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Biomedical subjects

E Dzierzak

Publications and source records attributed to E Dzierzak.

At least 19 recordsLinked to original sources
PubMed2003

Developmental origins of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are at the foundation of the hematopoietic hierarchy and give rise to all blood lineages in the adult organism. A thorough understanding of the molecular, cellular, and developmental biology of HSCs is of fundamental importance, but is also clinically relevant for the advancement of cell replacement therapies and transplantation protocols in blood-related genetic disease and leukemias. While the major anatomical sites of hematopoiesis change during ontogeny, it was long believed that the developmental origin of the adult mammalian hematopoietic system was the yolk sac. However, current studies have shown that the first adult-type HSCs are autonomously generated in the intrabody portion of the mouse embryo, the aorta-gonads-mesonephros (AGM) region, and sublocalize to the dorsal aorta. HSCs are also found in the other large embryonic vessels, the vitelline and umbilical arteries. The intraluminal hematopoietic clusters along these vessels, together with the role of the Runx1 transcription factor in cluster and HSC formation and the HSC/endothelial/mesenchymal Runxl expression pattern, strongly suggest a vascular endothelial/mesenchymal origin for the first HSCs. Moreover, a transgenic mouse line expressing the GFP marker under the control of the Sca-1 transcriptional regulatory elements (GFP expression marks all HSCs) shows a clear localization of GFP-expressing cells to the endothelial cell layer of the dorsal aorta. Thus, highly enriched GFP-positive AGM HSCs will serve as a basis for the future examination of the cellular and molecular factors involved in the induction and expansion of adult HSCs.

Animals↗
PubMed2001

Cloning of the Ly-6A (Sca-1) gene locus and identification of a 3' distal fragment responsible for high-level gamma-interferon-induced expression in vitro.

The Ly-6A and Ly-6E allelic genes encode the Sca-1 protein, which is one of the most widely used markers in haematopoietic stem cell isolation procedures. Identification of the specific gene regulatory elements that direct haematopoietic stem cell specific expression of Sca-1 is of current interest for purposes of stem cell manipulation. Both the Ly-6E and Ly-6A alleles have been examined for regions containing DNase I hypersensitive sites thought to be indicative of transcriptional regulatory elements. In these previous studies, the Ly-6E allele with its flanking regulatory sequences was cloned, and the region responsible for high-level gamma-interferon (gamma-IFN)-induced expression was localized to a 3' distal sequence containing two strong DNase1 hypersensitive sites. Because the Ly-6A allele is thought to provide higher levels of expression in haematopoietic stem cells, we isolated over 25 kb of the Ly-6A gene and flanking regulatory regions. We show here that sequences analogous to those in the Ly-6E allele are responsible for high-level gamma-IFN-induced expression in vitro. Furthermore, we show that this 3' distal Ly-6A fragment directs high-level gamma-IFN-induced expression from a heterologous promoter, suggesting that it is a potent enhancer that could be useful for expression in haematopoietic stem cells.

Alleles↗
PubMed2000

CFU-S(11) activity does not localize solely with the aorta in the aorta-gonad-mesonephros region.

The aorta-gonad-mesonephros (AGM) region is a potent hematopoietic site in the midgestation mouse conceptus and first contains colony-forming units-spleen day 11 (CFU-S(11)) at embryonic day 10 (E10). Because CFU-S(11) activity is present in the AGM region before the onset of hematopoietic stem cell (HSC) activity, CFU-S(11) activity in the complex developing vascular and urogenital regions of the AGM was localized. From E10 onward, CFU-S(11) activity is associated with the aortic vasculature, and is found also in the urogenital ridges (UGRs). Together with data obtained from organ explant cultures, in which up to a 16-fold increase in CFU-S(11) activity was observed, it was determined that CFU-S(11) can be increased autonomously both in vascular sites and in UGRs. Furthermore, CFU-S(11) activity is present in vitelline and umbilical vessels. This, together with the presence of CFU-S(11) in the UGRs 2 days before HSC activity, suggests both temporally and spatially distinct emergent sources of CFU-S(11). (Blood. 2000;96:2902-2904)

Animals↗
PubMed2000

Definitive hematopoietic stem cells first develop within the major arterial regions of the mouse embryo.

The aorta-gonad-mesonephros (AGM) region is a potent hematopoietic site within the mammalian embryo body, and the first place from which hematopoietic stem cells (HSCs) emerge. Within the complex embryonic vascular, excretory and reproductive tissues of the AGM region, the precise location of HSC development is unknown. To determine where HSCs develop, we subdissected the AGM into aorta and urogenital ridge segments and transplanted the cells into irradiated adult recipients. We demonstrate that HSCs first appear in the dorsal aorta area. Furthermore, we show that vitelline and umbilical arteries contain high frequencies of HSCs coincident with HSC appearance in the AGM. While later in development and after organ explant culture we find HSCs in the urogenital ridges, our results strongly suggest that the major arteries of the embryo are the most important sites from which definitive HSCs first emerge.

Animals↗
PubMed2000

Haploinsufficiency of AML1 affects the temporal and spatial generation of hematopoietic stem cells in the mouse embryo.

The AML1:CBFbeta transcription factor complex is essential for definitive hematopoiesis. Null mutations in mouse AML1 result in midgestational lethality with a complete lack of fetal liver hematopoiesis. While the cell autonomous nature and expression pattern of AML1 suggest an intrinsic role for this transcription factor in the developing hematopoietic system, no direct link to a functional cell type has been made. Here, we examine the consequences of AML1 loss in hematopoietic stem cells (HSC) of the mouse embryo. We demonstrate an absolute requirement for AML1 in functional HSCs. Moreover, haploinsufficiency results in a dramatic change in the temporal and spatial distribution of HSCs, leading to their early appearance in the normal position in the aorta-gonad-mesonephros region and also in the yolk sac.

Animals↗
PubMed1999

Embryonic beginnings of definitive hematopoietic stem cells.

The ability of the many cell types within the adult blood system to be constantly replenished and renewed from hematopoietic stem cells is an interesting problem in development and differentiation and has led to questions concerning how, when and where these stem cells for the adult hematopoietic system are generated within the embryo. During embryonic development many mature hematopoietic cells appear before adult-type hematopoietic stem cells thus the notion of a conventional hematopoietic hierarchy is challenged. Experiments probing the development of hematopoietic stem cells in the mouse embryo strongly suggest that at least two independent hematopoietic sites generate blood cells during development; the yolk sac, which produces the transient embryonic hematopoietic system, and the AGM (aorta-gonad-mesonephros) region, which initiates the long-lived adult hematopoietic system.

Animals↗
PubMed1998

High level inhibition of HIV replication with combination RNA decoys expressed from an HIV-Tat inducible vector.

Intracellular immunization, an antiviral gene therapy approach based on the introduction of DNA into cells to stably express molecules for the inhibition of viral gene expression and replication, has been suggested for inhibition of HIV infection. Since the Tat and Rev proteins play a critical role in HIV regulation, RNA decoys and ribozymes of these sequences have potential as therapeutic molecular inhibitors. In the present study, we have generated several anti-HIV molecules; a tat-ribozyme, RRE, RWZ6 and TAR decoys and combinations of decoys, and tested them for inhibition of HIV-1 replication in vitro. We used T cell specific CD2 gene elements and regulatory the HIV inducible promoter to direct high level expression and a 3' UTR sequence for mRNA stabilization. We show that HIV replication was most strongly inhibited with the combination TAR + RRE decoy when compared with the single decoys or the tat-ribozyme. We also show that the Tat-inducible HIV promoter directs a higher level of steady-state transcription of decoys and inhibitors and that higher levels of expression directly relate to increased levels of inhibition of HIV infection. Furthermore, a stabilization of the 3' end of TAR + RRE inhibitor transcripts using a beta-globin 3' UTR sequence leads to an additional 15-fold increase in steady-state RNA levels. This cassette when used to express the best combination decoy inhibitor TAR + RRE, yields high level HIV inhibition for greater than 3 weeks. Taken together, both optimization for high level expression of molecular inhibitors and use of combinations of inhibitors suggest better therapeutic application in limiting the spread of HIV.

Cell Line↗
PubMed1997

Expression of the Ly-6E.1 (Sca-1) transgene in adult hematopoietic stem cells and the developing mouse embryo.

The mouse hematopoietic marker Sca-1, encoded by the Ly-6E.1 and Ly-6A.2 genes, has been instrumental in the enrichment and characterization of the stem cell for the adult blood system. In the studies reported here, we use Ly-6E.1 genomic fragments to direct expression of a lacZ marker transgene in vivo to study Ly-6E.1 specific regulatory elements in the hematopoietic stem cell and to localize these cells in the developing mouse embryo. We demonstrate that a region approximately 9 kb downstream from the transcriptional start site is required for the distinct, restricted expression pattern of the Ly-6E.1-lacZ transgene within adult hematopoietic stem cells and embryos. We also demonstrate that viable and functional lacZ-expressing hematopoietic stem cells can be enriched by FDG staining and flow cytometric sorting. The Ly-6E.1-lacZ-mediated enrichment of hematopoietic stem cells from adult transgenic bone marrow in combination with the temporal expression pattern of the transgene in the pro/mesonephros suggest an intraembryonic site of development for these cells in the mouse.

Animals↗
PubMed1996

Definitive hematopoiesis is autonomously initiated by the AGM region.

The adult hematopoietic system of mammals is a dynamic hierarchy of cells with the hematopoietic stem cell at its foundation. During embryonic development, the source and expansion potential of this cell remain unclear. Two sites of hematopoietic activity, the yolk sac and aorta-gonad-mesonephros (AGM) region, function in mouse ontogeny at the pre-liver stage of hematopoiesis. However, cellular interchange between these tissues obscures the embryonic site of hematopoietic stem cell generation. Here we present the results of a novel in vitro organ culture system demonstrating that, at day 10 in gestation, hematopoietic stem cells initiate autonomously and exclusively within the AGM region. Furthermore, we provide evidence for the in vitro expansion of hematopoietic stem cells within the AGM region. These results strongly suggest that the AGM region is the source of the definitive adult hematopoietic system, which subsequently colonizes the liver.

Animals↗
PubMed1996

The Ly-6E.1 (Sca-1) gene requires a 3' chromatin-dependent region for high-level gamma-interferon-induced hematopoietic cell expression.

The Ly-6E.1/A.2 gene product recognized by the Sca-1 antibody has been found on murine hematopoietic stem cells and some hematopoietic precursors, T lymphocytes, and nonhematopoietic cell lineages, suggesting a complex array of gene regulatory elements. The ability to use the Ly6E.1/A.2 transcriptional regulatory elements to direct expression of heterologous genes will allow for the manipulation of these cells during development and in hematopoietic cell transplantations. To identify the elements necessary for high-level expression, we have made deletion constructs of Ly-6E.1 gene flanking regions containing DNase I hypersensitive sites, tested them for expression in hematopoietic cells, and have performed kinetic analyses to correlate the appearance of hypersensitive sites with gene transcription and protein expression. We show that a 3' region containing two DNase I hypersensitive sites at +8.7 and +8.9 kb is required for high-level, gamma-interferon (gamma-IFN)-induced expression of the Ly-6E.1 gene and that a consensus sequence for a gamma-IFN-responsive element localizes to the +8.7 site. We also provide a description of allele- and cell-specific DNase I hypersensitive site patterns of the Ly-6E.1 and Ly-6A.2 genes. Taken together, these data indicate that while both 5' and 3' hypersensitive sites are rapidly induced with gamma-IFN, the 3' most distal hypersensitive sites are involved in directing high levels of expression of Sca-1 in hematopoietic cells.

Animals↗
PubMed1996

Characterization of the first definitive hematopoietic stem cells in the AGM and liver of the mouse embryo.

At day 10 in mouse gestation, the intraembryonic aorta-gonads-mesonephros (AGM) region generates the first definitive hematopoietic stem cells (HSCs) of the adult blood system. By 11 days postcoitum, the liver contains such HSCs. While HSCs of the adult bone marrow and late-stage fetal liver have been extensively characterized for cell surface markers, there has been no phenotypic description of the first HSCs during embryo development. We report here the temporal cell surface phenotype of HSCs from the AGM region and early fetal liver and show that all HSCs reside in the c-kit+ population. c-kit+ HSCs from AGM and liver are mainly CD34+ and in the AGM are in both Mac-1+ and Mac-1 fractions. These results demonstrate that during mouse ontogeny the first definitive HSCs are similar in cell surface phenotype to the HSCs of adult bone marrow but that spatial localization and developmental time are critical factors in the phenotypic assessment of this functional cell population.

Animals↗
PubMed1995

Mouse embryonic hematopoiesis.

The hematopoietic system of vertebrates is derived from the mesodermal germ layer in early embryogenesis. Various animal models have been used for the study of hematopoiesis, from early stages in the visceral yolk sac or its analog, to the later stages where hematopoiesis is observed in intraembryonic areas surrounding the aorta, genital ridge and pro/mesonephros. Using the mouse as a model, we describe what is known about mammalian embryonic hematopoiesis and put it in the context of hematopoietic cell formation in avian, amphibian and fish embryos. Evolutionary comparisons and recent experimental evidence show that there are two embryonic sites of developing hematopoietic activity in the mouse before fetal liver hematopoiesis and suggest that, during ontogeny, two successive waves of hematopoietic activity may contribute to the blood system of the adult.

Animals↗
PubMed1995

Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis.

GATA-3 is one member of a growing family of related transcription factors which share a strongly conserved expression pattern in all vertebrate organisms. In order to elucidate GATA-3 function using a direct genetic approach, we have disrupted the murine gene by homologous recombination in embryonic stem cells. Mice heterozygous for the GATA3 mutation are fertile and appear in all respects to be normal, whereas homozygous mutant embryos die between days 11 and 12 postcoitum (p.c.) and display massive internal bleeding, marked growth retardation, severe deformities of the brain and spinal cord, and gross aberrations in fetal liver haematopoiesis.

Abnormalities, Multiple↗
PubMed1994

Isolation of tyrosine kinase related genes expressed in the early hematopoietic system.

Transmembrane tyrosine kinase receptors are involved in cellular interactions which promote proliferation and differentiation of many cell types. To identify receptor tyrosine kinases important in embryonic hematopoietic cell development we have utilized the polymerase chain reaction (PCR) and degenerate oligonucleotides for isolation of such genes from mouse yolk sac and fetal liver. Sequence analysis of PCR amplified cDNAs from these hematopoietic sites of day 8 and 14 embryos, resulted in the isolation of nine tyrosine kinase and three serine/threonine kinase related clones. Two of these receptors, tek and flk-1, are expressed in both yolk sac and fetal liver and have been shown previously to be important for endothelial cell development. Two other clones, 9B4 and 9A2 appeared novel upon isolation but have been recently described as ryk and SK2 (rat homologue). Here we describe the twelve isolated kinases, the specific expression patterns of flk-1, tek and ryk kinases and their potential relationship to the development of the hematopoietic system.

Animals↗