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Zaret K. Micrococcal nuclease analysis of chromatin structure. Curr Protoc Mol Biol. 2005 Feb;Chapter 21:Unit 21 1.
This unit describes methodology for using micrococcal nuclease to investigate the presence of nucleosomes at a particular location in chromatin and to map the positions of nucleosomes at various levels of resolution. The approaches are readily adaptable to other probes of chromatin structure that cause DNA cleavage. Results obtained from such chromatin studies provide a structural view of the molecular environment of gene in their native context in cells.
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Tremblay KD, Zaret KS. Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues. Dev Biol. 2005 Apr 1;280(1):87-99.
The location and movement of mammalian gut tissue progenitors, prior to the expression of tissue-specific genes, has been unknown, but this knowledge is essential to identify transitions that lead to cell type specification. To address this, we used vital dyes to label exposed anterior endoderm cells of early somite stage mouse embryos, cultured the embryos into the tissue bud phase of development, and determined the tissue fate of the dye labeled cells. This approach was performed at three embryonic stages that are prior to, or coincident with, foregut tissue patterning (1-3 somites, 4-6 somites, and 7-10 somites). Short-term labeling experiments tracked the movement of tissue progenitor cells during foregut closure. Surprisingly, we found that two distinct types of endoderm-progenitor cells, lateral and medial, arising from three spatially separated embryonic domains, converge to generate the epithelial cells of the liver bud. Whereas the lateral endoderm-progenitors give rise to descendants that are constrained in tissue fate and position along the anterior-posterior axis of the gut, the medial gut endoderm-progenitors give rise to descendants that stream along the anterior-posterior axis at the ventral midline and contribute to multiple gut tissues. The fate map reveals extensive morphogenetic movement of progenitors prior to tissue specification, it permits a detailed analysis of endoderm tissue patterning, and it illustrates that diverse progenitor domains can give rise to individual tissue cell types.
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Yoshitomi H, Zaret KS. Endothelial cell interactions initiate dorsal pancreas development by selectively inducing the transcription factor Ptf1a. Development. 2004 Feb;131(4):807-17.
Dorsal and ventral pancreatic bud development from the endoderm requires inductive interactions with diverse mesodermal cell types and the action of transcription factors expressed within the endoderm. Presently it is unclear which mesodermal interactions activate which pancreatic transcription factors, and whether such inductions are common for initiating dorsal and ventral pancreas development. Previous studies of Lammert et al. (Lammert, E., Cleaver, O. and Melton, D. (2001) Science 294, 564-567) showed that signaling from embryonic blood vessel cells, derived from the mesoderm, promotes pancreatic bud development. Using a combination of mouse Flk1(-/-) embryos lacking endothelial cells and tissue recombination experiments, we discovered that the initial induction of dorsal endoderm cells positive for the pancreatic and duodenal transcription factor Pdx1 does not require aorta or endothelial cell interactions, but dorsal pancreatic bud emergence and the maintenance of Pdx1 expression does. Aortal endothelial cells induce the crucial pancreatic transcription factor Ptf1a in the dorsal pancreatic endoderm; whereas the vitelline veins, which are normally adjacent to the emerging ventral pancreatic bud, are unnecessary for ventral Ptf1a induction or for ventral pancreatic bud initiation. We find that the aorta cells themselves, apart from the blood supply, cause the induction of Ptf1a in dorsal endoderm explants. Thus, endothelial cell interactions specifically promote early dorsal pancreatic development, at least in part, by inducing Ptf1a(+) pancreatic progenitors. Additionally, we find that endothelial cells are necessary for the induction of both the insulin and glucagon genes.
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Yoshitomi H, Zaret KS. Specification of liver from embryonic endoderm. Stem Cells Handbook. 2004;:345-52.
A review. During organogenesis, how do endoderm cells acquire their multipotency, become specified to different cell types, and give rise to tissue buds. The liver derives from the definitive gut endoderm, which expresses many genes in common with the visceral endoderm, which give rise to the yolk sac. The gut endoderm forms from epithelial sheets which form the foregut and hindgut, which elongate and converge at the midsection. During detn., different domains of endoderm are dependent on different groups of transcription factors, which appear to be controlled partially by preprogramming and partially by the influence of overlying mesoderm. When progenitor cells become specified, they proliferate more extensively, forming a tissue bud that extends into the surrounding mesenchymal domain. The liver tissue bud interacts with the adjacent septum transversum mesenchyme between the liver tissue bud and the developing heart (cardiogenic mesoderm). Coordinate signaling from both the cardiogenic mesoderm and septum transversum mesenchyme is required to induce liver differentiation in the ventral foregut endoderm. On the other hand, dorsal-posterior mesenchyme inhibits the induction of liver gene expression in endoderm outside the ventral foregut region. Liver progenitor cells in the ventral foregut also have the potential to undergo pancreatic differentiation. At the time of hepatic specification, there is a burst of expression of fibroblast growth factor-1 (FGF-1) and FGF-2 and persistent expression of FGF-8 in the cardiac mesoderm, and the adjacent ventral foregut expresses FGF receptor genes (FGF-R1 and FGF-R4). Inhibition of this signaling results in failure of hepatic gene expression; FGF signaling is sufficient to induce a hepatic fate, while suppressing a pancreatic fate. Bone morphogenetic proteins from the septum transversum, in conjunction with FGF signaling from the cardiogenic mesoderm, pattern the ventral foregut endoderm into liver and pancreatic cell domains. The induction of expression of liver genes is tightly coupled to morphol. changes in the cells from cuboidal to columnar and increased proliferation resulting in formation of the liver bud. The bud expands into the surrounding mesenchyme of the septum transversum with subsequent appearance of endothelial cells that will define the sinusoidal paths. Isolation and culture of uncommitted foregut endodermal cells of the mouse between the 2- and 6-somite stage is described. [on SciFinder (R)]
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Bort R, Martinez-Barbera JP, Beddington RS, Zaret KS. Hex homeobox gene-dependent tissue positioning is required for organogenesis of the ventral pancreas. Development. 2004 Feb;131(4):797-806.
In animal development, digestive tissues emerge from different positions of the endoderm as a result of patterning signals from overlying mesoderm. Although embryonic tissue movement during gastrulation generates an initial positional relationship between the endoderm and mesoderm, the role of subsequent endoderm movement against the mesoderm in patterning is unknown. At embryonic day 8.5 in the mouse, proliferation of cells at the leading edge of ventral-lateral endoderm, where the liver and ventral pancreas emerge, helps close off the foregut. During this time, the endoderm grows adjacent to and beyond the cardiogenic mesoderm, an inducer of the liver program and an inhibitor of the pancreas program. The homeobox gene Hex is expressed in this endoderm cell domain and in the liver and ventral pancreas buds, after organogenesis. We have found that in Hex(-/-) embryos, there is a complete failure in ventral pancreatic specification, while the liver program is still induced. However, when Hex-null ventral endoderm is isolated prior to its interaction with cardiogenic mesoderm and is cultured in vitro, it activates early pancreas genes. We found that Hex controls the proliferation rate, and thus the positioning, of the leading edge of endoderm cells that grow beyond the cardiogenic mesoderm, during gut tube closure. Thus, Hex-controlled positioning of endoderm cells beyond cardiogenic mesoderm dictates ventral pancreas specification. Other endodermal transcription factors may also function morphogenetically rather than by directly regulating tissue-specific programs.
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Chang J, Nicolas E, Marks D, Sander C, Lerro A, Buendia MA, Xu C, Mason WS, Moloshok T, Bort R, Zaret KS, Taylor JM. miR-122, a mammalian liver-specific microRNA, is processed from hcr mRNA and may downregulate the high affinity cationic amino acid transporter CAT-1. RNA Biol. 2004 Jul;1(2):106-13.
These studies show that miR-122, a 22-nucleotide microRNA, is derived from a liver-specific noncoding polyadenylated RNA transcribed from the gene hcr. The exact sequence of miR-122 as well as the adjacent secondary structure within the hcr mRNA are conserved from mammalian species back to fish. Levels of miR-122 in the mouse liver increase to half maximal values around day 17 of embryogenesis, and reach near maximal levels of 50,000 copies per average cell before birth. Lewis et al. (2003) predicted the cationic amino acid transporter (CAT-1 or SLC7A1) as a miR-122 target. CAT-1 protein and its mRNA are expressed in all mammalian tissues but with lower levels in adult liver. Furthermore, during mouse liver development CAT-1 mRNA decreases in an almost inverse correlation with miR-122. Eight potential miR-122 target sites were predicted within the human CAT-1 mRNA, with six in the 3'-untranslated region. Using a reporter construct it was found that just three of the predicted sites, linked in a 400-nucleotide sequence from human CAT-1, acted with synergy and were sufficient to strongly inhibit protein synthesis and reduce mRNA levels. In summary, these studies followed the accumulation during development of miR-122 from its mRNA precursor, hcr, through to identification of what may be a specific mRNA target, CAT-1.
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Mason
Taylor
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Taganov KD, Cuesta I, Daniel R, Cirillo LA, Katz RA, Zaret KS, Skalka AM. Integrase-specific enhancement and suppression of retroviral DNA integration by compacted chromatin structure in vitro. J Virol. 2004 Jun;78(11):5848-55.
Integration of viral DNA into the host chromosome is an obligatory step in retroviral replication and is dependent on the activity of the viral enzyme integrase. To examine the influence of chromatin structure on retroviral DNA integration in vitro, we used a model target comprising a 13-nucleosome extended array that includes binding sites for specific transcription factors and can be compacted into a higher-ordered structure. We found that the efficiency of in vitro integration catalyzed by human immunodeficiency virus type 1 (HIV-1) integrase was decreased after compaction of this target with histone H1. In contrast, integration by avian sarcoma virus (ASV) integrase was more efficient after compaction by either histone H1 or a high salt concentration, suggesting that the compacted structure enhances this reaction. Furthermore, although site-specific binding of transcription factors HNF3 and GATA4 blocked ASV DNA integration in extended nucleosome arrays, local opening of! H1-compacted chromatin by HNF3 had no detectable effect on integration, underscoring the preference of ASV for compacted chromatin. Our results indicate that chromatin structure affects integration site selection of the HIV-1 and ASV integrases in opposite ways. These distinct properties of integrases may also affect target site selection in vivo, resulting in an important bias against or in favor of integration into actively transcribed host DNA.
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Skalka
Katz
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Lemaigre F, Zaret KS. Liver development update: new embryo models, cell lineage control, and morphogenesis. Curr Opin Genet Dev. 2004 Oct;14(5):582-90.
The three phases of liver development that are the focus of this review are: the specification of hepatoblasts within the endoderm, the lineage split of hepatoblasts into hepatocytes and biliary cells, and the interaction of these cells with different mesodermal cell derivatives during liver morphogenesis. Advances in these areas include new genes and experimental models for studying liver development, the role of HNF6 and HNF1beta transcription factors and notch signaling in the hepatocyte-biliary cell lineage decision, the identification of genomic targets for HNF4, and HNF4's role in controlling hepatic epithelial structure and the sinusoidal organization of the liver.
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