scholarly journals Hypoxic stress underlies defects in erythroblast islands in the Rb-null mouse

Blood ◽  
2007 ◽  
Vol 110 (6) ◽  
pp. 2173-2181 ◽  
Author(s):  
Benjamin T. Spike ◽  
Benjamin C. Dibling ◽  
Kay F. Macleod
Keyword(s):  
Fetal Liver ◽  
Cell Maturation ◽  
Null Mouse ◽  
Hypoxic Stress ◽  
Red Cell ◽  
Exogenous Stress ◽  
Erythroid Maturation ◽  
Definitive Erythropoiesis

Abstract Definitive erythropoiesis occurs in islands composed of a central macrophage in contact with differentiating erythroblasts. Erythroid maturation including enucleation can also occur in the absence of macrophages both in vivo and in vitro. We reported previously that loss of Rb induces cell-autonomous defects in red cell maturation under stress conditions, while other reports have suggested that the failure of Rb-null erythroblasts to enucleate is due to defects in associated macrophages. Here we show that erythropoietic islands are disrupted by hypoxic stress, such as occurs in the Rb-null fetal liver, that Rb−/− macrophages are competent for erythropoietic island formation in the absence of exogenous stress and that enucleation defects persist in Rb-null erythroblasts irrespective of macrophage function.

Emp Null Mice Are Non-Viable and Exhibit Erythroid Differentiation Defect.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 806-806 ◽  
Author(s):  
Shivani Soni ◽  
Shashi Bala ◽  
Babette Gwynn ◽  
Kenneth E. Sahr ◽  
Luanne L. Peters ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Direct Sequencing ◽  
Embryonic Stem ◽  
Gene Trap ◽  
Physical Contact ◽  
Erythroid Cells ◽  
Wild Type ◽  
Definitive Erythropoiesis

Abstract Emp, erythroblast macrophage protein, was originally detected in erythroblasts and macrophages, which form erythroblastic islands during erythropoiesis in the human bone marrow. The physical contact between erythroblasts and macrophages was suggested to promote the terminal maturation of erythroblasts, leading to their enucleation in vitro. To evaluate the function of Emp in vivo, we employed gene targeting studies to develop an Emp(−/−) mouse model. Mouse embryonic stem cells containing a gene-trap insertion in Emp were obtained from BayGenomics. Insertion of the gene-trap vector into Emp was verified by direct sequencing of cDNA obtained by 5′RACE. Chimeric mice generated by blastocyst microinjection were intercrossed, and the offspring were genotyped by PCR and Southern hybridization. The Emp (+/−) mice were healthy and fertile. However, no live Emp (−/−) mice were found among the progeny of the Emp (+/−) intercrosses. Analysis of timed pregnancies revealed that Emp (−/−) embryos were present at a frequency roughly consistent with Mendelian inheritance throughout the embryonal stages. Homozygous Emp (−/−) embryos were small and pale compared to their littermates, and they survived embryonic development but died at birth. To determine the effect, if any, of Emp gene deletion on definitive hematopoiesis, livers of +/+, +/−, and −/− embryos at E15.5 were examined after H&E and Giemsa staining of paraffin-embedded serial sections, and cytospins. We found few mature erythroid cells in the sinusoids of homozygotes, in contrast to those of either wild-type or heterozygotes, where abundant enucleated red blood cells were observed. Although nucleated erythrocytes were found in both wild-type and mutant embryos, their relative proportions were very different: the less mature forms (proerythroblasts) predominated in the −/− embryos whereas the more mature forms (polychromatophilic/orthochromatic and enucleated erythrocytes) were most common in +/+ and +/− embryos. Furthermore, erythroblastic islands consisting of a central macrophage surrounded by developing erythroblasts were seen in the cytospin preparations of wild-type and heterozygote livers but not in those of homozygous null livers. Since fetal liver macrophages (FLMs) are indispensable for definitive erythropoiesis, we investigated the effect that Emp’s absence might have on development of FLMs. The E15.5 fetal liver sections were stained with the macrophage-specific F4/80 antigen. Numerous F4/80-positive macrophages were present throughout the liver of normal embryos whereas, the number was substantially reduced in Emp (−/−) liver. In summary, in the absence of Emp, FLMs are significantly reduced and terminal maturation of erythroid cells is negatively affected. Thus, the availability of Emp(−/−) embryos will provide a unique experimental model to study the function of macrophages in definitive erythropoiesis.

scholarly journals Enucleation of primitive erythroid cells generates a transient population of “pyrenocytes” in the mammalian fetus

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2409-2417 ◽  
Author(s):  
Kathleen E. McGrath ◽  
Paul D. Kingsley ◽  
Anne D. Koniski ◽  
Rebecca L. Porter ◽  
Timothy P. Bushnell ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Multispectral Imaging ◽  
Fetal Liver ◽  
Fetal Blood ◽  
Immunohistochemical Examination ◽  
Imaging Flow Cytometry ◽  
Transient Population ◽  
Erythroid Maturation ◽  
Definitive Erythropoiesis

Enucleation is the hallmark of erythropoiesis in mammals. Previously, we determined that yolk sac–derived primitive erythroblasts mature in the bloodstream and enucleate between embryonic day (E)14.5 and E16.5 of mouse gestation. While definitive erythroblasts enucleate by nuclear extrusion, generating reticulocytes and small, nucleated cells with a thin rim of cytoplasm (“pyrenocytes”), it is unclear by what mechanism primitive erythroblasts enucleate. Immunohistochemical examination of fetal blood revealed primitive pyrenocytes that were confirmed by multispectral imaging flow cytometry to constitute a distinct, transient cell population. The frequency of primitive erythroblasts was higher in the liver than the bloodstream, suggesting that they enucleate in the liver, a possibility supported by their proximity to liver macrophages and the isolation of erythroblast islands containing primitive erythroblasts. Furthermore, primitive erythroblasts can reconstitute erythroblast islands in vitro by attaching to fetal liver–derived macrophages, an association mediated in part by α4 integrin. Late-stage primitive erythroblasts fail to enucleate in vitro unless cocultured with macrophage cells. Our studies indicate that primitive erythroblasts enucleate by nuclear extrusion to generate erythrocytes and pyrenocytes and suggest this occurs in the fetal liver in association with macrophages. Continued studies comparing primitive and definitive erythropoiesis will lead to an improved understanding of terminal erythroid maturation.

EKLF-driven PIT1 expression is critical for mouse erythroid maturation in vivo and in vitro

Blood ◽  
2013 ◽  
Vol 121 (4) ◽  
pp. 666-678 ◽  
Author(s):  
Anne Forand ◽  
Laurent Beck ◽  
Christine Leroy ◽  
Alice Rousseau ◽  
Valérie Boitez ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Protein A ◽  
Erythroid Cell ◽  
Functional Link ◽  
Targeted Deletion ◽  
Proliferation And Apoptosis ◽  
Erythroid Maturation

AbstractThe PIT1/SLC20A1 protein, a well-described sodium/phosphate cotransporter and retrovirus receptor, has been identified recently as a modular of proliferation and apoptosis in vitro. The targeted deletion of the PIT1 gene in mice revealed a lethal phenotype due to severe anemia attributed to defects in liver development. However, the presence of immature erythroid cells associated with impaired maturation of the globin switch led us to investigate the role of PIT1 in hematopoietic development. In the present study, specific deletion of PIT1 in the hematopoietic system and fetal liver transplantation experiments demonstrated that anemia was associated with an erythroid cell– autonomous defect. Moreover, anemia was not due to RBC destruction but rather to maturation defects. Because Erythroid Krüppel-like Factor (EKLF)–knockout mice showed similar maturation defects, we investigated the functional link between PIT1 and EKLF. We demonstrated that EKLF increases PIT1 expression during RBC maturation by binding to its promoter in vivo and that shRNA-driven depletion of either PIT1 or EKLF impairs erythroid maturation of G1E cells in vitro, whereas reexpression of PIT1 in EKLF-depleted G1E cells partially restores erythroid maturation. This is the first demonstration of a physiologic involvement of PIT1 in erythroid maturation in vivo.

LACK OF EFFECT OF CORTICOSTEROIDS ON THE IN VIVO DISAPPEARANCE OF SULFHYDRYL-INHIBITED AND ANTIBODY-COATED ERYTHROCYTES

1964 ◽  
Vol 47 (3_Suppl) ◽  
pp. S28-S36
Author(s):  
Kailash N. Agarwal
Keyword(s):  
Red Cells ◽  
List Type ◽  
Red Cell

ABSTRACT Red cells were incubated in vitro with sulfhydryl inhibitors and Rhantibody with and without prior incubation with prednisolone-hemisuccinate. These erythrocytes were labelled with Cr51 and P32 and their disappearance in vivo after autotransfusion was measured. Prior incubation with prednisolone-hemisuccinate had no effect on the rate of red cell disappearance. The disappearance of the cells was shown to take place without appreciable intravascular destruction.

Neuropilin-1 on hematopoietic cells as a source of vascular development

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1801-1809 ◽  
Author(s):  
Yoshihiro Yamada ◽  
Yuichi Oike ◽  
Hisao Ogawa ◽  
Yasuhiro Ito ◽  
Hajime Fujisawa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Fetal Liver ◽  
Vascular Development ◽  
Hematopoietic Cells ◽  
Vegf Receptor ◽  
Knock Out ◽  
Neuropilin 1 ◽  
Vasculogenesis And Angiogenesis

Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-165 (VEGF165) and acts as a coreceptor that enhances the function of VEGF165 through VEGF receptor-2 (VEGFR-2). Studies using transgenic and knock-out mice of NP-1 indicated that this molecule is important for vascular development as well as neuronal development. We recently reported that clustered soluble NP-1 phosphorylates VEGFR-2 on endothelial cells with a low dose of VEGF165 and rescues the defective vascularity of the NP-1−/− embryo in vitro and in vivo. Here we show that NP-1 is expressed by CD45+ hematopoietic cells in the fetal liver, can bind VEGF165, and phosphorylates VEGFR-2 on endothelial cells. CD45+NP-1+ cells rescued the defective vasculogenesis and angiogenesis in the NP-1−/− P-Sp (para-aortic splanchnopleural mesodermal region) culture, although CD45+NP-1− cells did not. Moreover, CD45+NP-1+ cells together with VEGF165 induced angiogenesis in an in vivo Matrigel assay and cornea neovascularization assay. The extracellular domain of NP-1 consists of “a,” “b,” and “c” domains, and it is known that the “a” and “c” domains are necessary for dimerization of NP-1. We found that both the “a” and “c” domains are essential for such rescue of defective vascularities in the NP-1 mutant. These results suggest that NP-1 enhances vasculogenesis and angiogenesis exogenously and that dimerization of NP-1 is important for enhancing vascular development. In NP-1−/− embryos, vascular sprouting is impaired at the central nervous system (CNS) and pericardium where VEGF is not abundant, indicating that NP-1–expressing cells are required for normal vascular development.

Hypoxic Stress Upregulates the Expression of Slc38a1 in Brown Adipocytes via Hypoxia-Inducible Factor-1α

Pharmacology ◽  
2017 ◽  
Vol 101 (1-2) ◽  
pp. 64-71 ◽  
Author(s):  
Tetsuhiro Horie ◽  
Kazuya Fukasawa ◽  
Takashi Iezaki ◽  
Gyujin Park ◽  
Yuki Onishi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hypoxia Inducible Factor ◽  
Amino Acid Transporters ◽  
Hypoxic Stress ◽  
Gene Encoding ◽  
Brown Adipocytes ◽  
Hypoxia Inducible Factor 1Α ◽  
Brown Adipose

The availability of amino acid in the brown adipose tissue (BAT) has been shown to be altered under various conditions; however, little is known about the possible expression and pivotal role of amino acid transporters in BAT under physiological and pathological conditions. The present study comprehensively investigated whether amino acid transporters are regulated by obesogenic conditions in BAT in vivo. Moreover, we investigated the mechanism underlying the regulation of the expression of amino acid transporters by various stressors in brown adipocytes in vitro. The expression of solute carrier family 38 member 1 (Slc38a1; gene encoding sodium-coupled neutral amino acid transporter 1) was preferentially upregulated in the BAT of both genetic and acquired obesity mice in vivo. Moreover, the expression of Slc38a1 was induced by hypoxic stress through hypoxia-inducible factor-1α, which is a master transcription factor of the adaptive response to hypoxic stress, in brown adipocytes in vitro. These results indicate that Slc38a1 is an obesity-associated gene in BAT and a hypoxia-responsive gene in brown adipocytes.

Age-dependent changes in volume and haemoglobin content of erythrocytes in the carp (Cyprinus carpio L.)

1989 ◽  
Vol 141 (1) ◽  
pp. 133-149 ◽  
Author(s):  
W. Speckner ◽  
J. F. Schindler ◽  
C. Albers
Keyword(s):  
Gel Filtration ◽  
Linear Increase ◽  
Human Erythrocytes ◽  
Main Peak ◽  
Red Cell ◽  
Human Haemoglobin ◽  
Cell Fractions ◽  
Haemoglobin Content

Carp erythrocytes were fractionated by angle-head centrifugation which yielded fractions with a linear increase in density. Haematological examinations revealed that the heavier red blood cells of carp had greater volumes (MCV), more haemoglobin (MCH) and higher haemoglobin concentrations (MCHC) than light ones. The same experiments with human red cell fractions yielded a decrease in MCV, constant MCH and an increase in MCHC. Haemoglobin content in individual erythrocytes was also determined by scanning stage absorbance cytophotometry to establish the frequency distribution of the cellular haemoglobin contents. In carp, the distribution was symmetrical with the means increasing with density. No such change with cell density was found in human erythrocytes. Both carp and human erythrocytes incorporated [2-14C]glycine in vitro. After gel filtration, radioactivity was detected in carp, but not in human, haemoglobin fractions. 14C was found in all three haemoglobin fractions, obtained by isoelectric focusing, and was present in the haem and in the globin. [2-14C]glycine-labelled erythrocytes were reinjected into chronically cannulated carp and followed in vivo for several months. With time, the main peak of scintillation counts shifted from red cell fractions of low to high density. This is considered as evidence that density and age of red cells in carp are positively correlated and that erythrocytes can synthesize haemoglobin while circulating in the peripheral blood.

Fetal liver pro-B and pre-B lymphocyte clones: expression of lymphoid-specific genes, surface markers, growth requirements, colonization of the bone marrow, and generation of B lymphocytes in vivo and in vitro

1992 ◽  
Vol 12 (2) ◽  
pp. 518-530
Author(s):  
R Palacios ◽  
J Samaridis
Keyword(s):  
B Cell ◽  
B Lymphocytes ◽  
Stromal Cells ◽  
B Lymphocyte ◽  
Germ Line ◽  
Fetal Liver ◽  
Rag 1 ◽  
B Cell Precursors

We describe here the development and characterization of the FLS4.1 stromal line derived from 15-day fetal liver of BALB/c embryos and defined culture conditions that efficiently support the cloning and long-term growth of nontransformed B-220+ 14-day fetal liver cells at two stages of B-cell development, namely, pro-B lymphocytes (immunoglobulin [Ig] genes in germ line configuration) and pre-B cells (JH-rearranged genes with both light-chain Ig genes in the germ line state). All B-cell precursor clones require recombinant interleukin-7 (rIL-7) and FLS4.1 stromal cells for continuous growth in culture, but pro-B lymphocyte clones can also proliferate in rIL-3. None proliferate in rIL-1, rIL-2, rIL-4, rIL-5, rIL-6, or leukemia inhibitory factor. FLS4.1 stromal cells synthesize mRNA for Steel factor but not for IL-1 to IL-7; all pro-B and pre-B clones express c-Kit, the receptor for Steel factor, and a c-Kit-specific antibody inhibits the enhanced proliferative response of fetal liver B-220+ B-cell precursors supported by FLS4.1 stromal cells and exogenous rIL-7 but does not affect that promoted by rIL-7 alone. Northern (RNA) blot analysis of the expression of the MB-1, lambda 5, Vpre-B, c mu, RAG-1, and RAG-2 genes in pro-B and pre-B clones show that transcription of the MB-1 gene precedes IgH gene rearrangement and RNA synthesis from c mu, RAG-1, RAG-2, lambda 5, and Vpre-B genes. All clones at the pre-B-cell stage synthesize mRNA for c mu, RAG-1, and RAG-2 genes; transcription of the lambda 5 and Vpre-B genes seems to start after D-to-JH rearrangement in B-cell precursors, indicating that the proteins encoded by either gene are not required for B-cell progenitors to undergo D-to-JH gene rearrangement. These findings mark transcription of the MB-1 gene as one of the earliest molecular events in commitment to develop along the B-lymphocyte pathway. Indeed, both pro-B and pre-B clones can generate in vitro and in vivo B lymphocytes but not T lymphocytes; moreover, these clones do not express the CD3-gamma T-cell-specific gene, nor do they have rearranged gamma, delta, or beta T-cell antigen receptor genes.

scholarly journals Essential nucleotide- and protein-dependent functions of Actb/β-actin

2018 ◽  
Vol 115 (31) ◽  
pp. 7973-7978 ◽  
Author(s):  
Xiaobai Patrinostro ◽  
Pallabi Roy ◽  
Angus Lindsay ◽  
Christopher M. Chamberlain ◽  
Lauren J. Sundby ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Null Mouse ◽  
Cellular Functions ◽  
Embryonic Fibroblasts ◽  
High Frequency Hearing Loss ◽  
Phenotypic Differences ◽  
Functional Differences ◽  
Actin Protein

The highly similar cytoplasmic β- and γ-actins differ by only four functionally similar amino acids, yet previous in vitro and in vivo data suggest that they support unique functions due to striking phenotypic differences between Actb and Actg1 null mouse and cell models. To determine whether the four amino acid variances were responsible for the functional differences between cytoplasmic actins, we gene edited the endogenous mouse Actb locus to translate γ-actin protein. The resulting mice and primary embryonic fibroblasts completely lacked β-actin protein, but were viable and did not present with the most overt and severe cell and organismal phenotypes observed with gene knockout. Nonetheless, the edited mice exhibited progressive high-frequency hearing loss and degeneration of actin-based stereocilia as previously reported for hair cell-specific Actb knockout mice. Thus, β-actin protein is not required for general cellular functions, but is necessary to maintain auditory stereocilia.

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