Biomedical subjects

Andras Nagy

Publications and source records attributed to Andras Nagy.

At least 19 recordsLinked to original sources

PubMed2006

Enhanced natural-killer cell and erythropoietic activities in VEGF-A-overexpressing mice delay F-MuLV-induced erythroleukemia.

We have previously reported that VEGF-A, in combination with MCP-5, contributes to leukemia progression within the splenic microenvironment of mice infected with F-MuLV. To study the influence of constitutively elevated VEGF-A levels on the progression of erythroleukemia, mice heterozygous for a VEGF-A "hypermorphic" allele (Vegfhi/+) were inoculated with F-MuLV. Unexpectedly, a significant delay in erythroleukemia was observed in Vegfhi/+ mice when compared with wild-type controls. These results suggested an altered physiologic response arising from elevated VEGF-A levels that decelerated erythroleukemic progression. Characterization of hematopoiesis in Vegfhi/+ spleens showed a higher natural killer cell activity, elevated B cells, and a decrease in T-cell number. Furthermore, higher erythroid progenitors (ie, CD34+, CD36+, and Ter119+ cells) were evident in the bone marrow, spleen, and peripheral blood of Vegfhi/+ mice. The CFU-E levels were significantly elevated in Vegfhi/+ bone marrow cultures, and this elevation was blocked by a neutralizing antibody to VEGF-A receptor (VEGFR-2). Moreover, erythroleukemic mice were treated with recombinant erythropoietin and, similar to diseased Vegfhi/+ mice, showed a delay in disease progression. We propose that a compensatory erythropoietic response combined with increased natural killer (NK) cell activity account for the extended survival of erythroleukemic, Vegfhi/+ mice.

Animals↗

PubMed2006

Placental but not heart defects are associated with elevated hypoxia-inducible factor alpha levels in mice lacking prolyl hydroxylase domain protein 2.

PHD1, PHD2, and PHD3 are prolyl hydroxylase domain proteins that regulate the stability of hypoxia-inducible factor alpha subunits (HIF-alpha). To determine the roles of individual PHDs during mouse development, we disrupted all three Phd genes and found that Phd2(-/-) embryos died between embryonic days 12.5 and 14.5 whereas Phd1(-/-) or Phd3(-/-) mice were apparently normal. In Phd2(-/-) mice, severe placental and heart defects preceded embryonic death. Placental defects included significantly reduced labyrinthine branching morphogenesis, widespread penetration of the labyrinth by spongiotrophoblasts, and abnormal distribution of trophoblast giant cells. The expression of several trophoblast markers was also altered, including an increase in the spongiotrophoblast marker Mash2 and decreases in the labyrinthine markers Tfeb and Gcm1. In the heart, trabeculae were poorly developed, the myocardium was remarkably thinner, and interventricular septum was incompletely formed. Surprisingly, while there were significant global increases in HIF-alpha protein levels in the placenta and the embryo proper, there was no specific HIF-alpha increase in the heart. Taken together, these data indicate that among all three PHD proteins, PHD2 is uniquely essential during mouse embryogenesis.

Animals↗

PubMed2006

Vascular endothelial growth factor directly inhibits primitive neural stem cell survival but promotes definitive neural stem cell survival.

There are two types of neural stem cells (NSCs). Primitive NSCs [leukemia inhibitory factor (LIF) dependent but exogenous fibroblast growth factor (FGF) 2 independent] can be derived from mouse embryonic stem (ES) cells in vitro and from embryonic day 5.5 (E5.5) to E7.5 epiblast and E7.5-E8.5 neuroectoderm in vivo. Definitive NSCs (LIF independent but FGF2 dependent) first appear in the E8.5 neural plate and persist throughout life. Primitive NSCs give rise to definitive NSCs. Loss and gain of functions were used to study the role of vascular endothelial growth factor (VEGF)-A and its receptor, Flk1, in NSCs. The numbers of Flk1 knock-out mice embryo-derived and ES cell-derived primitive NSCs were increased because of the enhanced survival of primitive NSCs. In contrast, neural precursor-specific, Flk1 conditional knock-out mice-derived, definitive NSCs numbers were decreased because of the enhanced cell death of definitive NSCs. These effects were not observed in cells lacking Flt1, another VEGF receptor. In addition, the cell death stimulated by VEGF-A of primitive NSC and the cell survival stimulated by VEGF-A of definitive NSC were blocked by Flk1/Fc-soluble receptors and VEGF-A function-blocking antibodies. These VEGF-A phenotypes also were blocked by inhibition of the downstream effector nuclear factor kappaB (NF-kappaB). Thus, both the cell death of primitive NSC and the cell survival of definitive NSC induced by VEGF-A stimulation are mediated by bifunctional NF-kappaB effects. In conclusion, VEGF-A function through Flk1 mediates survival (and not proliferative or fate change) effects on NSCs, specifically.

Animals↗

PubMed2006

Vascular endothelial growth factor a signaling in the podocyte-endothelial compartment is required for mesangial cell migration and survival.

The glomerular filtration barrier separates the blood from the urinary space and consists of two major cell types: podocytes and fenestrated endothelial cells. Mesangial cells sit between the capillary loops and provide structural support. Proliferation and loss of mesangial cells both are central findings in a number of renal diseases, including diabetic nephropathy and mesangiolysis, respectively. Using cell-specific gene targeting, it was shown previously that vascular endothelial growth factor A (VEGF-A) production by podocytes is required for glomerular endothelial cell migration, differentiation, and survival. For further investigation of the effect of gene dose and VEGF-A knockdown within the glomerulus, mice that carry one hypomorphic VEGF-A allele and one podocyte-specific null VEGF-A allele (VEGFhypo/loxP,Neph-Cre+/-) were generated; in these mice, the "allelic dose" of VEGF-A is intermediate between glomerular-specific heterozygous and null states. VEGFhypo/loxP,Neph-Cre+/- mice die at 3 wk of age from renal failure. Although endothelial cell defects are observed, striking loss of mesangial cells occurs postnatally. In addition, differentiated mesangial cells cannot be found in glomeruli of podocyte-specific null VEGF-A mice (VEGFloxP/loxP,Cre+/-). Together, these results demonstrate a key role for VEGF-A production in the podocyte for mesangial cell survival and differentiation.

Animals↗

PubMed2006

Murine embryonic stem cells.

Embryonic stem (ES) cells are derived from preimplantation stage mouse embryos at the time when they have reached the blastocyst stage. It is at this point that the first steps of differentiation take place during mammalian embryonic development. The individual blastomeres now start to organize themselves into three distinct locations, each encompassing a different cell type: outside epithelial cells, trophectoderm; cells at the blastocele surface of the inner cell mass (ICM), the primitive endoderm; and inside cells of the ICM, the primitive ectoderm. ES cells originate from the third population, the primitive ectoderm, which is a transiently existing group of cells in the embryo. Primitive ectoderm cells diminish within a day as the embryo is entering into the next steps of differentiation. ES cells, however, while retaining the property of their origin in terms of developmental potential, also have the ability to self-renew. It is hence important to realize that ES cells do not exist in vivo; they should be regarded simply as tissue culture artifact. Nevertheless, these powerful cells have the potential to differentiate into all the cells of the embryo proper and postnatal animal. Furthermore, they retain the limitation of their origin through their inability to contribute to the trophectoderm lineage (the trophoblast of the placenta) and the lineages of the primitive endoderm, the visceral and parietal endoderm. Due to these unique features, we must admit that even if we regard ES cells as products of in vitro culture and should not compare them to true somatic stem cells found in the adult organism, they certainly offer us a fantastic tool for genetic, developmental, and disease studies.

Animals↗

PubMed2005

"Agouti NOD": identification of a CBA-derived Idd locus on Chromosome 7 and its use for chimera production with NOD embryonic stem cells.

Penetrance of the complex of genes predisposing the nonobese diabetic (NOD) mouse to autoimmune diabetes is affected by the maternal environment. NOD.CBALs-Tyr(+)/Lt is an agouti-pigmented Chromosome 7 congenic stock of NOD/Lt mice produced as a resource for embryo transfer experiments to provide the necessary maternal factors and allow the easy identification of NOD (albino) embryo donor phenotype. CBcNO6/Lt, a recombinant congenic agouti stock already containing approximately 50% NOD genome, was used as the donor source of a wild-type CBA tyrosinase allele. When the incidence of diabetes was assessed after nine generations of backcrossing and one generation of sib-sib mating, significant reduction in diabetes development was observed. No difference in diabetes development was observed in Tyr/Tyr(c) heterozygotes, showing that protection was recessive. Analysis of diabetes progression in another NOD stock congenic for C57BL/6 alleles on Chromosome 7 linked to the glucose phosphate isomerase (Gpi1(b)) locus provided no protection, indicating that the diabetes resistance (Idd) gene was distal to 34 cM (D7Mit346). Approximately 5 cM of the distal congenic region overlaps a region from C57L previously associated with protection when homozygous. The delayed onset and reduced frequency of diabetes in the NOD.CBALs-Tyr(+)/Lt stock is an advantage when females of this stock are used as surrogate mothers in studies involving hysterectomy or embryo transfers. Indeed, a newly developed NOD embryonic stem (ES) cell line injected into NOD.CBALs- Tyr(+)/Lt blastocysts produced approximately 50% live-born mice, of which approximately 11% were chimeric. Presumably because of high genomic instability, no germline transmission was observed.

Animals↗

PubMed2005

Tbx20 dose-dependently regulates transcription factor networks required for mouse heart and motoneuron development.

To elucidate the function of the T-box transcription factor Tbx20 in mammalian development, we generated a graded loss-of-function series by transgenic RNA interference in entirely embryonic stem cell-derived mouse embryos. Complete Tbx20 knockdown resulted in defects in heart formation, including hypoplasia of the outflow tract and right ventricle, which derive from the anterior heart field (AHF), and decreased expression of Nkx2-5 and Mef2c, transcription factors required for AHF formation. A mild knockdown led to persistent truncus arteriosus (unseptated outflow tract) and hypoplastic right ventricle, entities similar to human congenital heart defects, and demonstrated a critical requirement for Tbx20 in valve formation. Finally, an intermediate knockdown revealed a role for Tbx20 in motoneuron development, specifically in the regulation of the transcription factors Isl2 and Hb9, which are important for terminal differentiation of motoneurons. Tbx20 could activate promoters/enhancers of several genes in cultured cells, including the Mef2c AHF enhancer and the Nkx2-5 cardiac enhancer. The Mef2c AHF enhancer relies on Isl1- and Gata-binding sites. We identified a similar Isl1 binding site in the Nkx2-5 AHF enhancer, which in transgenic mouse embryos was essential for activity in a large part of the heart, including the outflow tract. Tbx20 synergized with Isl1 and Gata4 to activate both the Mef2c and Nkx2-5 enhancers, thus providing a unifying mechanism for gene activation by Tbx20 in the AHF. We conclude that Tbx20 is positioned at a critical node in transcription factor networks required for heart and motoneuron development where it dose-dependently regulates gene expression.

Animals↗

PubMed2005

Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction.

Here we describe a triple transgenic mouse system, which combines the tissue specificity of any Cre-transgenic line with the inducibility of the reverse tetracycline transactivator (rtTA)/tetracycline-responsive element (tet-O)-driven transgenes. To ensure reliable rtTA expression in a broad range of cell types, we have targeted the rtTA transgene into the ROSA26 locus. The rtTA expression, however, is conditional to a Cre recombinase-mediated excision of a STOP region from the ROSA26 locus. We demonstrate the utility of this technology through the inducible expression of the vascular endothelial growth factor (VEGF-A) during embryonic development and postnatally in adult mice. Our results of adult induction recapitulate several different hepatic and immune cell pathological phenotypes associated with increased systemic VEGF-A protein levels. This system will be useful for studying genes in which temporal control of expression is necessary for the discovery of the full spectrum of functions. The presented approach abrogates the need to generate tissue-specific rtTA transgenes for tissues where well-characterized Cre lines already exist.

Animals↗

PubMed2005

Phenotypic analyses of mouse embryos with ubiquitous expression of Oct4: effects on mid-hindbrain patterning and gene expression.

Oct4 is a transcription factor that has been associated with pluripotency and fate determination in the initial cell lineages of mammals. On the other hand, Pou2, the ortholog of Oct4 in zebrafish, serves additional later functions during brain development acting as a differentiation switch. In mice, Oct4 is expressed throughout the neural plate of embryos until embryonic day (E) 8.0. In this study, we produced transgenic mouse embryos that ubiquitously express Oct4 and analyzed the consequences during development. We show that, at E8.0, a higher dosage of Oct4 in the neuroectoderm is sufficient to transiently alter mid-hindbrain patterning and produced a strong up-regulation of Pax2, indicating that Oct4 can regulate this gene in vivo. After E9.5, ectopic Oct4 in this region produced cell death and affected the development of the forebrain, suggesting that, at these later stages, Oct4 down-regulation is necessary for normal development to proceed. The phenotype of the transgenic embryos was also accompanied with an increase of Fgf8 expression in several of its endogenous domains, suggesting the possibility that Oct4 can participate in the regulation of expression of this ligand. Our observations support the hypothesis that Oct4, like zebrafish Pou2, has a conserved function during early brain patterning in mouse.

Animals↗

PubMed2005

Ectopic expression of KitD814Y in spermatids of transgenic mice, interferes with sperm morphogenesis.

Kit is a receptor tyrosine kinase that plays a fundamental role during the development of germ cells. Additionally, a truncated product, tr-kit, expressed in haploid spermatids and mature spermatozoa can induce parthenogenetic activation when microinjected into mouse eggs, through the activation of PLCgamma-1. In this work, we induced ectopic expression of a mutated Kit protein, Kit(D814Y) during germ cell development. The in vivo expression of this mutant in spermatids produced malformations in mature spermatozoa, and in the most severe cases, sterility. Ultrastructural analysis indicated that condensing spermatids in the transgenic mouse presented a mislocalization of the manchette; a structure that has a crucial role during the elongation steps of spermiogenesis. This morphogenetic phenotype was accompanied by an increased phosphorylation of PLCgamma-1 in spermatogenic cells. Interestingly, we also found that, in wild-type testis, PLCgamma-1 is specifically phosphorylated in condensing spermatids, coincident with the timing of expression of tr-kit in spermiogenesis. We propose that alterations of PLCgamma-1 activity artificially promoted by ectopic Kit(D814Y) expression are related to the abnormalities of spermiogenesis. Our observations suggest that PLCgamma-1 activity could be involved in the shaping of spermatozoa.

Animals↗

PubMed2005

Donor hematopoietic cells from transgenic mice that express GFP are immunogenic in immunocompetent recipients.

OBJECTIVE: To study the immunogenicity of hematopoietic cells marked with green fluorescence protein (GFP) while avoiding the potentially confounding effects of viral gene transduction, marked cells from GFP+ transgenic mice were tracked after transplantation into unconditioned immunocompetent recipients. MATERIALS AND METHODS: Marrow was harvested from GFP+ transgenic mice that had been crossed onto a BALB/cByJ background. Unconditioned marrow transplantation involved infusion of sex-matched or sex-mismatched cells into female BALB/cByJ hosts. Engraftment and contribution to circulating nucleated blood cells were compared to recipients of donor cells that were not GFP-marked. Donor cells were detected by flow cytometry (GFP) and fluorescence in situ hybridization (FISH) for Y-chromosome sequences. RESULTS: Donor cells from mice of the same genetic background that did not express GFP were detected for more than four-weeks in unconditioned recipients. In contrast, GFP-marked cells in the blood peaked at one-week, declined to undetectable levels by two-weeks and were not detected in the marrow at sacrifice. In sex-mismatched studies, detection of male GFP+ donor cells by FISH yielded levels similar to those observed by flow cytometry, in contrast to the levels detected for many weeks in mice infused with male cells that did not express GFP. In immunocompetent recipients immunized with irradiated GFP-expressing cells, rechallenge with GFP+ cells resulted in the accelerated loss of donor cells. CONCLUSION: Donor marrow cells from GFP+ transgenic mice were lost after infusion into unconditioned immunocompetent mice and sensitization studies infer an immunologic mechanism. These results are similar to studies of virally transduced cells. Thus, infusion of cells with optimum engraftment potential could not compensate for the loss of donor cells due to immunogenicity.

Animals↗

PubMed2004

Vascular endothelial growth factor controls neuronal migration and cooperates with Sema3A to pattern distinct compartments of the facial nerve.

Developing neurons accurately position their somata within the neural tube to make contact with appropriate neighbors and project axons to their preferred targets. Taking advantage of a collection of genetically engineered mouse mutants, we now demonstrate that the behavior of somata and axons of the facial nerve is regulated independently by two secreted ligands for the transmembrane receptor neuropilin 1 (Nrp1), the semaphorin Sema3A and the VEGF164 isoform of Vascular Endothelial Growth Factor. Although Sema3A is known to control the guidance of facial nerve axons, we now show that it is not required for the pathfinding of their somata. Vice versa, we find that VEGF164 is not required for axon guidance of facial motor neurons, but is essential for the correct migration of their somata. These observations demonstrate, for the first time, that VEGF contributes to neuronal patterning in vivo, and that different compartments of one cell can be co-ordinately patterned by structurally distinct ligands for a shared receptor.

Animals↗

PubMed2004

The gut microbiota as an environmental factor that regulates fat storage.

New therapeutic targets for noncognitive reductions in energy intake, absorption, or storage are crucial given the worldwide epidemic of obesity. The gut microbial community (microbiota) is essential for processing dietary polysaccharides. We found that conventionalization of adult germ-free (GF) C57BL/6 mice with a normal microbiota harvested from the distal intestine (cecum) of conventionally raised animals produces a 60% increase in body fat content and insulin resistance within 14 days despite reduced food intake. Studies of GF and conventionalized mice revealed that the microbiota promotes absorption of monosaccharides from the gut lumen, with resulting induction of de novo hepatic lipogenesis. Fasting-induced adipocyte factor (Fiaf), a member of the angiopoietin-like family of proteins, is selectively suppressed in the intestinal epithelium of normal mice by conventionalization. Analysis of GF and conventionalized, normal and Fiaf knockout mice established that Fiaf is a circulating lipoprotein lipase inhibitor and that its suppression is essential for the microbiota-induced deposition of triglycerides in adipocytes. Studies of Rag1-/- animals indicate that these host responses do not require mature lymphocytes. Our findings suggest that the gut microbiota is an important environmental factor that affects energy harvest from the diet and energy storage in the host. Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AY 667702--AY 668946).

Adipocytes↗

PubMed2004

A specific requirement for PDGF-C in palate formation and PDGFR-alpha signaling.

PDGF-C is a member of the platelet-derived growth factor (PDGF) family, which signals through PDGF receptor (PDGFR) alphaalpha and alphabeta dimers. Here we show that Pdgfc(-/-) mice die in the perinatal period owing to feeding and respiratory difficulties associated with a complete cleft of the secondary palate. This phenotype was less severe than that of Pdgfra(-/-) embryos. Pdgfc(-/-) Pdgfa(-/-) embryos developed a cleft face, subepidermal blistering, deficiency of renal cortex mesenchyme, spina bifida and skeletal and vascular defects. Complete loss of function of both ligands, therefore, phenocopied the loss of PDGFR-alpha function, suggesting that both PDGF-A and PDGF-C signal through PDGFR-alpha to regulate the development of craniofacial structures, the neural tube and mesodermal organs. Our results also show that PDGF-C signaling is a new pathway in palatogenesis, different from, and independent of, those previously implicated.

Abnormalities, Multiple↗

PubMed2004

Regulation of murine telomere length by Rtel: an essential gene encoding a helicase-like protein.

Little is known about the genes that regulate telomere length diversity between mammalian species. A candidate gene locus was previously mapped to a region on distal mouse Chr 2q. Within this region, we identified a gene similar to the dog-1 DNA helicase-like gene in C. elegans. We cloned this Regulator of telomere length (Rtel) gene and inactivated its expression in mice. Rtel(-/-) mice died between days 10 and 11.5 of gestation with defects in the nervous system, heart, vasculature, and extraembryonic tissues. Rtel(-/-) embryonic stem cells showed telomere loss and displayed many chromosome breaks and fusions upon differentiation in vitro. Crosses of Rtel(+/-) mice with Mus spretus showed that Rtel from the Mus musculus parent is required for telomere elongation of M. spretus chromosomes in F1 cells. We conclude that Rtel is an essential gene that regulates telomere length and prevents genetic instability.

Abnormalities, Multiple↗

PubMed2004

Hypoxia and hypoxia-inducible factor-1 target genes in central nervous system radiation injury: a role for vascular endothelial growth factor.

PURPOSE: Microvascular permeability changes and loss of blood-brain barrier integrity are important features of central nervous system (CNS) radiation injury. Expression of vascular endothelial growth factor (VEGF), an important determinant of microvascular permeability, was examined to assess its role in CNS radiation damage. Because hypoxia mediates VEGF up-regulation through hypoxia-inducible factor-1alpha (HIF1alpha) induction, we studied the relationships of hypoxia, HIF1alpha expression, and expression of VEGF in this damage pathway. EXPERIMENTAL DESIGN: Expression of HIF1alpha, VEGF, and another hypoxia-responsive gene, glucose transporter-1, was assessed in the irradiated rat spinal cord using immunohistochemistry and in situ hybridization. Hypoxic areas were identified using the nitroimidazole 2-(2-nitro-1H-imidazole-L-yl)-N-(2,2,3,3,3,-pentafluoropropyl) acetamide. To determine the causal importance of VEGF expression in radiation myelopathy, we studied the response of transgenic mice with greater (VEGF-A(hi/+)), reduced (VEGF-A(lo/+)), and wild-type VEGF activity to thoracolumbar irradiation. RESULTS: In rat spinal cord, the number of cells expressing HIF1alpha and VEGF increased rapidly from 16 to 20 weeks after radiation, before white matter necrosis and forelimb paralysis. A steep dose response was observed in expression of HIF1alpha and VEGF. HIF1alpha and VEGF expressing cells were identified as astrocytes. Hypoxia was present in regions where up-regulation of VEGF and glucose transporter-1 and increased permeability was observed. VEGF-A(lo/+) mice had a longer latency to development of hindlimb weakness and paralysis compared with wild-type or VEGF-A(hi/+) mice. CONCLUSIONS: VEGF expression appears to play an important role in CNS radiation injury. This focuses attention on VEGF and other genes induced in response to hypoxia as targets for therapy to reduce or prevent CNS radiation damage.

Animals↗

PubMed2004

Cre recombinase specificity defined by the tau locus.

We generated a transgenic mouse line (tau::Cre) by targeting the Cre to the tau locus (Mapt). Based on previous reports on the expression of Tau during development, we expected the Cre recombinase to be expressed in a neuron-specific and pan-neuronal manner. However, intercrosses between the tau::Cre and the Cre-activatable reporter animals resulted in offspring with recombination either restricted to the nervous system or throughout the entire conceptus, indicating expression of Tau early in development. The percentage of neuron-specific excision was dependent on the Cre reporter used representing different Cre target sites in the mouse genome. In spite of the observed variability, our data suggest that the tau::Cre mouse line can be used for pan-neuronal recombination of floxed alleles when it is used with caution.

Animals↗

PubMed2004

Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals.

Spectral variants of green fluorescent protein are widely used in live samples for a broad range of applications: from visualization of protein interactions, through following gene expression, to marking particular cells in complex tissues. Higher wavelength emissions (such as red) are preferred due to the lower background-autofluorescence in tissues (Miyawaka et al., Nat Cell Biol Suppl S1-7, 2003). Until now, however, red fluorescent proteins (RFP) have displayed toxicity in murine embryos, which has hampered its application in this model. Here we report strong expression of a recently developed RFP variant, DsRed.T3, in mouse ES cells, embryos, and adult mice. Our results show that the red fluorescent wavelength has a superior tissue penetrance compared with spectral variants of lower wavelength. Furthermore, we have generated an ES cell line and a corresponding transgenic mouse line in which red fluorescence is activated upon Cre excision. Finally, we introduced cell type-specifically expressed Cre transgenes into this Cre recombinase reporter cell line, and by using the tetraploid embryo complementation assay, we could directly verify the Cre recombinase specificity on ES cell-derived embryos/animals.

Animals↗