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Erythropoietin receptor signalling is required for normal brain development

¹ Laboratory of Chemical Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA
² Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA
³ Laboratory of Cell Regulation and Carcinogenesis, NCI, National Institutes of Health, Bethesda, MD 20892, USA
⁴ Department of Cell Biology, Georgetown University Medical School, Washington, DC 20007, USA
⁵ Armed Forces Institute of Pathology, Washington, DC 20306, USA
⁶ Department of Genetics and Development, Columbia University, New York, NY 10032, USA
⁷ Cornel Medical College, New York, NY 10021, USA

View larger version (144K): [in a new window]   Fig. 1. Analysis of embryonic liver and heart. Foetal livers from Epor–/– (A) and Epor+/+ (B) embryos at E12.5 were analysed by TUNEL. Apoptotic cells are stained red. (C-E) E12.5 embryos from Epor+/+ mice were analysed for EpoR production in heart. Immunostaining of EpoR was detected in myocardium and endocardium (C,D); tenfold excess of blocking peptide completely blocked anti-EpoR antibody staining (E). (F,G,I,J) Von Willebrand factor (vWF) staining of E12.5 hearts Epor+/+ mice (F,G) and Epor–/– (I,J) mice. Arrowheads indicate vWF positive cells. (H,K) TUNEL assay of Epor+/+ (H) and Epor–/– (K) embryonic hearts show increased apoptosis in the Epor–/– embryo. Arrowhead indicates TUNEL positive cells. Bars, 0.025 mm (A,B,G,H,J,K) or 0.1 mm (C-F,I).

View larger version (111K): [in a new window]   Fig. 2. EpoR production in developing brain. (A-G) Immunostaining of Epor+/+ embryos for EpoR shows the production pattern of EpoR in the brain at E10.5 (A), E11.5 (B-F) and E12.5 (G). The inset shows transgene expression of an Epor-lacZ promoter-reporter gene construct that provides tissue-specific expression in regions corresponding to endogenous EpoR production at E10.5 (A) and E12.5 (G). (C-F) Increased magnifications of B. (H,I) Hypoplasia of neuroepithelium of the fourth ventricle was observed in the E12.5 Epor–/– embryo (I) compared with Epor+/+ (H). (J-Q) TUNEL analysis of embryonic brain of Epor–/– (J-M) and Epor+/+ (N-Q) mice at E10.5 (J,K,N,O) and E12.5 (L,M,P,Q). Increased apoptosis was observed for Epor–/– as early as E10.5. Increased apoptotic cells are shown in neuroepithelium of E10.5 midbrain (coronal section) (J,K) and E12.5 hindbrain (sagittal section) (L,M). Red blood cells (indicated by arrowhead and enlarged in the inset) were observed in the E10.5 neuroepithelium (K). (K,M,O,Q) Increased magnifications of J,L,N and P, respectively. Abbreviations: GE, ganglionic eminence; IV, fourth ventricle; VZ, ventricular zone; IZ, intermediate zone. Bars, 1 mm (B,G), 0.4 mm (A, inset A, inset G, H-J,L,N,P), 0.2 mm (K,O) and 0.05 mm (C-F,M,O,Q).

View larger version (31K): [in a new window]   Fig. 3. Neuronal progenitor cells from Epor–/– cortex. (A) At E9.5, nestin expression is comparable in cortices of the Epor+/+ and Epor+/– (n=5), and Epor–/– embryos (n=3). (B) Single-cell suspension isolated from E10.5 Epor–/– cerebral cortices (n=6) showed marked reduction in total cell numbers compared with Epor+/+ and Epor+/– cortices (n=13). (C) By E10.5, nestin mRNA level was downregulated by four times in the Epor–/– cortex (n=4) compared with Epor+/+ and Epor+/– (n=6). (D,E) A marked decrease in the numbers and proportion of nestin positive progenitor cells acutely isolated from the E10.5 Epor–/– cortex (n=4) was also observed compared with Epor+/+ and Epor+/– cortices (n=12). (F,J,K) Fewer neurons were produced from in vitro cultures of Epor–/– cortical cells. Cells were stained for MAP2 (F) or ß-tubulin III (red) (J,K) and with DAPI (blue) after 4 days of culture in NBM. The proportions of cells with MAP2 positive staining are indicated in (F) for Epor–/– (n=5) and for Epor+/+ and Epor+/– (n=6). Representative fields for Epor+/+ cultures with 41% of 200 cells (J) and for Epor–/– cultures with 23% of 199 cells (K) with ß-tubulin III positive staining are shown. (G-I,L,M) EpoR production improved neuronal cell survival under hypoxia. Six and 24 hours after cells were switched to Locke’s solution and cultured under the hypoxic condition of 2% oxygen tension, cells were stained with MAP2 (red), TUNEL (green) and DAPI (blue). After 6 hours, the number of surviving neurons was markedly decreased (G) and apoptotic neurons were significantly increased (H) in Epor–/– cultures (n=3). No surviving Epor–/– neurons were observed after 24 hours exposure (n=3) (G). About 10% of the surviving cells were neurons in the Epor+/+ cultures. Erythropoietin addition increased the survival of Epor+/+ neurons producing EpoR (I); no erythropoietin response was observed in Epor–/– cultures. In all experiments (E-M), cells were initially plated at the same density. For cell enumeration, the percentage of nestin (E) and MAP2 (F-I) positive cells isolated for each individual embryo were determined by an investigator blind to the genotypes by quantifying 20 microscopic fields documented by digital camera images. A total of 2000-4000 cells were counted. Scale bars: 0.025 mm in L,M; 0.05 mm in J,K. (B-F) P < 0.01. Open bars represent Epor+/+ or Epor+/+ and Epor+/– cultures, as indicated, and solid bars represent Epor–/– cultures.

View larger version (18K): [in a new window]   Fig. 4. Erythropoietin stimulation of neuronal cells. (A) Erythropoietin (5 U ml–1) stimulated NT2 cell proliferation when cultured under 2% oxygen tension for 3 days. (B) Epor expression was induced in NT2 cells cultured with 5% CO2 and 2% O2 compared with 20% O2, determined by quantitative RT-PCR using Taqman probes. (C) GATA3 production was induced by erythropoietin stimulation. (D) Epor expression was induced in NT2 cells overexpressing GATA3. (E) In transfection assays, the human Epor promoter was linked to a luciferase reporter gene to assess Epor promoter activity in NT2 cells. GATA3 production induced Epor promoter activity (EpoR). Mutation of the GATA motif (EpoR) markedly decreases promoter activity. (F) GATA3 expression was downregulated in E10.5 Epor–/– cortex (solid bar) compared with expression in the Epor+/+ cortex (open bar).

View larger version (21K): [in a new window]   Fig. 5. Effect of erythropoietin on the viability of embryonic neurons. (A) Cell viability was determined for primary embryonic rat cortical neurons supplement starved for 24 hours in Locke’s solution with or without erythropoietin (5 U ml–1) under normoxia (20% O2) and hypoxia (2% O2). (B) The percentage of cells undergoing apoptosis was determined by TUNEL analysis. (C-E) Real-time RT-PCR quantitation was used to determine the levels of Epor (C), erythropoietin (Epo) (D) and Bcl-xL (E) gene expression in the cells after 24 hours of culture in Locke’s solution. Expression is normalized to ribosomal protein S16 levels as control. (F) Induction of phosphorylation of Jak2 and STAT5 in cortical neurons treated with erythropoietin cultured at 20% or 2% oxygen tension was examined. (A-C) Cultures with and without supplemental erythropoietin (5 U ml–1) are represented by solid and open bars, respectively.

View larger version (13K): [in a new window]   Fig. 6. Effect of erythropoietin pretreatment on viability of embryonic neurons. (A) Primary embryonic rat cortical neurons were treated with (closed bars) and without (open bars) erythropoietin (5 U ml–1) on day 4 of culture and cell viability determined on day 8. (B,C) On day 8, cells were cultured in Locke’s solution without supplemental erythropoietin under normoxia (20% O2) (B) and hypoxia (2% O2) (C). After 24 hours, undamaged neurons were counted in premarked microscopic fields. At least six fields were observed for each well, at least three wells were studied in three separate experiments. A total of 300-900 neurons in each condition were counted.