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• W1996756747 abstract "Suppression of programmed cell death is critical for the final maturation of red blood cells and depends largely on the anti-apoptotic effects of EpoR–STAT5–Bcl-xL signaling. As the major eukaryotic serine/threonine phosphatase, protein phosphatase 2A (PP2A) regulates multiple cellular processes, including apoptosis. However, whether PP2A plays a role in preventing erythroid cells from undergoing apoptosis remains to be elucidated. We conditionally inactivated the catalytic subunit α of PP2A (PP2Acα), which is the predominant form of PP2Ac, during early embryonic hematopoiesis. Loss of PP2Acα in hematopoietic cells perturbed definitive erythropoiesis characterized by fetal liver atrophy, reduced Ter119+ cell number, abnormal expression patterns of molecular markers, less colony formation, and a reduction in definitive globin expression. Levels of erythropoiesis-promoting cytokines and initial seeding with hematopoietic progenitors remained unchanged in PP2AcαTKO fetal livers. We noted impaired expansion of the fetal erythroid compartment, which was associated with increased apoptosis of committed erythroid cells. Mechanistically, PP2Acα depletion markedly reduced Tyr694 phosphorylation of STAT5 and expression of Bcl-xL. Unexpectedly, PP2Acα-deficient embryos did not manifest any early embryonic vascular defects. Collectively, these data provide direct loss-of-function evidence demonstrating the importance of PP2Acα for the survival of committed erythroid cells during fetal liver erythropoiesis. Suppression of programmed cell death is critical for the final maturation of red blood cells and depends largely on the anti-apoptotic effects of EpoR–STAT5–Bcl-xL signaling. As the major eukaryotic serine/threonine phosphatase, protein phosphatase 2A (PP2A) regulates multiple cellular processes, including apoptosis. However, whether PP2A plays a role in preventing erythroid cells from undergoing apoptosis remains to be elucidated. We conditionally inactivated the catalytic subunit α of PP2A (PP2Acα), which is the predominant form of PP2Ac, during early embryonic hematopoiesis. Loss of PP2Acα in hematopoietic cells perturbed definitive erythropoiesis characterized by fetal liver atrophy, reduced Ter119+ cell number, abnormal expression patterns of molecular markers, less colony formation, and a reduction in definitive globin expression. Levels of erythropoiesis-promoting cytokines and initial seeding with hematopoietic progenitors remained unchanged in PP2AcαTKO fetal livers. We noted impaired expansion of the fetal erythroid compartment, which was associated with increased apoptosis of committed erythroid cells. Mechanistically, PP2Acα depletion markedly reduced Tyr694 phosphorylation of STAT5 and expression of Bcl-xL. Unexpectedly, PP2Acα-deficient embryos did not manifest any early embryonic vascular defects. Collectively, these data provide direct loss-of-function evidence demonstrating the importance of PP2Acα for the survival of committed erythroid cells during fetal liver erythropoiesis. The production of red blood cells (RBCs) is normally maintained at a constant level by well-tuned regulation of erythropoiesis. During terminal maturation, mammalian erythroblasts accumulate hemoglobin, assemble the RBC cytoskeleton, extrude their nuclei, and give rise to RBCs.1Richmond T.D. Chohan M. Barber D.L. Turning cells red: signal transduction mediated by erythropoietin.Trends Cell Biol. 2005; 15: 146-155Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar Suppression of programmed cell death is considered to be critical for the final maturation of RBCs and depends strongly on anti-apoptotic effect of erythropoietin (EPO) stimulation and the intracellular EpoR-STAT5-Bcl-xL signaling axis.2Koury M.J. Bondurant M.C. Erythropoietin retards DNA breakdown and prevents programmed death in erythroid progenitor cells.Science. 1990; 248: 378-381Crossref PubMed Scopus (718) Google Scholar EPO−/− and EpoR−/− mice die at embryonic day (E) 13.5 owing to a failure in definitive erythropoiesis.3Wu H. Liu X. Jaenisch R. Lodish H.F. Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor.Cell. 1995; 83: 59-67Abstract Full Text PDF PubMed Scopus (851) Google Scholar STAT5▵N mice are embryonic anemic due to decreased survival of RBC progenitors.4Socolovsky M. Nam H. Fleming M.D. Haase V.H. Brugnara C. Lodish H.F. Ineffective erythropoiesis in Stat5a(−/−)5b(−/−) mice due to decreased survival of early erythroblasts.Blood. 2001; 98: 3261-3273Crossref PubMed Scopus (577) Google Scholar, 5Socolovsky M. Fallon A.E. Wang S. Brugnara C. Lodish H.F. Fetal anemia and apoptosis of red cell progenitors in Stat5a−/−5b−/− mice: a direct role for Stat5 in Bcl-X(L) induction.Cell. 1999; 98: 181-191Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar Bcl-x is a member of the BclII gene family, the members of which share homology in four conserved regions (BH1-4 domains), which control their dimerization and function. The expression of Bcl-x is increased in terminally differentiated erythroblasts,6Gregoli P.A. Bondurant M.C. The roles of Bcl-X(L) and apopain in the control of erythropoiesis by erythropoietin.Blood. 1997; 90: 630-640Crossref PubMed Google Scholar during which stage it positively regulates the survival of these cells.7Motoyama N. Kimura T. Takahashi T. Watanabe T. Nakano T. Bcl-x prevents apoptotic cell death of both primitive and definitive erythrocytes at the end of maturation.J Exp Med. 1999; 189: 1691-1698Crossref PubMed Scopus (119) Google Scholar Indeed, Bcl-x–deficient mice are embryonic lethal owing to massive apoptosis of immature erythroid cells in the fetal liver.8Motoyama N. Wang F. Roth K.A. Sawa H. Nakayama K. Negishi I. Senju S. Zhang Q. Fujii S. et al.Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice.Science. 1995; 267: 1506-1510Crossref PubMed Scopus (1021) Google Scholar Factors that regulate the survival of maturing erythroblasts are also relevant to clinical anemia induced by chemotherapy and chronic diseases, such as renal disorders, myeloma, and myelodysplasic syndromes.9Boogaerts M. Mittelman M. Vaupel P. Beyond anaemia management: evolving role of erythropoietin therapy in neurological disorders, multiple myeloma and tumour hypoxia models.Oncology. 2005; 69: 22-30Crossref PubMed Scopus (12) Google Scholar, 10List A. Kurtin S. Roe D.J. Buresh A. Mahadevan D. Fuchs D. Rimsza L. Heaton R. Knight R. Zeldis J.B. Efficacy of lenalidomide in myelodysplastic syndromes.N Engl J Med. 2005; 352: 549-557Crossref PubMed Scopus (784) Google Scholar, 11Lacombe C. Resistance to erythropoietin.N Engl J Med. 1996; 334: 660-662Crossref PubMed Scopus (30) Google Scholar In eukaryotic cells, at least 30% of proteins can be modulated by reversible phosphorylation. Controlled protein phosphorylation, mediated by protein kinases and phosphatases, regulates multiple cellular processes, including apoptosis.12Hunter T. Signaling–2000 and beyond.Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2260) Google Scholar, 13Janssens V. Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling.Biochem J. 2001; 353: 417-439Crossref PubMed Scopus (1530) Google Scholar Deregulation of apoptosis can lead to many human diseases, including cancer, Alzheimer's disease, cardiac dysfunction, and inflammation,14Thompson C.B. Apoptosis in the pathogenesis and treatment of disease.Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6184) Google Scholar, 15Su J.H. Deng G. Cotman C.W. Bax protein expression is increased in Alzheimer's brain: correlations with DNA damage Bcl-2 expression, and brain pathology.J Neuropathol Exp Neurol. 1997; 56: 86-93Crossref PubMed Scopus (223) Google Scholar most of which have been reported to involve deregulation of protein phosphatase 2A (PP2A),13Janssens V. Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling.Biochem J. 2001; 353: 417-439Crossref PubMed Scopus (1530) Google Scholar, 16Mumby M. PP2A: unveiling a reluctant tumor suppressor.Cell. 2007; 130: 21-24Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 17Marshall M. Anilkumar N. Layland J. Walker S.J. Kentish J.C. Shah A.M. Cave A.C. Protein phosphatase 2A contributes to the cardiac dysfunction induced by endotoxemia.Cardiovasc Res. 2009; 82: 67-76Crossref PubMed Scopus (21) Google Scholar the major eukaryotic serine/threonine phosphatase. However, whether PP2A also plays a role in preventing erythroid cells from undergoing programmed cell death is still largely unknown. The only previously reported function of PP2A in erythropoiesis is associated with activation of K-Cl cotransport.18Bize I. Guvenc B. Robb A. Buchbinder G. Brugnara C. Serine/threonine protein phosphatases and regulation of K-Cl cotransport in human erythrocytes.Am J Physiol. 1999; 277: C926-C936PubMed Google Scholar Heterotrimeric PP2A is composed of a scaffold subunit (A subunit), a catalytic subunit (PP2Ac), and a regulatory subunit (B subunit).19Virshup D.M. Protein phosphatase 2A: a panoply of enzymes.Curr Opin Cell Biol. 2000; 12: 180-185Crossref PubMed Scopus (291) Google Scholar The scaffold subunit is flexible and structurally links PP2Ac with various regulatory subunits to form different holoenzymes. Molecular cloning has revealed the existence of two mammalian PP2Ac isoforms: PP2Acα (encoded by the Ppp2ca gene) and PP2Acβ (encoded by the Ppp2cb gene). These two isoforms share 97% amino acid identity, and seven of the eight residues that differ between them are located within the first 30 amino acids (encoded by exon I).13Janssens V. Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling.Biochem J. 2001; 353: 417-439Crossref PubMed Scopus (1530) Google Scholar, 20Arino J. Woon C.W. Brautigan D.L. Miller Jr., T.B. Johnson G.L. Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes.Proc Natl Acad Sci U S A. 1988; 85: 4252-4256Crossref PubMed Scopus (117) Google Scholar Both PP2Ac isoforms are ubiquitously expressed, and PP2Acα transcripts are generally 10-fold more abundant than are PP2Acβ transcripts owing to transcriptional regulation.21Khew-Goodall Y. Hemmings B.A. Tissue-specific expression of mRNAs encoding α- and β-catalytic subunits of protein phosphatase 2A.FEBS Lett. 1988; 238: 265-268Crossref PubMed Scopus (78) Google Scholar, 22Khew-Goodall Y. Mayer R.E. Maurer F. Stone S.R. Hemmings B.A. Structure and transcriptional regulation of protein phosphatase 2A catalytic subunit genes.Biochemistry. 1991; 30: 89-97Crossref PubMed Scopus (83) Google Scholar However, detailed interpretation of the specific contribution of these two isoforms has long been hampered owing to the lack of reliable antibodies or specific chemical inhibitors that can distinguish between them. We sought to address a role for PP2Acα in erythropoiesis using a genetic approach. Conventional deletion of the Ppp2ca allele in mice results in embryonic lethality after E6 due to absent mesoderm formation,23Gotz J. Probst A. Ehler E. Hemmings B. Kues W. Delayed embryonic lethality in mice lacking protein phosphatase 2A catalytic subunit Cα.Proc Natl Acad Sci U S A. 1998; 95: 12370-12375Crossref PubMed Scopus (183) Google Scholar making it impossible to determine the functional importance of Ppp2ca in embryonic erythropoiesis. Herein, we conditionally inactivated the Ppp2ca allele during early embryonic hematopoiesis by using Tie2Cre transgenic mice, which are ideal models to induce early gene recombination in hematopoietic and endothelial cells.24Schlaeger T.M. Mikkola H.K. Gekas C. Helgadottir H.B. Orkin S.H. Tie2Cre-mediated gene ablation defines the stem-cell leukemia gene (SCL/tal1)-dependent window during hematopoietic stem-cell development.Blood. 2005; 105: 3871-3874Crossref PubMed Scopus (92) Google Scholar We discovered that loss of the Ppp2ca allele perturbed fetal liver erythropoiesis. PP2Acα was not required to establish the fetal liver hematopoietic stem and progenitor cells (HSCs/Ps) pool; however, it enhanced the cell survival function of EPO. It acted, at least in part, by promoting the STAT5–Bcl-xL axis and, thereby, inhibiting apoptosis of committed erythroid cells. Mice carrying the conditional Ppp2ca allele (Ppp2cafl/fl) were bred with Tie2Cre mice,25Koni P.A. Joshi S.K. Temann U.A. Olson D. Burkly L. Flavell R.A. Conditional vascular cell adhesion molecule 1 deletion in mice: impaired lymphocyte migration to bone marrow.J Exp Med. 2001; 193: 741-754Crossref PubMed Scopus (409) Google Scholar which were purchased from The Jackson Laboratory (Bar Harbor, ME). To visualize cells with recombined alleles, mice bearing the Tie2Cre transgene were crossed with ROSA26 reporter mice.26Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain.Nat Genet. 1999; 21: 70-71Crossref PubMed Scopus (4152) Google Scholar All mice used in this study were of a mixed 129/B6 background. Mice were maintained in an Association for Assessment and Accreditation of Laboratory Animal Care International–credited specific pathogen–free animal facility, and animal welfare and experimental procedures were approved by the Animal Care and Use Committee of the Model Animal Research Center, the host for the National Resource Center for Mutant Mice in China, Nanjing University. Genotyping was performed by PCR analyses of genomic DNA isolated from mouse tails or yolk sacs. Genotyping primer sets and PCR reaction programs are listed in Table 1 and in Supplemental Table S1 (available at http://ajp.amjpathol.org), respectively.Table 1Genotyping PrimersPrimer namePrimer sequenceFirst loxPF: 5′-AAGTTACTGAGTGCAGTGTGCCTTG-3′R: 5′-TTATACCCTTCCTCATTCGCTCTGC-3′Second loxPF: 5′-TAGCCCATGCCTTTAATCTCAGAGC-3′R: 5′-CACTCGTCGTAGAACCCATAAACC-3′CreF: 5′-GCCTGCATTACCGGTCGATGC-3′R: 5′-CAGGGTGTTATAAGCAATCCC-3′DNA-EXdelF: 5′-AACCCACCCTTGAGAAACAGC-3′R: 5′-TACCCTTCCTCATTCGCTCTG-3′RNA-EXdelF: 5′-ATTACCTGTTTATGGGAGACTATGT-3′R: 5′-AAGTGGTCACGGCTGTTGATG-3′F, forward; R, reverse. Open table in a new tab F, forward; R, reverse. Female Ppp2cafl/fl mice were mated with male Tie2Cre+/Ppp2cafl/+ mice for embryo collection. Fetal livers were dissociated mechanically by pipetting for fluorescence-activated cell sorting (FACS) analyses. Single-cell suspensions were prepared by drawing medium and cells up and down through a 1-mL syringe and 27-gauge needle. Fetal liver cells from E12.5 embryos were cultured in Iscove's modified Dulbecco's medium with 2% fetal bovine serum. Cells were maintained at 37°C and 5% CO2 in the presence or absence of 10 U/mL recombinant human EPO. For the annexin V binding assay, stimulation lasted 18 hours.27Zang H. Sato K. Nakajima H. McKay C. Ney P.A. Ihle J.N. The distal region and receptor tyrosines of the Epo receptor are non-essential for in vivo erythropoiesis.EMBO J. 2001; 20: 3156-3166Crossref PubMed Scopus (101) Google Scholar For the Western blot assay, stimulation lasted 15 minutes. Freshly isolated fetal liver cells were stained with different combinations of Ter119-PE, CD45-FITC, Gr-1–FITC, CD41-FITC, c-Kit–APC, CD71-biotin, and streptavidin-PECy5. Megakaryocyte progenitors and megakaryocytes were sorted as Lin−c-Kit+CD41+ and Lin−c-Kit−CD41+ cells, respectively.28Perez L.E. Desponts C. Parquet N. Kerr W.G. SH2-inositol phosphatase 1 negatively influences early megakaryocyte progenitors.PLoS One. 2008; 3: e3565Crossref PubMed Scopus (14) Google Scholar For analysis of Lin−Sca-1+c-Kit+ (LSK) cells, fetal liver cells were stained with c-Kit–APC, Sca-1–FITC, and a lineage marker cocktail containing CD3-PE, CD5-PE, B220-PE, Gr-1–PE, and Ter119-PE. Apoptotic cells were verified using annexin V–FITC and Ter119-PE double staining. Endothelial cells were selected as CD31+CD45−.29Kutcher M.E. Klagsbrun M. Mamluk R. VEGF is required for the maintenance of dorsal root ganglia blood vessels but not neurons during development.FASEB J. 2004; 18: 1952-1954PubMed Google Scholar Stained cells were analyzed using a FACSCalibur flow cytometer equipped with CellQuest software or were sorted using LSR II and four-laser FACSAria II sorters (all from Becton Dickinson, San Jose, CA). Sorted cells were collected in buffer containing RNase inhibitor and were stored at −70°C. The calculated absolute fetal liver cell numbers and the percentages of Ter119+, CD45+, Gr-1+, Lin−c-Kit+CD41+, Lin−c-Kit−CD41+, LSK, and CD31+CD45− cells allowed for the determination of absolute cell numbers of these particular cell lineages in whole fetal liver samples. Fetal liver cells from E12.5 embryos were harvested in Iscove's modified Dulbecco's medium with 2% fetal bovine serum. For the erythroid colony-forming unit (CFU-E) assay, 2 × 104 cells were plated in 1 mL of methylcellulose medium supplemented with EPO (MethoCult M3334; STEMCELL Technologies Inc., Vancouver, BC, Canada) and were cultured for 2 days. For the erythroid blast-forming unit (BFU-E) assays, 1 × 105 cells were plated in 1 mL of methylcellulose medium and were cultured for 7 days. Erythroid colonies were stained for hemoglobin using benzidine. CFU-E contained 8 to 32 benzidine-positive cells and BFU-E contained 3 or more clusters of CFU-E. The reported values are mean ± SEM as determined for three or four embryos from each genotype. Tissue protein was extracted in a phosphatase extraction buffer containing 20 mmol/L imidazole-HCl, 2 mmol/L EDTA, 2 mmol/L EGTA (pH 7.0), 1 mmol/L benzamidine, 1 mmol/L phenylmethylsulfonyl fluoride, and protein inhibitor cocktails. Phosphatase activity was quantified using a malachite green–based PP2A Assay Kit (Upstate Biotechnology, Waltham, MA). Briefly, total proteins were immunoprecipitated with anti-PP2Ac, and PP2Ac-bound beads were incubated with synthetic phosphopeptide for the dephosphorylation reaction. The reaction supernatant was then mixed with malachite green reagent for color development. Changes in absorbance were measured at 650 nm. RNA from sorted cells was extracted using RNAspin Mini Kit (GE Healthcare Bio-Sciences Corp, Piscataway, NJ), whereas RNA from fetal livers was isolated using RNAiso (Takara Bio Inc., Shiga, Japan). Reverse transcription was performed using a first-strand cDNA synthesis kit. Quantitative PCR–based measurements of RNA abundance were performed using SYBR green reagents in an ABI 7300 sequence detector (Applied Biosystems, Foster City, CA). Amplification of the GAPDH gene served as an input control of cDNA templates. Primers used in this study are listed in Table 2. Efficiency of amplification for all primers was validated by determining the slope of CT versus dilution series.Table 2Primers Used for Quantitative PCRGeneForward primerReverse primerPpp2cb5′-GAGGGTACTACTCTGTGGAGAC-3′5′-CCGGCTTTCGTGATTTCCT-3′βmaj/min5′-ATGGCCTGAATCACTTGGAC-3′5′-ACGATCATATTGCCCAGGAG-3′βH15′-TCCTTGGGCTTGGGGGTTA-3′5′-TGTGGGACAGAGCATTGGC-3′ϵy5′-TGGCCTGTGGAGTAAGGTCAA-3′5′-GAAGCAGAGGACAAGTTCCCA-3′EPO5′-CAGGCCCTGCTAGCCAATT-3′5′-ACGTAGACCACTGATGGCTTTGT-3′SCF5′-CCCTGAAGACTCGGGCCTA-3′5′-CAATTACAAGCGAAATGAGAGCC-3′Bclx5′-ACTGTGCGTGGAAAGCGTAGA-3′5′-TGCTGCATTGTTCCCGTAGAG-3′Pim15′-TTCTGGACTGGTTCGAGAGG-3′5′-GCTCCTCGTTCGGTGATAAA-3′Cis15′-CCACTGGCTTTGTCAAGAAGG-3′5′-AGGCCACATAGTGCTGCACAA-3′Socs35′-CCGCTTCGACTGTGTACTCAAG-3′5′-TCTTCTCGCCCCCAGAATAGAT-3′Osm5′-AACTGAGCAAGCCTCACTTCC-3′5′-ATGCCGAGGATATTGTGCCG-3′GAPDH5′-TGCCCAGAACATCATCCCT-3′5′-GGTCCTCAGTGTAGCCCAAG-3′ Open table in a new tab Tissue or cell proteins were extracted using 1% Nonidet P-40 (Caledon Laboratories Ltd., Georgetown, ON, Canada), 20 mmol/L Tris · HCl (pH 8.0), 5 mmol/L EDTA, 0.5 mmol/L EGTA, 150 mmol/L NaCl, 10% glycerol, 20 mmol/L β-glycerophosphate, protein inhibitors, and phosphatase inhibitors. Primary antibodies used in this study included anti-PP2Ac (Upstate Biotechnology), anti-PP2A-Aα/β (Santa Cruz Biotechnology, Santa Cruz, CA), anti-pTyr694–STAT5 (Cell Signaling Technology Inc., Beverly, MA), anti-STAT5 (Cell Signaling Technology Inc.), and anti–Bcl-xL (eBioscience Inc., San Diego, CA). Embryos were fixed on ice in fixation buffer containing 0.2% glutaraldehyde, 5 mmol/L EGTA (pH 8.0), and 2 mmol/L MgCl2 in PBS. Samples were washed with rinse buffer containing 0.01% sodium deoxycholate, 0.02% Nonidet P-40, 5 mmol/L EGTA (pH 8.0), and 2 mmol/L MgCl2 in PBS. Samples were then stained with lacZ staining buffer containing 1 mg/mL 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal), 10 mmol/L K3Fe(CN)6, 10 mmol/L K4Fe(CN)6, and 20 mmol/L Tris · HCl (pH 7.3) in rinse buffer. Stained tissues were paraffin embedded, sectioned, and counterstained with nuclear fast red. Data were analyzed using two-tailed t-tests and are presented as mean ± SEM. For this study, we generated the Ppp2cafl/fl mice, in which two loxP sites were introduced into the 3′ and 5′ ends of the region spanning exons 3 to 5 of the Ppp2ca gene (data not shown). To conditionally inactivate the Ppp2ca allele during embryonic hematopoiesis, we used two mouse lines, Ppp2cafl/fl mice and Tie2Cre mice, in which the Cre transgene is directed under the receptor tyrosine kinase Tek promoter/enhancer. Because Tie2Cre mice also exhibit Cre expression in the female germline, the Cre allele was maintained only in male mice in this study. To generate PP2AcαTKO (Tie2Cre+/Ppp2cafl/fl) mice, we first set up mating between male Tie2Cre+ and female Ppp2cafl/+ mice. Male Tie2Cre+/Ppp2cafl/+ mice were subsequently mated with female Ppp2cafl/fl mice to generate PP2AcαTKO mice (see Supplemental Figure S1A at http://ajp.amjpathol.org). PCR analyses of genomic DNA from mouse tails were performed to verify the expected genotypes (see Supplemental Figure S1, B and C, at http://ajp.amjpathol.org). In addition to endothelial cells, HSCs/Ps in mouse embryos have also been reported to express Tie2.30Hamaguchi I. Huang X.L. Takakura N. Tada J. Yamaguchi Y. Kodama H. Suda T. In vitro hematopoietic and endothelial cell development from cells expressing TEK receptor in murine aorta-gonad-mesonephros region.Blood. 1999; 93: 1549-1556PubMed Google Scholar, 31Hsu H.C. Ema H. Osawa M. Nakamura Y. Suda T. Nakauchi H. Hematopoietic stem cells express Tie-2 receptor in the murine fetal liver.Blood. 2000; 96: 3757-3762PubMed Google Scholar, 32Takakura N. Huang X.L. Naruse T. Hamaguchi I. Dumont D.J. Yancopoulos G.D. Suda T. Critical role of the TIE2 endothelial cell receptor in the development of definitive hematopoiesis.Immunity. 1998; 9: 677-686Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar On initiation of the hematopoietic program, Tie2 regulatory elements are transiently active in hematopoietic progenitors. To determine the extent of Cre expression in the fetal liver, we bred Tie2Cre mice with ROSA26-LacZ reporter mice. The relatively sporadic blue staining in fetal livers of mice carrying the Cre and the floxed Ppp2ca alleles indicated the distribution pattern of hematopoietic cells having undergone Tie2Cre-mediated gene recombination (Figure 1A). To verify the excision efficiency of the Ppp2ca allele, we harvested genomic DNA, cDNA, and protein from E12.5 fetal livers. Figure 1B shows data confirming that recombination occurred in the floxed Ppp2ca genomic locus in Tie2Cre+/Ppp2cafl/+ and Tie2Cre+/Ppp2cafl/fl embryos. To confirm that deletion of the targeted exons indeed altered Ppp2ca expression, we measured mRNA and protein levels of PP2Acα and PP2Acβ in fetal livers. RT-PCR analyses showed that transcription of the Ppp2ca gene was truncated in total fetal liver cells (Figure 1C) and sorted HSCs (LSK) of PP2AcαTKO embryos (Figure 1D), although intact transcripts were still observed. Transcription of Ppp2cb was not altered by excision of the Ppp2ca allele (Figure 1E). Owing to the high sequence similarity between PP2Acα and PP2Acβ, we did not have a reliable antibody to distinguish between these two isoforms. However, total quantity of catalytic subunit was still substantially reduced in PP2AcαTKO fetal livers (Figure 1G). PP2A phosphatase activity in PP2AcαTKO samples was approximately 36.4% of the activity observed in Tie2Cre+/Ppp2cafl/+ fetal livers (Figure 1F). Expression of the scaffolding subunit of PP2A remained unchanged despite loss of the Ppp2ca allele (Figure 1H). PP2Ac protein quantity and PP2A phosphatase activity were similar between Tie2Cre+/Ppp2ca+/+ and Tie2Cre+/Ppp2cafl/+ fetal livers (see Supplemental Figure S2 at http://ajp.amjpathol.org). In subsequent experiments, we used Tie2Cre+/Ppp2cafl/+ mice as controls (CTRs). PP2AcαTKO embryos were pale, indicating defective hematopoiesis (Figure 2A). The CTR fetal liver showed a bright red appearance compared with the pale fetal liver of PP2AcαTKO embryos (Figure 2B). The total cellularity of PP2AcαTKO fetal liver was dramatically reduced at E12.5 and E14.5 (Figure 2C). PP2AcαTKO fetal liver cells were also larger (macrocytic) than CTR cells (Figure 2D). As determined by histologic analyses, CTR livers contained numerous hematopoietic elements. In contrast, PP2AcαTKO fetal livers consisted primarily of hepatic cells and, infrequently, hematopoietic progenitors or nucleated primitive RBCs (Figure 2E). To confirm and clarify hematopoietic defects in PP2Acα-deficient embryos, we analyzed the expression of several hematopoietic lineage markers in E14.5 fetal livers, such as Ter119+ for erythrocytes, CD45+ for leukocytes,33Springer T. Galfre G. Secher D.S. Milstein C. Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens.Eur J Immunol. 1978; 8: 539-551Crossref PubMed Scopus (519) Google Scholar Gr-1+ for granulocytes, Lin−c-Kit+CD41+ for megakaryocyte progenitors, and Lin−c-Kit−CD41+ for megakaryocytes28Perez L.E. Desponts C. Parquet N. Kerr W.G. SH2-inositol phosphatase 1 negatively influences early megakaryocyte progenitors.PLoS One. 2008; 3: e3565Crossref PubMed Scopus (14) Google Scholar (Figure 3A). The absolute number of Ter119+ cells in PP2AcαTKO fetal liver was dramatically reduced to approximately 11.8% of that in CTRs (CTR versus PP2AcαTKO: 3.39 × 107 ± 5.79 × 106 versus 4.02 × 106 ± 1.86 × 106 cells per fetal liver), whereas no statistically significant differences were observed in the mean ± SEM absolute number of CD45+ cells (CTR versus PP2AcαTKO: 1.28 × 106 ± 1.51 × 105 versus 1.15 × 106 ± 1.91 × 105 cells per fetal liver), Gr-1+ cells (CTR versus PP2AcαTKO: 4.94 × 105 ± 1.52 × 105 versus 3.90 × 105 ± 1.34 × 105 cells per fetal liver), Lin−c-Kit+CD41+ cells (CTR versus PP2AcαTKO: 1.29 × 104 ± 0.26 × 104 versus 1.49 × 104 ± 0.34 × 104 cells per fetal liver), and Lin−c-Kit−CD41+ cells (CTR versus PP2AcαTKO: 1.66 × 104 ± 0.20 × 104 versus 1.75 × 104 ± 0.78 × 104 cells per fetal liver). These findings suggest that the severe anemia observed in PP2AcαTKO embryos was mainly due to erythropoietic abnormalities, with no apparent impairment in other hematopoietic lineages. RT-PCR analyses of sorted Ter119+, CD45+, Gr-1+, and CD41+ cells revealed incomplete knockout of PP2Acα in these particular hematopoietic lineages (Figure 3B). To explain the erythroid impairment phenotype, we hypothesize that considering their similar deletion efficiency, the maturation of erythroid and other hematopoietic lineages are differentially regulated. Flow cytometry was performed to further verify the maturation stage of fetal liver erythroid cells. Expression of Ter119 marks committed erythropoietic precursors beyond the CFU-E stage,34Ikuta K. Kina T. MacNeil I. Uchida N. Peault B. Chien Y.H. Weissman I.L. A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells.Cell. 1990; 62: 863-874Abstract Full Text PDF PubMed Scopus (514) Google Scholar, 35Neubauer H. Cumano A. Muller M. Wu H. Huffstadt U. Pfeffer K. Jak2 deficiency defines an essential developmental checkpoint in definitive hematopoiesis.Cell. 1998; 93: 397-409Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar whereas c-Kit marks HSCs capable of long-term reconstitution.36Ikuta K. Weissman I.L. Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation.Proc Natl Acad Sci U S A. 1992; 89: 1502-1506Crossref PubMed Scopus (499) Google Scholar Proerythroblasts, characterized as the c-Kit+Ter119− cell fraction, differentiate into c-Kit−Ter119+ erythroblasts during maturation. However, this process was severely impaired in PP2AcαTKO embryos (Figure 3C). CD71 antibodies bind to the transferring receptor, which is highly expressed in proerythroblasts and early erythroblasts and decays in late erythroblasts and reticulocytes. Cells from CTR livers contained at least four distinct cell populations, defined by the following characteristic staining patterns: CD71medTer119low, CD71highTer119low, CD71highTer119high, and CD71medTer119high (Figure 3D, left, regions R1-R4, respectively). However, cell populations from PP2AcαTKO livers showed far fewer mature erythroblasts (Figure 3D, right, regions R1-R4, respectively). Tie2Cre+/Ppp2ca+/+ and Tie2Cre+/Ppp2cafl/+ fetal livers had a similar, bright red appearance and c-Kit–Ter119 and CD71-Ter119 staining patterns, confirming that Tie2Cre+/Ppp2cafl/+ fetal livers had normal embryonic erythropoiesis (see Supplemental Figure S3 at http://ajp.amjpathol.org). The generation of mature RBCs involves the commitment of pluripotent HSCs that progress through the BFU-E and CFU-E stages and the proerythroblast and erythroblast stages and, finally, differentiate into enucleated erythrocy" @default.
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