but only when stably integrated into chromatin and not when transiently transfected. The results suggest a complex regulatory interplay between distinct genetic regulatory sequences that function specifically in chromatin.

Nucleosome positioning by the winged helix transcription factor HNF3.

Nucleosome positioning at genetic regulatory sequences is not well understood. The transcriptional enhancer of the mouse serum albumin gene is active in liver, where regulatory factors occupy their target sites on three nucleosome-like particles designated N1, N2, and N3. The winged helix transcription factor HNF3 binds to two sites near the center of the N1 particle. We created dinucleosome templates using the albumin enhancer sequence and found that site-specific binding of HNF3 protein resulted in nucleosome positioning in vitro similar to that seen in liver nuclei. Thus, binding of a transcription factor can position an underlying nucleosome core.

Binding of the winged-helix transcription factor HNF3 to a linker histone site on the nucleosome.

The transcription factor HNF3 and linker histones H1 and H5 possess winged-helix DNA-binding domains, yet HNF3 and other fork head-related proteins activate genes during development whereas linker histones compact DNA in chromatin and repress gene expression. We compared how the two classes of factors interact with chromatin templates and found that HNF3 binds DNA at the side of nucleosome cores, similarly to what has been reported for linker histone. A nucleosome structural binding site for HNF3 is occupied at the albumin transcriptional enhancer in active and potentially active chromatin, but not in inactive chromatin in vivo. While wild-type HNF3 protein does not compact DNA extending from the nucleosome, as does linker histone, site-directed mutants of HNF3 can compact nucleosomal DNA if they contain basic amino acids at positions previously shown to be essential for nucleosomal DNA compaction by linker histones. The results illustrate how transcription factors can possess special nucleosome-binding activities that are not predicted from studies of factor interactions with free DNA.

In vivo footprinting with limiting amounts of embryo tissues: a role for C/EBP beta in early hepatic development.

Regulatory factors important for the developmental control of genes have been identified by genetic studies or by examining the ontological expression profiles of proteins that were originally characterized in adult tissues; direct biochemical studies of transcription factors within small amounts of embryo tissues have been limited. We have found that the ligation-mediated PCR (LM-PCR) technique can detect specific dimethylsulfate modifications in genomic DNA from as few as several thousand cells, making it technically feasible to identify protein-DNA interactions in pools of nascent embryo tissues. Herein we show that LM-PCR can reveal methylation protections on the albumin gene enhancer in embryonic mouse hepatocytes, indicating occupancy of a C/EBP factor binding site. Comparison of the in vivo protection pattern with that obtained from the in vitro analysis of different C/EBP isoforms suggests that in embryonic hepatocytes, C/EBP beta is bound to the albumin gene enhancer. Detailed protocols are provided so that the approach can be used to study other genes in developing embryos.