Faculty Publications

Studies of the formation of pancreas and liver progenitors have focused on individual inductive signals and cellular responses. Here, we investigated how bone morphogenetic protein, transforming growth factor-beta (TGFbeta), and fibroblast growth factor signaling pathways converge on the earliest genes that elicit pancreas and liver induction in mouse embryos. The inductive network was found to be dynamic; it changed within hours. Different signals functioned in parallel to induce different early genes, and two permutations of signals induced liver progenitor domains, which revealed flexibility in cell programming. Also, the specification of pancreas and liver progenitors was restricted by the TGFbeta pathway. These findings may enhance progenitor cell specification from stem cells for biomedical purposes and can help explain incomplete programming in stem cell differentiation protocols.

Several recent reports, including two Cell Stem Cell papers (Zhu et al., 2009 [this issue]; Borowiak et al., 2009), screened small molecule libraries for compounds that promote embryonic stem cell differentiation. Their combined success helps bypass challenges associated with using natural protein factors and has revealed insights into controlling stem cell differentiation.

The development of functional cell populations such as hepatocytes and pancreatic beta cells from embryonic stem cell (ESC) is dependent on the efficient induction of definitive endoderm early in the differentiation process. To monitor definitive endoderm formation in mouse ESC differentiation cultures in a quantitative fashion, we generated a reporter cell line that expresses human CD25 from the Foxa3 locus and human CD4 from the Foxa2 locus. Induction of these reporter ESCs with high concentrations of activin A led to the development of a CD25-Foxa3(+) CD4-Foxa2(+) population within 4-5 days of culture. Isolation and characterization of this population showed that it consists predominantly of definitive endoderm that is able to undergo hepatic specification under the appropriate conditions. To develop reagents that can be used for studies on endoderm development from unmanipulated ESCs, from induced pluripotent stem cells, and from the mouse embryo, we generated monoclonal antibodies against the CD25-Foxa3(+) CD4-Foxa2(+) population. With this approach, we identified two antibodies that react specifically with endoderm from ESC cultures and from the early embryo. The specificity of these antibodies enables one to quantitatively monitor endoderm development in ESC differentiation cultures, to study endoderm formation in the embryo, and to isolate pure populations of culture- or embryo-derived endodermal cells. STEM CELLS 2009; 27: 2103-2113

A review. Newly specified hepatic and pancreatic progenitors, which originate from common endodermal domains, are able to reverse their course and develop into gut progenitors. Understanding what underlies such programming reversal and intrinsic regenerative capacities should illuminate the basis of cellular plasticity and facilitate targeted programming of stem cells. The liver and pancreas arise from a common multi-potent population of endoderm cells and share many aspects of their early development. Yet each tissue originates from multiple spatial domains of the endoderm, under the influence of different genes and inductive cues, and obtains different regenerative capacities. Emerging genetic evidence is illuminating the ability of newly specified hepatic and pancreatic progenitors to reverse their course and develop into gut progenitors. Understanding how tissue programming can be reversed and how intrinsic regenerative capacities are detd. should facilitate the discovery of the basis of cellular plasticity and aid in the targeted programming and growth of stem cells. [on SciFinder (R)]

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.

The Forkhead box (Fox) transcription factors play diverse roles in differentiation, development, hormone responsiveness, and aging. A pioneer activity of the Forkhead factors in developmental processes has been reported, but how this may apply to other contexts of Forkhead factor regulation remains unexplored. In this study, we address the pioneer activity of the thyroid-specific factor FoxE1 during thyroid differentiation. In response to hormone induction, FoxE1 binds to the compacted chromatin of the inactive thyroperoxidase (TPO) promoter, which coincides with the appearance of strong DNase I hypersensitivity at the FoxE1 binding site. In vitro, FoxE1 can bind to its site even when this is protected by a nucleosome, and it creates a local exposed domain specifically on H1-compacted TPO promoter-containing nucleosome arrays. Furthermore, nuclear factor 1 binds to the TPO promoter simultaneously with FoxE1, and this binding has an additive effect on FoxE1-mediated chromatin structure alteration. On the basis of our findings, we propose that FoxE1 is a pioneer factor whose primary mechanistic role in mediating the hormonal regulation of the TPO gene is to enable other regulatory factors to access the chromatin. The presented model extends the reported pioneer activity of the Forkhead factors to processes involved in hormone-induced differentiation.

The critical pancreatic transcription factor Pdx1 is expressed throughout the pancreas early, but selectively in insulin-producing beta cells postnatally. Previous studies showed that 5' conserved promoter regions Areas I and II (Pdx1(PB)) direct endocrine cell expression, while an adjacent region (Pdx1(XB)) containing the conserved Area III directs transient beta cell expression. Here we use Cre-mediated lineage tracing to track cells that activated these regions. Pdx1(PB)Cre mediated only endocrine cell recombination, while Pdx1(XB)Cre directed broad and early recombination in the developing pancreas. Also, a reporter transgene containing Areas I-II-III was expressed throughout the E10.5 pancreas, and gradually became beta cell selective, similar to endogenous Pdx1. These data suggested that sequences within Area III mediate early pancreas-wide Pdx1 expression. Area III contains a binding site for PTF1, a transcription factor complex essential for pancreas development. This site contributed to Area III-dependent reporter gene expression in the acinar AR42J cell line, while PTF1 specifically trans-activated Area III-containing reporter expression in a non-pancreatic cell line. Importantly, Ptf1a occupied sequences spanning the endogenous PTF1 site in Area III of E11.5 pancreatic buds. These data strongly suggest that PTF1 is an important early activator of Pdx1 in acinar and endocrine progenitor cells during pancreas development.

Different steps of embryonic pancreas and liver development require inductive signals from endothelial cells. During liver development, interactions between newly specified hepatic endoderm cells and nascent endothelial cells are crucial for the endoderm's subsequent growth and morphogenesis into a liver bud. Reconstitution of endothelial cell stimulation of hepatic cell growth with embryonic tissue explants demonstrated that endothelial signalling occurs independent of the blood supply. During pancreas development, midgut endoderm interactions with aortic endothelial cells induce Ptf1a, a crucial pancreatic determinant. Endothelial cells also have a later effect on pancreas development, by promoting survival of the dorsal mesenchyme, which in turn produces factors supporting pancreatic endoderm. A major goal of our laboratory is to determine the endothelial-derived molecules involved in these inductive events. Our data show that cultured endothelial cells induce Ptf1a in dorsal endoderm explants lacking an endogenous vasculature. We are purifying endothelial cell line product(s) responsible for this effect. We are also identifying endothelial-responsive regulatory elements in genes such as Ptf1a by genetic mapping and chromatin-based assays. These latter approaches will allow us to track endothelial-responsive signal pathways from DNA targets within progenitor cells. The diversity of organogenic steps dependent upon endothelial cell signalling suggests that cross-regulation of tissue development with its vasculature is a general phenomenon.

Poly(A) signals are required for efficient 3' end formation and transcriptional termination of most protein-encoding genes transcribed by RNA polymerase II. However, transcription can extend far beyond the poly(A) site before termination occurs. This implies the existence of further downstream termination signals. In mammals, a variety of sequence elements, in addition to the poly(A) site, have been implicated in the termination process. For example, termination of the human beta- and epsilon-globin genes is mediated by a sequence downstream of the poly(A) site that promotes an RNA cotranscriptional cleavage (CoTC). Here we report the identification of multiple termination sequences in the mouse serum albumin (MSA) 3' flanking region. Many transcripts from this region are cleaved cotranscriptionally, implying that such cleavage of pre-mRNA may be a more general feature of transcriptional termination.

In a recent issue of Developmental Cell, Nikolova et al. (2006) make the unusual discovery that, unlike most epithelial cells, pancreatic beta cells do not produce a basement membrane of extracellular matrix. Rather, the matrix is provided to a subset of beta cells by local capillary endothelial cells, explaining in part how the endothelium helps promote optimal levels of insulin production.

Understanding the tissue interactions that induce pancreatic progenitor cells from the embryonic endoderm provides insights into congenital malformations, tissue repair, and differentiating stem cells to a pancreatic fate. The specification of pancreatic progenitors within the dorsal endodermal epithelium has been thought to involve two phases of mesodermal interactions; first with the lateral plate mesoderm and notochord and then with aortic endothelial cells. Afterwards, branching morphogenesis of the pancreatic bud is induced by Isl-1-positive dorsal mesenchyme cells, whose growth is stimulated by factors in the circulation. Using mouse genetic models and embryo tissue explants, we show that the aortic endothelium and dorsal mesenchyme each possess an additional role in pancreatic induction, prior to the branching morphogenesis step. Specifically, we find that aortic endothelial cells promote the survival of nearby, Isl-1-positive dorsal mesenchyme, independently of factors from the circulation. Furthermore, we find that FGF10 signaling from the mesenchyme cells maintains Ptf1a expression in the dorsal pancreatic bud and appears genetically redundant with a role for the transcription factor gene HNF6 in promoting the induction of Pdx-1-positive dorsal endoderm. Together, these studies reveal a relay pathway from aortic endothelium to dorsal mesenchyme and then to the endoderm, along with functions of the dorsal mesenchyme that promote the initial differentiation of the dorsal pancreatic endoderm, prior to organ morphogenesis.

Novel binding sites for the forkhead transcription factor family member Forkhead box A (FoxA), previously referred to as Hepatocyte Nuclear Factor 3 (HNF3), were found within the mouse mammary tumor virus long terminal repeat (MMTV LTR). The effect of FoxA1 on MMTV LTR chromatin structure, and expression was evaluated in Xenopus laevis oocytes. Mutagenesis of either of the two main FoxA binding sites showed that the distal site, -232/-221, conferred FoxA1-dependent partial inhibition of glucocorticoid receptor (GR) driven MMTV transcription. The proximal FoxA binding segment consisted of two individual FoxA sites at -57/-46 and -45/-34, respectively, that mediated an increased basal MMTV transcription. FoxA1 binding altered the chromatin structure of both the inactive- and the hormone-activated MMTV LTR. Hydroxyl radical foot printing revealed FoxA1-mediated changes in the nucleosome arrangement. Micrococcal nuclease digestion showed the hormone-dependent sub-nucleosome complex, containing approximately 120 bp of DNA, to be expanded by FoxA1 binding to the proximal segment into a larger complex containing approximately 200 bp. The potential function of the FoxA1-mediated expression of the MMTV provirus for maintenance of expression in different tissues is discussed.