ARTICLE Received 11 Nov 2012 | Accepted 7 Nov 2013 | Published 6 Dec 2013 DOI: 10.1038/ncomms3902 Erythropoietin production in neuroepithelial and neural crest cells during primitive erythropoiesis Norio Suzuki1,*, Ikuo Hirano2,*, Xiaoqing Pan2,3, Naoko Minegishi2,3 & Masayuki Yamamoto2,3 Erythropoietin (Epo) supports both primitive erythropoiesis in the yolk sac and definitive erythropoiesis in the fetal liver and bone marrow. Although definitive erythropoiesis requires kidney- and liver-secreted Epo, it is unclear which cells produce Epo for primitive erythropoiesis. Here we find neural Epo-producing (NEP) cells in mid-gestational stage embryos using mouse lines that express green fluorescent protein (GFP) under the Epo gene regulation. In these mice, GFP is expressed exclusively in a subpopulation of neural and neural crest cells at embryonic day 9.0 when Epo-deficient embryos exhibit abnormalities in primitive erythropoiesis. The GFP-positive NEP cells express Epo mRNA and the ex vivo culture of embryonic day 8.5 neural tubes results in the secretion of Epo, which is able to induce the proliferation and differentiation of yolk sac-derived erythroid cells. These results thus suggest that NEP cells secrete Epo and might support the development of primitive erythropoiesis. 1 Division of Interdisciplinary Medical Science, United Centers for Advanced Research and Translational Medicine, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. 2 Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. 3 Tohoku Medical Megabank Organization, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to N.S. (email: [email protected]). NATURE COMMUNICATIONS | 4:2902 | DOI: 10.1038/ncomms3902 | www.nature.com/naturecommunications 1 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3902 rythropoietin (Epo) is one of the best-characterized observed in Epo-KO embryos at E8.5 (5–9 somite pair stages cytokines that stimulates the proliferation and differentia- (sp); Fig. 1a) or E9.0 (16–20 sp; Fig. 1a). Nonetheless, the yolk Etion of erythroid cells1. Epo binds to the Epo receptor sacs of Epo-KO mice were moderately and severely anaemic at (EpoR) on the surface of erythroid progenitor (EP) cells and E10.5 and E11.5, respectively (Fig. 1a). We found that Epo-KO stimulates the proliferation and maturation of progenitor cells embryos died by E12.5 due to anaemia, and this is consistent with via the phosphorylation of EpoR and associated signalling the previous observations18. molecules2. Tissue hypoxia that is caused by anaemia or To characterize the yolk sac cells of Epo-KO embryos, we altitude sickness induces Epo gene expression in the kidney and conducted flow cytometry analyses. In wild-type yolk sacs, liver of adult humans and mice1,3. This hypoxia-induced Epo transferrin receptor (CD71) expression in Ter119 þ erythroblasts gene expression is transcriptionally regulated by hypoxia- (EBs) gradually increased during embryonic development inducible transcription factors (HIFs). The first characterized (Fig. 1b), whereas the increase was not so significant for Epo- HIF-binding sequence (that is, hypoxia responsive element or HRE) resides in the 30-region of the Epo gene4–6. The first wave of murine erythropoiesis begins in the blood islands of the visceral yolk sac at embryonic day 7.5 (E7.5). This Wild typeEpo-KO Wild type Epo-KO erythropoiesis is referred to as primitive erythropoiesis7,8. The fetal liver subsequently becomes the principal erythropoietic tissue in late-stage embryos. Around birth, erythropoiesis moves to the bone marrow and spleen6. The latter type of erythropoiesis is referred to as definitive erythropoiesis7,8. Yolk sac-derived erythrocytes express embryonic b-globin (ey-globin), whereas E8.5 E8.5 definitive erythrocytes express adult-type b-globin (primarily bmaj-globin)8. This shift of erythropoiesis from one tissue to another and the change in globin-gene expression are common features of vertebrate erythropoiesis. Mouse and human definitive erythrocytes in the peripheral blood are enucleated, whereas primitive erythrocytes have a nucleus when they are released from E9.0 E9.0 yolk sac and complete their maturation (enucleation) while circulating9,10. Tissues other than the liver and kidney also secrete Epo, but whether Epo makes critical contributions to non-haematopoietic tissues or cells remains to be clarified11–16. In contrast, transgenic complementation rescue analyses of EpoR gene revealed that EpoR expression in non-haematopoietic tissues is dispensable for E10.5 E10.5 mouse survival and fertility17. Thus, while Epo also has non- haematopoietic functions under pathological or stress conditions, the principal role of Epo is to support erythropoiesis. Epo or EpoR gene knockout mice are lethal at BE12.5 because 18 of a defect in definitive erythropoiesis . We have identified renal CD71 Epo-producing (REP) cells in the interstitial space of the 19–21 E11.5 kidney . REP cells secrete Epo into the bloodstream where E11.5 Ter119 it acts as an endocrine factor for bone marrow EPs6,22. While adult mouse hepatocytes also produce low levels of Epo, this Wild type hepatic Epo production is dispensable for erythropoiesis in adult E8.5 mice6,23. In contrast, fetal liver-derived Epo stimulates liver Epo-KO 6 E9.0 erythropoiesis in a paracrine manner . * Importantly, Epo or EpoR gene knockout mice also exhibit an E10.5 inefficient production of primitive erythrocytes, suggesting that * ** 18 E11.5 Epo plays important roles in primitive erythropoiesis . However, * ** Epo-producing cells in the early stages of mouse development 60 40 20 0 0 1,000 2,000 3,000 have not been identified. Therefore, in the present study we CD71+ Ter119+ cells (%) CD71 expression level address Epo production in early-stage embryos. We observe Epo production in neural and neural crest cells of E8.5–E11.5 Figure 1 | Epo is required for primitive erythropoiesis in mouse embryos embryos. We also determine that this Epo production is able to after E9.0. (a) Whole-mount images of wild-type and Epo-KO embryos at induce the proliferation and differentiation of yolk sac-derived the indicated stages (E8.5, E9.0, E10.5 and E11.5). Scale bars, 1.0 mm. erythroid cells under ex vivo co-culture conditions. The major (b) FACS analysis of yolk sac cells from wild-type and Epo-KO embryos source of Epo for primitive erythropoiesis might be a at E8.5 (5–9 sp), E9.0 (16–20 sp), E10.5 and E11.5. The relative abundance subpopulation of neural and neural crest cells, which we refer of the CD71 þ Ter119 þ fraction is indicated in each panel as a percentage. to as neural Epo-producing (NEP) cells. The blue and red lines indicate the mean fluorescent intensities of CD71 expression in the Ter119 þ CD71 þ fractions of wild-type and Epo-KO embryos, respectively. (c) The relative abundance (left) and CD71 Results expression levels (right) of the CD71 þ Ter119 þ fraction in the yolk sac cells The indispensable roles of Epo in primitive erythropoiesis.To from wild-type and Epo-KO embryos at the indicated stages. FACS data elucidate the contribution of Epo to primitive erythropoiesis, we from more than three embryos in each group are presented with s.d. analysed an Epo-knockout (Epo-KO or Epo À / À ) mouse line *Po0.05, **Po0.01 when compared with the wild-type controls, based on (Supplementary Fig. S1)20,21. No apparent abnormality was Student’s t-test. 2 NATURE COMMUNICATIONS | 4:2902 | DOI: 10.1038/ncomms3902 | www.nature.com/naturecommunications & 2013 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3902 ARTICLE KO yolk sacs. The CD71 fluorescence intensity was markedly E9.0, although Epo-producing tissues or cells have not been lower in Epo-KO yolk sacs than that in wild-type yolk sacs after identified in early-stage embryos. E9.0 (Fig. 1c). Moreover, the relative abundance of CD71 þ To identify Epo-producing tissues or cells in mid-gestational stage Ter119 þ cells was decreased in Epo-KO yolk sacs after E10.5 embryos, we dissected embryos (Supplementary Fig. S2a–c) at E8.5 compared with those in wild-type yolk sacs (Fig. 1c). These (5–9 sp), E9.5 (22–25 sp) and E10.5, and analysed Epo messenger results support our contention that Epo contributes to primitive RNA expression using reverse transcription (RT)–quantitative PCR erythropoiesis and is required for the differentiation of (qPCR). To our surprise, Epo mRNA was exclusively expressed in primitive erythroid cells towards the CD71-positive stage in yolk the head region at E8.5 (Fig. 2a). As Epo mRNA was not detected in sacs after E9.0. samples from whole embryos at E7.5, the head region of E8.5 embryos appeared to be the first Epo-producing tissue during mouse embryogenesis. Epo mRNA expression was detected in the head region and in the fetal liver in E9.5 and E10.5 embryos Epo expression in developing neural tissues. To our current 6,24 knowledge, the earliest time of Epo expression is at E9.5 in the (Fig. 2a) .TherelativeEpo mRNA expression level in the head liver24. However, the impaired primitive erythropoiesis in region gradually decreased during development and was Epo-KO mice implies that Epo is produced in embryos around undetectable at E11.5, whereas Epo mRNA expression in the fetal liver gradually increased over this period (Fig. 2a). 12 E8.5 E9.5 E10.5 10 8 mRNA 6 Epo 4 2 0 PI YS Hd Ht Bd PI YS Hd Ht Lv PI YS Hd Ht Lv NT NT NT E8.5 E8.5 E9.0 E EE EE E E NT NT NT E9.5 E9.5 E10.0 Figure 2 | Embryonic neural tissues in the mid-gestation embryo transiently express Epo.