Endogenous Erythropoietin Signaling Is Required for Normal Neural Progenitor Cell Proliferation
2007; Elsevier BV; Volume: 282; Issue: 35 Linguagem: Inglês
10.1074/jbc.m701988200
ISSN1083-351X
AutoresZhi-Yong Chen, Pundit Asavaritikrai, Josef T. Prchal, Constance Tom Noguchi,
Tópico(s)Glioma Diagnosis and Treatment
ResumoErythropoietin (Epo) and its receptor (EpoR), critical for erythropoiesis, are expressed in the nervous system. Prior to death in utero because of severe anemia EpoR-null mice have fewer neural progenitor cells, and differentiated neurons are markedly sensitive to hypoxia, suggesting that during development Epo stimulates neural cell proliferation and prevents neuron apoptosis by promoting oxygen delivery to brain or by direct interaction with neural cells. Here we present evidence that neural progenitor cells express EpoR at higher levels compared with mature neurons; that Epo stimulates proliferation of embryonic neural progenitor cells; and that endogenous Epo contributes to neural progenitor cell proliferation and maintenance. EpoR-null mice were rescued with selective EpoR expression driven by the endogenous EpoR promoter in hematopoietic tissue but not in brain. Although these mice exhibited normal hematopoiesis and erythrocyte production and survived to adulthood, neural cell proliferation and viability were affected. Embryonic brain exhibited increased neural cell apoptosis, and neural cell proliferation was reduced in the adult hippocampus and subventricular zone. Neural cells from these animals were more sensitive to hypoxia/glutamate neurotoxicity than normal neurons in culture and in vivo. These observations demonstrate that endogenous Epo/EpoR signaling promotes cell survival in embryonic brain and contributes to neural cell proliferation in adult brain in regions associated with neurogenesis. Therefore, Epo exerts extra-hematopoietic function and contributes directly to brain development, maintenance, and repair by promoting cell survival and proliferation independent of insult, injury, or ischemia. Erythropoietin (Epo) and its receptor (EpoR), critical for erythropoiesis, are expressed in the nervous system. Prior to death in utero because of severe anemia EpoR-null mice have fewer neural progenitor cells, and differentiated neurons are markedly sensitive to hypoxia, suggesting that during development Epo stimulates neural cell proliferation and prevents neuron apoptosis by promoting oxygen delivery to brain or by direct interaction with neural cells. Here we present evidence that neural progenitor cells express EpoR at higher levels compared with mature neurons; that Epo stimulates proliferation of embryonic neural progenitor cells; and that endogenous Epo contributes to neural progenitor cell proliferation and maintenance. EpoR-null mice were rescued with selective EpoR expression driven by the endogenous EpoR promoter in hematopoietic tissue but not in brain. Although these mice exhibited normal hematopoiesis and erythrocyte production and survived to adulthood, neural cell proliferation and viability were affected. Embryonic brain exhibited increased neural cell apoptosis, and neural cell proliferation was reduced in the adult hippocampus and subventricular zone. Neural cells from these animals were more sensitive to hypoxia/glutamate neurotoxicity than normal neurons in culture and in vivo. These observations demonstrate that endogenous Epo/EpoR signaling promotes cell survival in embryonic brain and contributes to neural cell proliferation in adult brain in regions associated with neurogenesis. Therefore, Epo exerts extra-hematopoietic function and contributes directly to brain development, maintenance, and repair by promoting cell survival and proliferation independent of insult, injury, or ischemia. Epo 2The abbreviations used are:EpoerythropoietinEpoRerythropoietin receptorNPCsneural progenitor cellsWTwild typebFGFbasic fibroblast growth factorBrdUrdbromodeoxyuridineCrecyclic recombinaseRTreverse transcriptionTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labelingDAPI4′,6-diamidino-2-phenylindoleSVZsubventricular zone. 2The abbreviations used are:EpoerythropoietinEpoRerythropoietin receptorNPCsneural progenitor cellsWTwild typebFGFbasic fibroblast growth factorBrdUrdbromodeoxyuridineCrecyclic recombinaseRTreverse transcriptionTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end-labelingDAPI4′,6-diamidino-2-phenylindoleSVZsubventricular zone. is a hypoxia responsive cytokine required for production of erythroid cells. It triggers erythroid progenitor cell proliferation and differentiation by binding to its specific membrane receptor EpoR. EpoR-null mice and Epo-null mice die in utero because of lack of mature red blood cell production (1Lin C.S. Lim S.K. D'Agati V. Costantini F. Genes Dev. 1996; 10: 154-164Crossref PubMed Scopus (349) Google Scholar, 2Wu H. Liu X. Jaenisch R. Lodish H.F. Cell. 1995; 83: 59-67Abstract Full Text PDF PubMed Scopus (848) Google Scholar). However, functional EpoR has been identified in non-erythroid cells such as endothelial, muscle, and neural cells, and there is increasing evidence that Epo can act to stimulate cell proliferation, cell-specific function or promote cell survival in these tissues (3Beleslin-Cokic B.B. Cokic V.P. Yu X. Weksler B.B. Schechter A.N. Noguchi C.T. Blood. 2004; 104: 2073-2080Crossref PubMed Scopus (250) Google Scholar, 4Jelkmann W. Wagner K. Ann. Hematol. 2004; 83: 673-686Crossref PubMed Scopus (127) Google Scholar, 5Ogilvie M. Yu X. Nicolas-Metral V. Pulido S.M. Liu C. Ruegg U.T. Noguchi C.T. J. Biol. Chem. 2000; 275: 39754-39761Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 6Studer L. Csete M. Lee S.H. Kabbani N. Walikonis J. Wold B. McKay R. J. Neurosci. 2000; 20: 7377-7383Crossref PubMed Google Scholar, 7Shingo T. Sorokan S.T. Shimazaki T. Weiss S. J. Neurosci. 2001; 21: 9733-9743Crossref PubMed Google Scholar, 8Yu X. Shacka J.J. Eells J.B. Suarez-Quian C. Przygodzki R.M. Beleslin-Cokic B. Lin C.S. Nikodem V.M. Hempstead B. Flanders K.C. Costantini F. Noguchi C.T. Development (Camb.). 2002; 129: 505-516Crossref PubMed Google Scholar). In culture, astrocytes and neurons up-regulate Epo and EpoR expression in response to hypoxia (8Yu X. Shacka J.J. Eells J.B. Suarez-Quian C. Przygodzki R.M. Beleslin-Cokic B. Lin C.S. Nikodem V.M. Hempstead B. Flanders K.C. Costantini F. Noguchi C.T. Development (Camb.). 2002; 129: 505-516Crossref PubMed Google Scholar, 9Gassmann M. Heinicke K. Soliz J. Ogunshola O.O. Marti H.H. Hofer T. Grimm C. Heinicke I. Egli B. Adv. Exp. Med. Biol. 2003; 543: 323-330Crossref PubMed Scopus (103) Google Scholar, 10Knabe W. Knerlich F. Washausen S. Kietzmann T. Siren A.L. Brunnett G. Kuhn H.J. Ehrenreich H. Anat. Embryol. (Berl.). 2004; 207: 503-512Crossref PubMed Scopus (73) Google Scholar). Epo activates Jak2/Stat5 and NF-κB pathways to protect neurons from glutamate and hypoxic damage (8Yu X. Shacka J.J. Eells J.B. Suarez-Quian C. Przygodzki R.M. Beleslin-Cokic B. Lin C.S. Nikodem V.M. Hempstead B. Flanders K.C. Costantini F. Noguchi C.T. Development (Camb.). 2002; 129: 505-516Crossref PubMed Google Scholar, 11Digicaylioglu M. Lipton S.A. Nature. 2001; 412: 641-647Crossref PubMed Scopus (856) Google Scholar, 12Dzietko M. Felderhoff-Mueser U. Sifringer M. Krutz B. Bittigau P. Thor F. Heumann R. Buhrer C. Ikonomidou C. Hansen H.H. Neurobiol. Dis. 2004; 15: 177-187Crossref PubMed Scopus (123) Google Scholar). In EpoR-null embryonic mice, increased apoptosis in brain is observed and EpoR-null embryonic cortical neurons in culture do not survive after 24-h exposure to hypoxia (8Yu X. Shacka J.J. Eells J.B. Suarez-Quian C. Przygodzki R.M. Beleslin-Cokic B. Lin C.S. Nikodem V.M. Hempstead B. Flanders K.C. Costantini F. Noguchi C.T. Development (Camb.). 2002; 129: 505-516Crossref PubMed Google Scholar). Prior to death because of severe anemia in EpoR-null mice and Epo-null mice, the number of embryonic neural progenitor cells (NPCs) is reduced, and brain development appears underdeveloped, although no major structures are absent (8Yu X. Shacka J.J. Eells J.B. Suarez-Quian C. Przygodzki R.M. Beleslin-Cokic B. Lin C.S. Nikodem V.M. Hempstead B. Flanders K.C. Costantini F. Noguchi C.T. Development (Camb.). 2002; 129: 505-516Crossref PubMed Google Scholar, 13Tsai P.T. Ohab J.J. Kertesz N. Groszer M. Matter C. Gao J. Liu X. Wu H. Carmichael S.T. J. Neurosci. 2006; 26: 1269-1274Crossref PubMed Scopus (348) Google Scholar). In vitro, Epo can stimulate the differentiation of NPCs toward neurons. Animal models suggest that exogenous Epo may also be active in adult central nervous system. Epo has been shown to be neuroprotective for ischemia blunt force trauma and UV light damage (14Brines M.L. Ghezzi P. Keenan S. Agnello D. de Lanerolle N.C. Cerami C. Itri L.M. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10526-10531Crossref PubMed Scopus (1276) Google Scholar, 15Grimm C. Wenzel A. Stanescu D. Samardzija M. Hotop S. Groszer M. Naash M. Gassmann M. Reme C. J. Neurosci. 2004; 24: 5651-5658Crossref PubMed Scopus (112) Google Scholar, 16Sakanaka M. Wen T.C. Matsuda S. Masuda S. Morishita E. Nagao M. Sasaki R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4635-4640Crossref PubMed Scopus (892) Google Scholar). Direct infusion of Epo into the lateral ventricles in gerbils provided neuroprotection to hippocampal CA1 neurons in experimental cerebral ischemia and prevented ischemia-induced learning disability, whereas infusion of soluble EpoR promoted neuronal degeneration (16Sakanaka M. Wen T.C. Matsuda S. Masuda S. Morishita E. Nagao M. Sasaki R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4635-4640Crossref PubMed Scopus (892) Google Scholar). The protection to CA1 neurons and cognitive impairment is dose-dependent on the local Epo administration (17Catania M.A. Marciano M.C. Parisi A. Sturiale A. Buemi M. Grasso G. Squadrito F. Caputi A.P. Calapai G. Eur. J. Pharmacol. 2002; 437: 147-150Crossref PubMed Scopus (90) Google Scholar).The lack of mature erythrocyte production in EpoR-null mice results in severe anemia and death in utero before day 13.5 (1Lin C.S. Lim S.K. D'Agati V. Costantini F. Genes Dev. 1996; 10: 154-164Crossref PubMed Scopus (349) Google Scholar, 2Wu H. Liu X. Jaenisch R. Lodish H.F. Cell. 1995; 83: 59-67Abstract Full Text PDF PubMed Scopus (848) Google Scholar). To determine whether the increased apoptosis in embryonic brain and affected brain development are due to anemia in utero and lack of oxygen delivery or due to endogenous Epo signaling in brain or neural cells, we developed mice that expressed EpoR driven by the endogenous EpoR promoter in hematopoietic tissue but not in the central nervous system. Homozygous mice can survive to adulthood, have normal hematocrit, and exhibit no gross morphologic defects. Mice with selective rescue of EpoR showed undetectable EpoR expression in brain. We present evidence for novel functions of endogenous Epo on NPCs and neural cell survival. In culture, EpoR from wild type (WT) mice is expressed at higher levels on NPCs than mature neurons in culture, and NPC proliferation is elevated in the presence of Epo. Examination of brain during development of the mice with selective EpoR expression revealed increased apoptosis during embryogenesis, reduced proliferation in the hippocampus, and increased sensitivity of neurons to hypoxia and glutamate toxicity. In addition, injection of glutamate showed increased toxicity in these mice. These observations exemplify the neural protective activity of endogenous Epo and indicate that Epo directly stimulates proliferation in the hippocampus and subventricular zone, regions associated with adult neurogenesis.EXPERIMENTAL PROCEDURESPrimary Hippocampal Cell/Neurosphere Culture and Immunocytochemical Staining—The hippocampus from embryonic day 16 (E16) mouse embryos were dissected in phosphate-buffered saline and mechanically dissociated. For primary hippocampal cell cultures, dissociated cells (105/well) were plated in 6-well dishes pre-coated with poly-d-lysine and cultured in serum-free medium containing 0.6% d-glucose, 100 μg/ml transferrin, 25 μg/ml insulin, 20 nm progesterone, 6 μm putrescine, 30 nm selenium, 0.5 units/ml Pen-Strep, 1 mm l-glutamine, 50% minimum Eagle's medium, and 50% F-12. Without additional trophic factors, these cultures are predominantly mature neurons (MAP-2 immunopositive cells) with only small proportions of NPCs (nestin immunopositive cells) and glia.For neurosphere cultures, dissociated cells were plated in uncoated T25 flask with supplemental basic fibroblast growth factor (bFGF) (PeproTech, Inc., Rocky Hill, NJ) until NPCs aggregated and proliferated to form neurospheres (2-3 days) (18Ray J. Peterson D.A. Schinstine M. Gage F.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3602-3606Crossref PubMed Scopus (403) Google Scholar). To test the mitogenic effect of Epo on NPCs, Epo was used in place of bFGF. The neurospheres were then collected for Western blotting. Bromodeoxyuridine (BrdUrd) (Sigma) at 25 μm was added to the medium for proliferation assay. The cultures were maintained for 24-48 h, fixed with 4% paraformaldehyde in phosphate-buffered saline, then immunocytochemically stained with rabbit anti-mouse EpoR (M-20) (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-nestin antibodies (1:100; Chemicon, Temecula, CA) using fluorescence-conjugated secondary antibodies (1:200; Chemicon). The stained neurons were examined under a Nikon Eclipse TE-2000-U fluorescent microscope, and the images were captured using PerkinElmer confocal image system. Fluorescence density was analyzed by Image-Pro Plus program (Media Cybernetics Inc., Silver Spring, MD).Western Blotting—Tissues or cells were lysed with radioimmune precipitation assay buffer buffer (10 mm Tris-HCl, 1 mm EDTA, 0.1% SDS, 0.1% Na3VO4, 1% Triton X-100) and protease inhibitor (Roche Diagnostics, Indianapolis, IN), incubated on ice for 30 min, and centrifuged at 17,000 × g for 10 min. The protein sample was run on NuPAGE 4-12% BisTris gel (Invitrogen) for 1 h at 200 V. Protein was transferred to nitrocellulose by standard methods. The blot was blocked with 5% nonfat milk in Tween 20 Tris-buffered saline at room temperature for 1 h, probed with primary antibodies for EpoR, nestin, and β-actin (1:1000; Santa Cruz Biotechnology) at 4 °C overnight, washed in Tris-buffered saline, probed with corresponding horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h, and rinsed in Tris-buffered saline for chemiluminescent detection.Development of Conditional Rescue Mice—The mouse EpoR gene was replaced by the human EpoR gene by substituting the genomic region between exon 1 to exon 8 of the endogenous mouse EpoR gene with exon 1 to exon 8 of the human EpoR gene. The human EpoR gene was inactivated using a neo cassette flanked by LoxP sites inserted into intron 7 (19Divoky V. Liu Z. Ryan T.M. Prchal J.F. Townes T.M. Prchal J.T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 986-991Crossref PubMed Scopus (45) Google Scholar). Mouse and human EpoR are functionally similar and in vivo the EpoR-null mouse can be rescued with a human transgene (8Yu X. Shacka J.J. Eells J.B. Suarez-Quian C. Przygodzki R.M. Beleslin-Cokic B. Lin C.S. Nikodem V.M. Hempstead B. Flanders K.C. Costantini F. Noguchi C.T. Development (Camb.). 2002; 129: 505-516Crossref PubMed Google Scholar). Only mice heterozygous for the disrupted EpoR gene survive, as homozygous mice lack EpoR expression and die in utero. These heterozygous mice were crossed with transgenic mice expressing Cre recombinase under the direction of the endothelial cell-specific receptor tyrosine kinase Tek (Tie2) promoter/enhancer (20Koni P.A. Joshi S.K. Temann U.A. Olson D. Burkly L. Flavell R.A. J. Exp. Med. 2001; 193: 741-754Crossref PubMed Scopus (406) Google Scholar). Expression of Cre in cells that normally express Tie2, such as embryonic endothelium, which give rise to hematopoietic stem cells, results in recombination of the two LoxP sites and excision of the neo cassette that restores appropriately regulated expression of the EpoR gene in Cre-expressing cells and subsequent generations of cells derived from these cells. Mice from resultant litters were screened and crossed to obtain mice homozygous for the disrupted EpoR gene that carry the Tie2-Cre transgene (designated ΔEpoR). All animal protocols were approved and carried out according to the guidelines of the NIDDK Animal Care and Use Committee.Reverse Transcription-PCR (RT-PCR)—Adult brains were harvested and put in Qiagen RNAlater™ RNA stabilization reagent (Qiagen, Germantown, MD). Total RNA was isolated using the Qiagen RNeasy. First-strand cDNA was synthesized using murine leukemia virus reverse transcriptase and oligo(dT)16 (PE Applied Biosystems, Foster City, CA). The EpoR transcripts in the brain were determined with RT-PCR using primers m-hEpoR-F2, 5′-GCAGTGAGCATGCCCAGGA-3′ and m-hEpoR-R2, 5′-GCTTCACCAATCCCGTTCAAG-3′. The primers are specific for both mouse and human EpoR. Reaction conditions were optimized to give amplification of PCR product for even low levels of expression. PCR reaction was performed for 40 cycles of 30-s denaturation at 94 °C, 1 min annealing at 58 °C, and 1-min extension at 72 °C.Blood Analysis—For analysis of red cell indices, blood was drawn into a K2EDTA coated micro-hematocrit tube from the orbital sinus of anesthetized adult mice. The red blood cell count, hemoglobin, and hematocrit were determined for each sample.Physical Characteristics and Gross Morphology—Adult wild type and ΔEpoR (six-month-old) male mice (n = 5 each) were weighed, deeply anesthetized, and perfused. Brains were dissected, weighed, length and width measured, and frozen sections prepared.TUNEL Analysis of Conditional Rescue Embryonic Brains—Wild type and ΔEpoR E16 embryonic brains were dissected and fixed with 4% paraformaldehyde in phosphate-buffered saline, prepared for frozen section, sliced, and then analyzed with TUNEL labeling reagent (Roche Diagnostics) followed by DAPI counter staining of nuclei. Serial sections were examined under a Nikon Eclipse TE-2000-U fluorescent microscope, and the images were captured using PerkinElmer confocal Image system.BrdUrd Immunohistochemistry and Quantification—Three-month-old mice were injected intraperitoneally with BrdUrd (100 mg/kg body weight). After 2 weeks of injection, mice were perfused, and the brains were dissected. Coronal sections (12 μm in 2:20 series) were cut with a cryostat and mounted on Superfrost®/Plus slides. Sections containing the hippocampal and subventricular zone (SVZ) regions were selected and treated sequentially with trypsin (0.1%) and HCl (2 m) followed by overnight incubation with sheep anti-BrdUrd antibody (1:100; BD Biosciences) at 4 °C. After exposure to fluorescence-conjugated secondary antibody (1:100; BD Biosciences), the sections were mounted with vector DAPI mounting medium (Vector Laboratories Inc., Burlingame, CA). Serial sections were then examined with a fluorescent microscope, and the images were captured. All BrdUrd-positive cells in the hippocampus and SVZ were counted as described previously (21Cheng Y. Black I.B. DiCicco-Bloom E. Eur. J. Neurosci. 2002; 15: 3-12Crossref PubMed Scopus (99) Google Scholar).Glutamate Exposure—To assess glutamate toxicity in adult animals, 100 μl of glutamate solution (25 m) was stereotaxically injected into the right ventricle of the mice. After 24 h the mice were euthanized, and the brains were dissected. Coronal sections (12 μm in 2:20 series) were collected on Superfrost®/Plus slides for apoptosis analysis by an indirect TUNEL labeling assay (Roche Diagnostics). For in vitro studies, cultured neural cells were exposed to different dose (0-500 μm) glutamate for 5 min. The glutamate was washed out completely after the exposure. The culture was examined under phase-contrast microscope after 24 h of glutamate exposure.Statistical Analysis—Standard deviations and p values determined by the Student's t test were calculated by standard methods (Microsoft Excel; Microsoft Corporation, Redmond, WA).RESULTSNeural Progenitor Cells Express Higher Level of Epo Receptors than Mature Neurons—We used a primary hippocampal cell culture system to evaluate whether EpoR is expressed in mature neurons or NPCs. Immunohistochemical colocalization studies showed that EpoR was colocalized with both mature neurons (MAP-2+) (Fig. 1) and NPCs (Fig. 2, A-D). Qualitatively, we found that NPCs gave more intense staining for EpoR than mature neurons (Fig. 2E). Thus, we compared the level of EpoR in two cell culture systems; one system contained predominantly neurons, and the other contained predominantly a NPC population (primary hippocampal culture and neurosphere culture, respectively). EpoR level was found higher in neurospheres than in primary cultures (Fig. 2F). These data suggest that EpoR is down-regulated as NPC terminally differentiates to mature neurons. We have reported that the expression of EpoR in the mouse brain peaks at mid gestation and then subsequently decreases to modest levels in the adult (22Liu Z.Y. Chin K. Noguchi C.T. Dev. Biol. 1994; 166: 159-169Crossref PubMed Scopus (78) Google Scholar, 23Liu C. Yu K. Shen K. Liu Z. Noguchi C.T. Proc. Assoc. Am. Physicians. 1996; 108: 449-454PubMed Google Scholar). This difference in EpoR expression level on NPC and mature neurons may account in part for the decrease of EpoR during development (22Liu Z.Y. Chin K. Noguchi C.T. Dev. Biol. 1994; 166: 159-169Crossref PubMed Scopus (78) Google Scholar, 23Liu C. Yu K. Shen K. Liu Z. Noguchi C.T. Proc. Assoc. Am. Physicians. 1996; 108: 449-454PubMed Google Scholar) because of the reduction of the NPC ratio.FIGURE 2NPC expression of EpoR. A-D, cultures of E16 hippocampal cells were double-immunostained with (A) EpoR (red) and (B) nestin (green) antibodies. The merged image (D) shows all nestin positive cells are also EpoR positive. The bright field image is shown in C. Scale bar, 100 μm. E, relative fluorescence intensity corresponding to EpoR staining was determined for EpoR positive and nestin positive cells (Nes+/EpoR+), for EpoR positive and nestin negative cells (Nes-/EpoR+) and for background staining of EpoR negative and nestin negative cells (Nes-/EpoR-). The EpoR density indicated by the pixel values of the fluorescence intensity was measured using ImagePro image analysis software. *, p < 0.001 compared with nestin negative cells. F, EpoR immunoblot of cultured neurospheres (lane 1) compared with cultured primary cells (lane 2). When β-actin levels are normalized, EpoR expression is found higher in neurosphere cultures than in primary cultures. Top panel, EpoR; bottom panel, β-actin control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Epo Stimulates Neural Progenitor Cell Proliferation—To investigate whether NPCs exhibit a direct growth response to Epo, Epo was added to NPC culture medium (Fig. 3). The number of NPCs is increased in the presence of Epo, and dose response indicated that 10 units/ml of Epo gave the optimum effect. Epo at 10 units/ml increased the number of nestin positive cells by 3-fold (Fig. 3, E and G). To test the proliferation response to Epo, the culture medium was supplemented with BrdUrd, a thymidine analogue incorporated into newly synthesized DNA of replicating cells. Double labeling of BrdUrd and nestin of 5 day cultures showed that about 50% NPC proliferated in response to 10 units/ml Epo (Fig. 3, E and H). The Epo response is about half as strong as that of bFGF (Fig. 3, F, H, and I). In the presence of bFGF (20 ng/ml), the number of NPC increased by 4-fold, and almost all NPC show BrdUrd incorporation. However, the combination of Epo and bFGF treatment showed no additive response on NPC proliferation (cell number) compared with bFGF alone.FIGURE 3Increased NPC proliferation by Epo stimulation. A-F, primary hippocampal cells cultured in medium containing 25 μm BrdUrd. Neural cells in control medium (A and D), 10 units/ml Epo (B and E), and 20 ng/ml bFGF (C and F) were subsequently fixed and double-immunostained with BrdUrd (green) and nestin (red). A-C, bright field images of the cultures. D-F, BrdUrd and nestin double-immunostaining. Scale bar, 100 μm. G, number of NPCs was determined after 2 days in culture without and with Epo (2.5, 5, 10, 20, and 40 units/ml as indicated), and results are normalized to the control value. H, number of NPCs was determined after 2 days in culture without and with bFGF (bFGF at 5, 10, and 20 ng/ml as indicated) and with bFGF (20 ng/ml) and Epo (10 units/ml), and results are normalized to the control value. I, the proliferation rate is the ratio between BrdUrd-labeled cells and the total number of NPCs. The proliferation rate of NPC in the presence of Epo and/or bFGF was determined. *, p < 0.001 compared with control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Generation of Brain EpoR-null Mice—Insertion of the neo cassette in intron 7 of the EpoR gene disrupts EpoR expression, and homozygous mice exhibit a phenotype similar to mEpoR-/- mice and die in utero because of lack of mature red blood cell production (19Divoky V. Liu Z. Ryan T.M. Prchal J.F. Townes T.M. Prchal J.T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 986-991Crossref PubMed Scopus (45) Google Scholar). Control mice were obtained by unconditional deletion of the neo cassette using a cytomegalovirus/Cre transgene (19Divoky V. Liu Z. Ryan T.M. Prchal J.F. Townes T.M. Prchal J.T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 986-991Crossref PubMed Scopus (45) Google Scholar). Conditional expression of EpoR in hematopoietic tissue but not in brain was obtained by breeding mice carrying the disrupted EpoR genetic background with the Tie2-Cre transgenic mouse (ΔEpoR) (20Koni P.A. Joshi S.K. Temann U.A. Olson D. Burkly L. Flavell R.A. J. Exp. Med. 2001; 193: 741-754Crossref PubMed Scopus (406) Google Scholar). Embryonic expression of Tie2-Cre in endothelium that gave rise to endothelial cells and hematopoietic stem cells rescued the EpoR-null phenotype by allowing for EpoR expression in early erythroid progenitor cells and restoring production of mature erythrocytes. ΔEpoR mice exhibited no gross morphologic defects. Red cell indices demonstrated that the ΔEpoR selective rescued mice had similar red blood cell content as their control peers. The red blood cell count was 8.8 ± 0.9 m/μl for ΔEpoR mice and 8.8 ± 1.6 m/μl for control mice. The hemoglobin values were 13.2 ± 1.0 g/dL for ΔEpoR mice and 14.2 ± 2.4 g/dL for control mice, and the hematocrit was 42.3 ± 1.5% for ΔEpoR mice and 44.2 ± 7.1% for control mice. RT-PCR confirmed the down-regulation of EpoR expression in brain in the ΔEpoR mice (Fig. 4A). We used Western blotting to confirm the marked reduction of EpoR protein in brain as expected for the selective expression of EpoR only in tissue derived from Tie2-expressing cells such as hematopoietic and endothelial cells (Fig. 4A).FIGURE 4Deletion of brain EpoR expression and increased apoptosis in ΔEpoR embryonic brain. A, left, RT-PCR analysis for brain EpoR expression in ΔEpoR mice. Top panel, EpoR; bottom panel, β-actin control; lane 1, DNA ladder; lane 2, RT-PCR analysis for WT mouse; lane 3, RT-PCR analysis for mice with targeted inactivation of EpoR in brain (ΔEpoR). Right, Western blotting for E16 brain EpoR, nestin, and β-actin in WT and ΔEpoR mice. B-C, apoptosis analysis of the cortex from E16 embryo brain from WT (B) and ΔEpoR (C) mice. Blue indicates DAPI staining to visualize nuclear DNA, and red indicates TUNEL positive cells. Scale bar, 100 μm. D, the % of apoptotic cells in the region of the cortex from E16 embryos is shown for ΔEpoR and wild type mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Epo Signaling Affects Neuron Development—Previously we demonstrated that embryonic brain of EpoR-null mice exhibited increased apoptosis, and neural cultures from these mice at E10.5 showed increased sensitivity to hypoxia suggesting an intrinsic defect during embryonic neural development as an indirect consequence of disrupted EpoR expression in hematopoietic/erythroid progenitor cells or possibly due to loss of Epo signaling in neural cells (8Yu X. Shacka J.J. Eells J.B. Suarez-Quian C. Przygodzki R.M. Beleslin-Cokic B. Lin C.S. Nikodem V.M. Hempstead B. Flanders K.C. Costantini F. Noguchi C.T. Development (Camb.). 2002; 129: 505-516Crossref PubMed Google Scholar). The selective expression of EpoR in the ΔEpoR mice restores normal hematopoiesis and the mice appear normal confirming that whereas EpoR expression in hematopoietic tissue is required for production of mature red blood cells and survival, EpoR expression in the central nervous system is not critical for life. We observed that during embryogenesis, ΔEpoR mice showed increased apoptosis in the brain compared with control mice (Fig. 4, B and C). The extent of apoptosis indicated by TUNEL positive cells was 2-fold greater in ΔEpoR embryonic brain (Fig. 4D). These data indicate that endogenous Epo signaling is required for normal brain development. The increased apoptosis in brain observed in EpoR-null mice prior to death in utero is not only a consequence of disrupted erythroid progenitor cell differentiation and lack of oxygen delivery (8Yu X. Shacka J.J. Eells J.B. Suarez-Quian C. Przygodzki R.M. Beleslin-Cokic B. Lin C.S. Nikodem V.M. Hempstead B. Flanders K.C. Costantini F. Noguchi C.T. Development (Camb.). 2002; 129: 505-516Crossref PubMed Google Scholar). A reduction of nestin protein in E16 ΔEpoR mouse brain detected by Western blotting (
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