Erythropoietin Production in Neuroepithelial and Neural Crest Cells During Primitive Erythropoiesis

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

mRNA 6

Epo 4

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. (a) RT–qPCR analyses of Epo mRNA expression in the placenta (Pl), the yolk sac (YS), the head (Hd), the heart (Ht), the body proper (Bd) and the liver (Lv) of wild-type embryos at E8.5 (5–9 sp), E9.5 (22–25 sp) and E10.5. The data indicate the averages of the relative expression levels with s.d. The levels were normalized using Gapdh mRNA expression levels, and three embryos were analysed in each sample. Note that Epo mRNA expression is detectable in heads (green) and livers (orange) of the embryos. The grey arrows indicate undetectable levels. (b–g) The GFP expression proﬁle in 22K-Epo-GFP transgenic mouse embryos at E8.5 (b,c), E9.0 (d), E9.5 (e,f) and E10.0 (g). GFP-positive cells are observed in or around the neural folds (arrowheads in b–d) at E8.5 and E9.0. Dotted circle, fetal liver; orange arrow, optic vesicle; E, otic vesicle; NT, neural tube. Scale bars, 0.5 mm (b,e,f) and 0.2 mm (c,d,g).

NATURE COMMUNICATIONS | 4:2902 | DOI: 10.1038/ncomms3902 | www.nature.com/naturecommunications 3 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3902

Table 1 | Epo-GFP transgene expression in E9.5 embryos. transgenic mouse lines (two lines for each mutation) were utilized to assess GFP expression in E9.5 embryos. The 50-GATA motif was not required for GFP expression in NEP cells but was Transgene Mutation Lines Head Liver Epidermis essential to repress ectopic GFP expression in the epidermis wt-Epo-GFP –2þþ À (Table 1). Similarly, analyses of 30-HRE-mutant transgenes (m3- 22K-Epo-GFP –3þþ À 0 17KXh-Epo-GFP –2þþ À Epo-GFP) revealed that 3 -HRE was dispensable for reporter m1-Epo-GFP 50-GATA 2 þþ þ expression in NEP cells (Table 1). These results indicate that the m2-Epo-GFP 50-GATA 2 þþ þ cis-acting regulatory elements for Epo gene expression in NEP 0 m3-Epo-GFP 30-HRE 2 þþ À cells reside in the 35-kb region but are independent of 5 -GATA Epo þ / À or Epo À / À GFP knock-in þþ À and 30-HRE.

Epo-GFP transgene expression in developing neuroepithelium. We found that GFP expression in the head region decreased in To identify which cell types express Epo in the embryonic head Epo-GFP transgenic mouse embryos after E10.5, while the fetal region, we next examined transgenic mouse lines that express liver consistently expressed GFP after E9.5 (Fig. 3a–d). Since GFP green fluorescent protein (GFP) under the control of the Epo expression was observed in the neuroepithelium around hind- regulatory region. To this end, we used transgenic mouse lines brain (Fig. 3a,b), we conducted immunofluorescent analyses that carried various bacterial artificial chromosome-based GFP using antibodies against GFP and Sox2, a marker for developing reporter transgenes that we previously established (Table 1; 25 þ 6,15,19,22 neuroepithelial cells . There are GFP cell fractions in the Supplementary Fig. S1) . Importantly, it has been shown neuroepithelium and nuclear staining of Sox2 in the GFP þ cells, that GFP fluorescence in these mice faithfully recapitulates the demonstrating that a part of neural lineage cells express the Epo endogenous Epo gene expression profile in adult livers and gene at E9.5 (Fig. 3e,f). kidneys. We isolated the GFP þ cells from the head region of 17KXh- We analysed seven lines of transgenic mice that carried wt- 6,15,19,22 Epo-GFP transgenic mouse embryos at E9.5 using fluorescence- Epo-GFP, 22K-Epo-GFP or 17KXh-Epo-GFP transgenes activated cell sorter (FACS; Supplementary Fig. S2e). RT–qPCR and found that all these mice exhibited green fluorescence in analyses of the isolated cells showed co-expression of neural rostral neural tissues and liver at E9.5 (22–25 sp) with a conserved marker mRNAs (Sox2 and Nestin)25,26 with Epo mRNA in NEP expression profile (Table 1). The representative GFP expression cells (Supplementary Fig. S3a). Epo-GFP expression was also patterns in 22K-Epo-GFP transgenic mouse embryos are shown observed in neuroepithelia of the otic and optic vesicles (Figs 2e,f in Fig. 2b–g. In E8.5–E10.0 embryos, green fluorescence was and 3a,b), and the expression was detectable in the retinal detected in rostral neural tissues, and the expression in the neuroepithelium until E11.5 (Fig. 3g). These results thus demon- rhombomeres (especially in r1, r3 and r5) was strong (Fig. 2f,g). strate that Epo is expressed transiently in a subpopulation of E9.5–E11.5 embryos also expressed GFP in the liver (Fig. 2e; developing neural cells. Supplementary Fig. S2c,d). The expression profile and GFP intensity in the transgenic mouse embryos were comparable with the RT–qPCR data, revealing high-level expression in the head Strong Epo-GFP expression in a neural crest cell cluster.We region at E8.5 and a decrease in this expression during detected a cell cluster expressing very bright green fluorescence development. Thus, these results demonstrate that cells in posterior to the otic vesicle in the 17KXh-Epo-GFP and 22K-Epo- neural tissues of E8.5–E11.5 embryos express Epo. We refer to GFP transgenic embryos at E9.0 (arrowheads in Figs 2d and these cells as NEP cells. 3a,h,i). The cell cluster formed a line from the dorsal neural tube to the developing heart (Supplementary Fig. S3b), which was considered to be a migration stream of cardiac neural crest cells27, and the cells seemed to be derived from the initially emerged NEP Regulatory elements for Epo gene expression in NEP cells.To high examine cis-acting regulatory elements of the Epo gene using cells in E8.5 embryos (arrowheads in Fig. 2b,c). These GFP transgenic reporter expression assays, we assessed GFP reporter cells were observed in all Epo-GFP transgenic mouse lines examined (Table 1). Since Epo is a secreted factor, it is plausible expression in two bacterial artificial chromosome transgenic high mouse lines that carry the wt-Epo-GFP, three lines that carry the that these GFP cells dominantly produce Epo among NEP 22K-Epo-GFP and two lines that carry the 17KXh-Epo-GFP cells. (transgene structures are shown in Supplementary Fig. S1)15,19,22. At E9.5, GFP-positive cells were detected in intersomitic spaces (Fig. 3j,k). These GFP-positive cells resided posterior to the Transgenic mouse embryos of all seven lines expressed GFP in þ NEP cells at E9.5 (Table 1), strongly supporting the notion that intersomitic vessels, which were lined with CD31 endothelial the GFP reporter expression, and hence Epo gene expression, in cells (Fig. 3j,k). It seems plausible that the close proximity NEP cells is reproducible and physiological. We also conclude between NEP cells and the vessels (Fig. 3g,j,k) is effective for that the transactivation event that induces Epo gene expression in prompt secretion of Epo into the bloodstream. Since trunk neural crest cells migrate into the intersomitic space from dorsal to NEP cells resides within a 32-kb region that spans the 17-kb 27 upstream and 15-kb downstream of the Epo gene (Supplementary ventral side of embryos , we conclude that NEP cells consist of a Fig. S1). subpopulation of neural crest cells and a subpopulation of neural Of the regulatory motifs within this 32-kb region, we have lineage cells. identified important roles such as (i) the GATA-binding motif (50-GATA) in the proximal promoter19 and (ii) the HIF-binding A subpopulation of neural crest cells produces Epo. To further motif in the 30-enhancer (30-HRE)6 play. These findings were verify that NEP cells include neural crest cells, we crossed the drawn by using mouse lines that carry the wt-Epo-GFP and its 17KXh-Epo-GFP mice with the neural crest cell-tracer mouse mutant transgene constructs. Taking advantage of these line Wnt1-Cre::Rosa26R-tdTomato (Supplementary Fig. S3c)28. transgenic mouse lines, we examined the contribution of these We found that a subpopulation of tdTomato-positive cells around cis-acting elements to Epo gene expression in NEP cells. Two the neural tube expressed GFP in E9.5 embryos (Fig. 4a,b). types of mutations in the 50-GATA motif in a total of four We conducted similar experiments using two additional neural

4 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

E E E

E9.0 E9.5 E10.5 E11.5

E9.5 GFP Sox2

E E L E NT R NT A

P E11.5 E9.0 NT

V I I

E9.5 E9.5 E9.0

Figure 3 | The expression of the Epo-GFP transgenes in mouse embryos. (a–d) GFP expressions in 22K-Epo-GFP (a,d) and 17KXh-Epo-GFP (b,c) transgenic mouse embryos at E9.0 (a), E9.5 (b), E10.5 (c) and E11.5 (d). Orange arrow, optic vesicle; E, otic vesicle; dotted circle, fetal liver. (e,f) Immunofluorescence of GFP (green) and Sox2 (red) in the transverse section of hindbrain (location is indicated by the dotted arrow in b) from a 17KXh-Epo-GFP transgenic mouse embryo at E9.5. The right panel of e is a merged image of left (GFP) and middle (Sox2) panels with counterstaining by 40,6-diamidino-2- phenylindole (DAPI; blue). GFP is expressed in Sox2-positive neuroepithelial cells (f, the higher magnification image of the dotted quadrangle in e). The dotted arrow indicates the dorsal direction. (g) The section of eye (R, retina; L, lens) from a 22K-Epo-GFP embryo at E11.5 shows that GFP is expressed in the retina surrounded by CD31 þ endothelial cells (red). (h) GFP expression in a three-dimensional image of a 22K-Epo-GFP embryo at E9.0. A dorsal view of the posterior (P)–anterior (A) axis was obtained using a laser confocal microscope with Amira software. GFP-positive cell clusters (arrowheads) are observed around the otic vesicle (E) and the neural tube (NT). (i) GFP expression in transverse sections around the rostral neural tube (indicated by the yellow line in a) of an E9.0 22K-Epo-GFP transgenic embryo. Serial sections are exhibited in a rostral-caudal order (upper-lower). (j,k) GFP expression in the intersomitic space of a 17KXh-Epo-GFP transgenic embryo at E9.5. GFP (green) and CD31 (red) immunofluorescence in sagittal sections of somites (yellow line in b) is shown with counterstaining by DAPI (blue) at low (j) and high (k) magnifications. V, aorta; I, intersomitic artery. Scale bars, 100 mm(e,h–k), 400 mm(a–c,g) and 1.0 mm (d). crest tracer lines of mice, Pax3-Cre::Rosa26R-tdTomato29 and The GFP-positive cells in E9.5 17KXh-Epo-GFP embryos were P0-Cre::Rosa26R-tdTomato30. The results demonstrated that separated with FACS, and the expressions of GFP reporter GFP-positive cells were partially overlapped with tdTomato- mRNA and endogenous Epo mRNA were examined using positive cells in these tracer mouse lines (Supplementary RT–qPCR. Consistent with the transgenic GFP expression, Fig. S3d–i). endogenous Epo mRNA expression was exclusively detected in

NATURE COMMUNICATIONS | 4:2902 | DOI: 10.1038/ncomms3902 | www.nature.com/naturecommunications 5 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3902

NT NT

E E

Head Liver Head Liver 5.0 2.5 Epo Wnt1 4.0 2.0 GFP Foxd3 3.0 1.5 Sox10

2.0 1.0 mRNA levels mRNA levels 1.0 0.5

0.0 0.0 GFP+ –T+ – GFP+ –T+ –

Head Liver 18 180 150 Albumin Normoxia 120 EpoR 12 Hypoxia 90

(pg per day) 6

60 Epo secretion mRNA levels 30 0 0 GFP+ –T+ – Hd Ht YS

Figure 4 | A subpopulation of neural crest cells express the Epo gene. (a,b) Both GFP (green, arrowheads) and tdTomato (red) expression is detected in transverse sections of the neural tube (NT) from E9.5 17KXh-Epo-GFP:Wnt1-Cre:Rosa26R-tdTomato embryos using confocal microscopy. DAPI (blue) was used for immunofluorescent counterstaining. Scale bars, 100 and 20 mmina and b, respectively. Note that a cell cluster in the proximal posterior region from the otic vesicle (E) co-expresses GFP and tdTomato (left panel in a). In contrast, the majority of the GFP-positive cells are negative for tdTomato in a section of the distal posterior region of the otic vesicle (right panel). The bottom panel in b is a merged image from the top (GFP) and middle (tdTomato) panels and shows a high magnification view of GFP-tdTomato double-positive cells (arrows). (c–e) RT–qPCR analysis of the GFP-positive ( þ ) and -negative ( À ) cell fractions that were sorted from the head and the liver regions of E9.5 (23–25 sp) 22K-Epo-GFP transgenic embryos. ‘T’ indicates unsorted total cells from the head region. Epo and GFP mRNA expression (c), neural crest marker (Wnt, Foxd3 and Sox10) mRNA expression (d) and Albumin and EpoR mRNA expression (e) are shown as averages of relative expression levels with s.d. (n ¼ 5 in each group). The mRNA levels in the GFP- positive fractions from the head are set to 1 in each experiment after normalization using Gapdh mRNA expression levels. (f) The Epo concentrations in the culture supernatants of E8.5 embryonic organ. E8.5 embryonic organs (the head, heart and yolk sac (YS)) were incubated for 24 h under normoxic or hypoxic (1% oxygen) conditions. The Epo concentrations in the supernatants were measured using ELISA. The arrows indicate undetectable levels. The data were calculated from the levels of Epo that were produced by each organ from a given embryo and are reported (with s.d.) as the averages of the measurements from four embryos. the GFP-positive cell fraction of both the head and the liver In the head region, the expression levels of the neural crest regions (Fig. 4c; Supplementary Fig. S3a). This result confirmed markers (Wnt1, Foxd3 and Sox10)31–33 were enriched in the the faithful recapitulation of endogenous Epo gene expression by GFP-positive cell fraction compared with the GFP-negative cell the 17KXh-Epo-GFP transgene. fraction (Fig. 4d). This indicates that a subpopulation of neural

6 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 crest cells express the Epo gene. In contrast, very low-level expression of neural crest markers was observed in GFP-positive cells of the liver that expressed the Albumin gene, a hepatocyte Hd Hd (Tg–) or Ht (Tg–) marker (Fig. 4d,e). This result conclusively demonstrates that + YS the Epo-expressing head cells are a different type of cells from (Gata1-HRD-GFP Tg+) Epo-expressing fetal hepatocytes6,19. Significant EpoR mRNA Ht YS 5 days in 1% O expression was detectable in GFP-negative head cells, which were 2 circulating erythroid cells. Moreover, EpoR expression was strong in the GFP-negative liver cell fraction; the majority of these cells were definitive haematopoietic cells (Fig. 4e). When E8.5 embryos (5–9 sp) were dissected and each portion of the embryo was incubated for 24 h under normoxic or hypoxic (1% oxygen) conditions, we detected a significant concentration of Epo in the culture supernatant of the E8.5 head region, which contained GFP-positive cells. However, no No Ht E8.5 Epo was observed in the supernatant from the heart or yolk sac regions (Fig. 4f). Epo production was not altered by hypoxic exposure of the neural tissue culture (Fig. 4f). These results demonstrate the consistency of GFP expression and Epo secretion in NEP cells.

Epo Hd E9.5 NEP cell-derived Epo induces primitive erythrocyte growth. We then examined the activity of NEP cell-derived Epo by 104 No Epo establishing a novel bioassay system (Fig. 5a,b). In this assay, we 3 Mk 103 collected haematopoietic cells from yolk sacs of E8.5 Gata1-HRD ) 48.1 2.3 46.3 3.8 3 EP (haematopoietic regulatory domain)-GFP transgenic mice, which 102 10 contain GFP-positive primitive erythroid/megakaryocytic lineage × EB 2 1 cells34,35. The yolk sac cells were incubated with the dissected Ery 10 0 0.0 17.8 cells ( 10 head region that contained NEP cells or heart region from E8.5 + 104 littermates without the transgene as a non-Epo-producing CD45 Hd Hd+Ab 103 control. We performed cultures under hypoxic conditions (1% 1 52.4 6.5 53.8 3.0 oxygen) to inhibit megakaryopoiesis, which is stimulated by 102 36,37 ambient oxygen tension in yolk sac cell cultures . 1 Following 5 days of incubation, we observed that more than Number of GFP 10 5.1 1.0 0 100 half of the cells in the head region culture were morphologically Hd Ht No 0 1 2 3 4 0 1 2 3 4 erythroid cells (Fig. 5c, Hd), which exhibited a small and 10 10 10 10 10 10 10 10 10 10 Ter119 mononuclear appearance that was similar to that of primitive erythroid cells that can normally be observed in E9.5 yolk sacs Figure 5 | NEP-derived Epo supports the growth of primitive erythroid (Fig. 5d). These erythroid-lineage cells were predominant in the cells. (a) GFP expression in a Gata1-HRD-GFP transgenic embryo at E8.5 culture that was supplemented with recombinant Epo, and this (6 sp). The whole embryo with the yolk sac (left) was dissected into the yolk culture condition was used as a positive control (Fig. 5c, Epo). In sac (YS), the head region (Hd) and the heart region (Ht). Scale bar, 1.0 mm. contrast, no apparent stimulation of erythropoiesis was observed (b) A schematic representation of the strategy that was employed to in the culture without supplementation (Fig. 5c, No). In the investigate the bioactivity of the Epo produced by the neural crest cells. E8.5 co-culture with the heart region, megakaryocyte-like cells (Fig. 5c, embryos that were derived from Gata1-HRD-GFP transgenic male and Ht, arrowheads) were increased in number, but erythroid cells wild-type female mice were separated into GFP-expressing (Tg þ ) and -non- were not. expressing (Tg À ) embryos. The yolk sac cells of Tg þ embryos were Using Gata1-HRD-GFP expression (Supplementary Fig. S4), co-cultured with the Hd or Ht from a Tg– embryo in a 96-well round bottom the primitive haematopoietic cells in the 5-day cultures were plate for 5 days under 1% oxygen conditions. (c,d) Wright–Giemsa staining of divided into four ﬂow cytometric fractions: a CD45 À Ter119 À cells that were derived from the yolk sac. Scale bars, 10 mm. The morphology fraction containing megakaryocytes, a CD45 þ Ter119 À fraction of the cells that were incubated for 5 days (c) and of E8.5 (6 sp) and E9.5 (25 containing EP, a CD45 þ Ter119 þ fraction containing EBs sp) primary cells before incubation (d) are shown. Note that whereas most and a CD45 À Ter119 þ fraction containing mature erythrocytes cells from the culture with Epo or Hd are erythroblastic cells that stain dark (Ery)38. The number of Gata1-HRD-GFP-positive cells was purple, many megakaryoblastic cells (arrowheads) with multiple nuclei are B10-fold higher in the culture with the head region than that observed in the cells that were incubated with Ht or without supplementation in the control culture without supplementation (Fig. 5e). The (No). (e) The total numbers of GFP-positive cells following a 5-day culture. number of Gata1-HRD-GFP-positive cells at the beginning of The cultured cells were counted, and the expression levels of CD45, Ter119 culture was estimated to be 500–1,000. Therefore, according to and GFP were analysed using FACS. Mk, megakaryocytes (CD45 À Te r1 19 À ); our previous ﬁnding that 10–20% of the 5,000 yolk sac cells at EP, erythroid progenitors (CD45 þ Te r1 19 À ); EB, erythroblast E8.5 were Gata1-HRD-GFP positive35, we estimated that a three- (CD45 þ Te r1 19 þ ); Ery, mature erythrocytes (CD45 À Te r1 19 þ ). (f) The anti- to sixfold increase in the number of Gata1-HRD-GFP-positive Epo antibody blocks the effect of the Hd region on the differentiation of yolk cells was induced during the 5-day culture with the head region. sac cells. FACS analysis of GFP-positive cells was conducted with CD45 and Although Gata1-HRD-GFP-positive cells were also increased in Ter119 antibodies following 5 days of culture. The percentages of each the culture with the heart region, the majority of the growing quadrangle are given. ‘No’ indicates the control in which no supplementation GFP-positive cells belonged to the megakaryocytic lineage was used. All the experiments were performed at least three times using (Fig. 5e). more than four transgenic embryos, and representative data are presented.

NATURE COMMUNICATIONS | 4:2902 | DOI: 10.1038/ncomms3902 | www.nature.com/naturecommunications 7 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3902

Recombinant Epo stimulates the maturation and anti-apoptosis activity of primitive erythrocytes in this culture system, and Circulation 17.8% of the GFP þ cells were CD45 À Ter119 þ Ery in the cultures that were supplemented with Epo (Fig. 5f, Epo). In Neuroepithelium, neural crest contrast, virtually no CD45 À Ter119 þ cells were observed in the Hepatocytes culture without supplementation (Fig. 5f, No). In the culture with NEP cells À þ Liver

the head region, 5.1% of the cells were CD45 Ter119 (Fig. 5f; Epo production Epo Epo Hd), and the addition of a neutralizing antibody against Epo signiﬁcantly inhibited erythroid differentiation (Fig. 5f, Hd þ Ab). These results demonstrate that NEP cells in the embryonic head Liver region secrete functional Epo that can stimulate the proliferation and maturation of primitive erythroid cells. Yolk sac, bloodstream Erythropoiesis

E7.5 E8.5 E9.0 E9.5 E10.5 E11.5 NEP cell-derived Epo stimulates erythroid cells in yolk sac. Necessity of NEP cells for primitive erythropoiesis should be E8.5 deﬁned by NEP cell-speciﬁc deletion of the Epo gene. However, Yolk sac Head E9.0 currently available Cre transgenic mouse lines are not suitable for 4 1.5 (Embryo proper at E8.5) E9.5 the loss-of-function analyses of Epo secretion from NEP cells Body because only a small number of NEP cells are labelled in any of ) 3 three mouse lines (Wnt1-Cre, Pax3-Cre and P0-Cre), which are –1 1.0 2 known for Cre expression in neural crest cells (see Fig. 4 and ** ** Supplementary Fig. S3). 0.5 ** 1 mRNA levels We then hypothesized that Epo secreted from NEP cells Epo (pg ng ** reached the yolk sac through circulation to support the primitive 0 0.0 erythropoiesis (Fig. 6a), as true heartbeats initiate around E9.0 E8.5 E9.0 E9.5 Gata1 Stat5a Stat5b (refs 7,8) and Epo-KO embryos exhibit abnormalities in the primitive erythropoiesis at this timing (Fig. 1b,c). To verify this 7 ** 10 E8.5 ** hypothesis, we examined whether Epo from NEP cells reach the 6 8 E9.0 yolk sac and primitive erythroid cells are stimulated at E9.0. 5 ** E9.5 We analysed Epo levels in the embryonic parts using enzyme- 4 6 linked immunosorbent assay (ELISA). Consistent with the above 3 4 ** RT–qPCR results (Fig. 2a; Supplementary Fig. S5), we detected ** **

2 mRNA levels

Phospho-Stat5 2 significant concentrations of Epo in the embryo proper at E8.5, amount) (Relative 1 and in the head regions at E9.0 and E9.5 (Fig. 6b). In the yolk sac, 0 0 while Epo was below the detectable level at E8.5, Epo started EpoRStat5a Stat5b Cish Tfrc E8.5 reaching the yolk sac after E9.0 and became detectible (Fig. 6b). E9.0 E9.5 These results are consistent with NEP cells in the head region Figure 6 | NEP-derived Epo stimulates erythroid cells in the yolk sac beginning to produce Epo at E8.5, and the Epo being delivered through circulation. (a) A schematic model of the relationship between into yolk sac through the blood flow after E9.0 when the Epo-producing tissues and erythropoietic tissues during the ontogeny of circulatory system is activated7,8. mouse erythropoiesis. The first EPs emerge in the yolk sac at BE7.5, and We next examined downstream pathway of the Epo-EpoR their maturation occurs in the yolk sac and the bloodstream. The first Epo system in the yolk sac. Gata1 mRNA level was decreased in the production is initiated at E8.5 in NEP cells locating in the neural crest and yolk sac from E8.5 to E9.5, when the level was normalized with the neuroepithelium. At BE9.0, a true heartbeat can be observed, and NEP the housekeeping Gapdh mRNA level (Fig. 6c), suggesting that cell-derived Epo may stimulate erythroid cells in the yolk sac through the the ratio of erythroid cells decreased in the yolk sac that is rapidly bloodstream. After E9.5, the hepatocytes begin to produce Epo for erythroid expanding during these embryonic stages35. The expression of cells in the fetal liver. Note that secreted Epo from NEP cells seems to Stat5 genes (Stat5a and Stat5b) was detected at E8.5 and the levels support the growth of erythroid cells in an endocrine manner before the were decreased at E9.0 and E9.5, similar to the Gata1 gene erythropoietic site moves to the liver. (b) Amounts of Epo protein in the expression (Fig. 6c). In contrast, phosphorylated Stat5 level was indicated parts of embryos at E8.5 (6–9 sp), E9.0 (16–20 sp) and E9.5 very low at E8.5 but the amount in each yolk sac was significantly (22–25 sp) were measured by ELISA. Dissection of embryos was carried increased after E9.0 (Fig. 6d). Since Epo-binding to EpoR out as described in Supplementary Fig. S2a–c. Data were presented as the immediately induces phosphorylation and transcriptional averages of four groups of pooled samples from more than five embryos activity of Stat5 (ref. 39), these results are consistent with NEP after normalization with the total protein amount. The Epo amount in E8.5 cell-derived Epo activating the primitive erythropoiesis at E9.0. YS was undetectable (arrow). (c–e) Changes in mRNA expression profiles We also found that the Cish gene, one of the early response genes and in phospho-Stat5 amounts from E8.5 to E9.5 in the yolk sac. Relative to Epo stimulation2,21, was dramatically induced at E9.0 (Fig. 6e). expression levels of Gata1, Stat5a and Stat5b mRNAs in the yolk sac were The gene for CD71 (Tfrc), which is known as a target of Stat5 determined after normalization with the Gapdh mRNA expression levels (c). (ref. 39), was also upregulated after E9.0, showing a very good Relative amounts of phospho-Stat5 in a yolk sac were measured and agreement with the decreased expression level of CD71 in yolk calculated by ELISA using the phosphorylated Stat5(Y699)-specific sac of E9.0 Epo-KO embryos (Fig. 1c). antibody (d). mRNA levels of the indicated genes in the yolk sac erythroid These results are thus consistent with NEP cell-derived Epo cells are represented after normalization with the Gata1 mRNA expression reaching EpoR on the yolk sac erythroid cells through the levels to be compensated by erythroid population in the yolk sac (e). Data bloodstream after E9.0, and inducing Stat5 activation and from E8.5 samples is set to 1, and the data are shown as the averages of expressions of the Cish and Tfrc genes (Supplementary Fig. S6). four to six samples with s.d. (c–e). **Po0.01 when compared with the E8.5 Since NEP cells are the only source of Epo in E9.0 embryos samples based on Student’s t-test (c–e).

8 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

(Supplementary Fig. S5), it is possible to think that defects in the neuronal and cardiac cells11–13. To address the non- primitive erythropoiesis of Epo-KO embryos at E9.0 (Fig. 1b,c) haematopoietic roles of Epo, we have developed a mouse line are provoked by the loss of Epo production in NEP cells. that expresses EpoR only in haematopoietic cells; however, these mice exhibit no apparent abnormality during embryogenesis17. We have also observed that Epo-KO embryos exhibit no apparent Discussion abnormality until E11.5 in non-haematopoietic organs. In Although Epo or EpoR deficiency appears to affect primitive zebrafish, the overexpression and knockdown of Epo erythropoiesis, the roles that Epo plays in primitive erythropoiesis dramatically alter erythropoiesis in the embryonic intermediate or the cells that supply Epo for primitive erythropoiesis have not cell mass and the adult kidney, but these alterations in Epo levels been elucidated. As summarized in Fig. 6a, in the present study do not affect the development of non-haematopoietic systems41. we demonstrate that Epo is indeed required for the progression of Moreover, NEP cells express low EpoR mRNA levels compared primitive erythropoiesis in mouse embryos. We further have with haematopoietic cells. On the basis of these observations, we discovered that a major source of Epo for primitive erythropoiesis conclude that NEP cell-secreted Epo is dispensable for the might be a subpopulation of neural and neural crest cells, which morphogenesis of non-haematopoietic tissues and for the we refer to as NEP cells. Consistent with our observation, the migration of neural and neural crest cells. principal Epo-producing tissue in zebrafish larvae is neural tissue, In summary, we have determined that a subpopulation of and Epo production moves to the heart and liver during neural cells and a subpopulation of neural crest cells produce Epo adulthood40,41. We also observed that a subpopulation of NEP to support primitive erythropoiesis during ontogeny. Of the many cells migrate along the blood vessels in the intersomitic space. It important issues that remain to be clarified, the following two are therefore appears that NEP cell-derived Epo may efficiently particularly intriguing. One is whether Epo signalling is required stimulate the differentiation of primitive erythroid cells in the yolk for the stress response of embryonic tissues, as observed in adult sac and bloodstream (see Fig. 6a). Indeed, defects in the heartbeat tissues. Another is whether neural crest-derived cells in adults of mouse embryos cause the maturation arrest of primitive have the potential to produce Epo in response to anaemic stress erythroid cells42. These results firmly support the contention that conditions. Further studies are necessary to elucidate the nature NEP cells support primitive erythropoiesis in the yolk sac. of NEP cells and the contributions of NEP cell-derived Epo to CD71, a transferrin receptor, plays important roles in iron embryonic haematopoiesis. uptake for haemoglobin synthesis43. As CD71 expression in primitive EBs is reduced significantly after E9.0 in Epo-deficient Methods mice, Epo appears to be indispensable for the CD71 expression Mice. We previously established the Epo-deficient mouse line in which enhanced and late-stage primitive erythropoiesis. The CD71 expression is GFP complementary DNA (BD Biosciences) was inserted into the Epo locus20,21. The Epo-GFP transgenic mouse lines and the Gata1-HRD-GFP transgenic mouse also impaired in the definitive erythroid cells of EpoR-deficient 6,15,19,22,34 17 line were also previously generated .The17KXh-Epo-GFP transgene mice , indicating that the Epo signal is essential for the contains the upstream 17-kb and downstream 15-kb sequences of the Epo gene upregulation of CD71 expression during erythroid maturation (Supplementary Fig. S1). The 50-GATA (50-TGATAA-30) motif in the wt-Epo-GFP in both primitive and definitive erythropoiesis. The loss of Stat5, construct was mutated to 50-TTATAA-30 and 50-TTTTAA-30 in the m1-Epo-GFP 19 an important transcription factor in the Epo signalling cascade, in and the m2-Epo-GFP constructs, respectively .Them3-Epo-GFP transgene has a mutation in the 30-HRE (from 50-ACGTG-30 to 50-AAAAG-30)6. To trace the definitive erythroid cells causes the downregulation of CD71 neural crest cells, Rosa26R-tdTomato mice46 were crossed with Wnt1-Cre28, Pax3- 39 expression . Thus, our finding that CD71 expression is decreased Cre29 or P0-Cre30 transgenic mouse lines, all of which except P0-Cre mice (the in primitive erythroid cells of Epo-KO embryos suggests that the Kumamoto University) were supplied by Jackson Laboratory. The genotype of Epo-mediated induction of CD71 expression via Stat5 activation these mice was determined using genomic PCR analysis with the primers that are listed in Supplementary Table S1. The animal experiments were performed may play an indispensable role in primitive erythroid cells. according to the Regulations for Animal Experiments and Related Activities of The neural crest is considered to be the fourth germ layer of Tohoku University and the regulations of the Standards for Human Care and Use vertebrates. The neural crest appears transiently in embryos at the of Laboratory Animals of the University of Tsukuba. mid-gestational stage, and neural crest cells differentiate into a variety of cell types, including neuronal, cardiac and skeletal Flow cytometry and cell sorting. For the cell surface marker analyses, single-cell cells27,31. Many studies have sought to delineate the nature of the suspensions of yolk sacs were prepared by passing the cells through a needle. neural crest cells and their derivatives, including studies in Antibody-stained cells were also stained with propidium iodide to gate out dead cells and were analysed using a FACS. We used the FACSCalibur with CellQuest avian embryos. However, many aspects of mammalian neural programme (Becton Dickinson). For the cell sorting analysis, single-cell suspen- crest cells that are related to cell fate and other properties remain sions were prepared by digesting embryonic tissues with Trypsin-EDTA (0.05%, unknown. This study provides evidence that a subpopulation of Invitrogen) for 30 min at 37 °C. The GFP-positive cells were sorted using FACS- mouse neural crest cells, which express Wnt1, Foxd3 and Sox10, Vantage SE and FACSAria II (Becton Dickinson). The mean fluorescent intensity and the percentage of the normal distribution of the GFP þ and GFP À fractions produce Epo. were calculated. The phycoerythrin-conjugated anti-Ter119, allophycocyanin- In this regard, the origin of the REP cells is an intriguing conjugated anti-CD71 antibodies, allophycocyanin-conjugated anti-CD45 anti- question and remains unknown20. REP cells express the bodies were obtained from BD Biosciences and used at 1:100 dilution. interstitial fibroblast marker CD73 and neuronal markers, such as microtubule-associated protein 2 and neurofilament light RT–qPCR. The RNA samples were purified from dissected embryonic organs and polypeptide19–21,44,45. Neural crest-derived cells distribute in the FACS-sorted cells using the RNeasy kit (Qiagen). The total RNA was reverse interstitial space of renal tubules in adult mice45. These transcribed using SenscriptRT (Qiagen). For the RT–qPCR analyses, Epo mRNA levels were measured using the ABI PRISM 7300 or StepOne real-time PCR observations support the notion that REP cells are derived from systems (Applied Biosystems). Gapdh mRNA was used as an internal standard6,19. neural crest cells. This hypothesis is further supported by the All primer sequences are listed in Supplementary Table S1. similarity in the regulatory mechanisms for the Epo gene in NEP and REP cells. Epo gene expression levels in both NEP and REP Histological analyses. The fluorescence of transgenic embryos was observed cells do not require the GATA motif or the HIF-binding motif, using a MZ16FA microscope (Leica) and a LMS510 confocal imaging system (Carl both of which are essential for the regulation of Epo in Zeiss). For the immunohistochemical analyses, the embryos were fixed in 4% 6,19 paraformaldehyde for 15 min. Frozen sections (25 mm thickness) were collected hepatocytes . from the fixed embryos after embedding them in blocks of OCT (Sakura Several reports have demonstrated that the Epo-EpoR signal- Finetechnical). The following antibodies were used for the immunohistochemical ling plays important roles in non-haematopoietic cells, including analyses: anti-GFP rabbit polyclonal antibody (Medical & Biological Laboratories,

NATURE COMMUNICATIONS | 4:2902 | DOI: 10.1038/ncomms3902 | www.nature.com/naturecommunications 9 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3902

1:500 dilution), anti-CD31 rat monoclonal antibody (e-Bioscience, 1:100 dilution), 19. Obara, N. et al. Repression via the GATA box is essential for tissue-specific anti-Sox2 rabbit polyclonal antibody (Abcam, 1:1000 dilution) and Alexa fluor erythropoietin gene expression. Blood 111, 5223–5232 (2008). 488- or 555-conjugated secondary antibodies (Molecular Probes, 1:500 dilution). 20. Pan, X. et al. Isolation and characterization of renal erythropoietin-producing cells from genetically produced anemia mice. PLoS One 6, e25839 (2011). Epo bioassay. GFP-expressing yolk sacs were selected from E8.5 littermates that 21. Yamazaki, S. et al. A mouse model of adult-onset anaemia due to were obtained from the mating of Gata1-HRD-GFP transgenic male mice31 with erythropoietin deficiency. Nat. Commun. 4, 1950 (2013). wild-type female mice. Single-cell suspensions were prepared from the yolk sacs by 22. Suzuki, N., Obara, N. & Yamamoto, M. Use of gene-manipulated mice in the incubating them in 0.05% Trypsin-EDTA (Invitrogen) for 5 min. Five thousand study of erythropoietin gene expression. Methods Enzymol. 435, 157–177 (2007). cells were incubated in 0.1 ml RPMI medium (Sigma) that was supplemented with 23. Zanjani, E. D., Ascensao, J. L., McGlave, P. B., Banisadre, M. & Ash, R. C. 10% fetal bovine serum (Hyclone), 2-mercaptoethanol (0.1 mM) and antibiotics. Studies on the liver to kidney switch of erythropoietin production. J. Clin. The incubations were performed with or without human recombinant Epo Invest. 67, 1183–1188 (1981). (4 mU ml À 1, Chugai Pharmaceutical) in 96-well round bottom plates in a multi- 24. Makita, T., Duncan, S. A. & Sucov, H. M. Retinoic acid, hypoxia, and GATA gas incubator (Astec) at 5% CO2 and 1% O2. GFP-negative embryos from the same factors cooperatively control the onset of fetal liver erythropoietin expression litters were dissected as shown in Supplementary Fig. S2a and added to these yolk and erythropoietic differentiation. Dev. Biol. 280, 59–72 (2005). sac cell cultures. An anti-Epo antibody (10 mgmlÀ 1, R&D Systems) was used to 25. Wood, H. B. & Episkopou, V. Comparative expression of the mouse Sox1, Sox2 block Epo function in the culture medium. Following a 5-day incubation, the cells and Sox3 genes from pre-gastrulation to early somite stages. Mech. Dev. 86, were harvested by pipetting and the GFP-expressing cells were analysed. 197–201 (1999). 26. Regad, T., Roth, M., Bredenkamp, N., Illing, N. & Papalopulu, N. The neural progenitor-specifying activity of FoxG1 is antagonistically regulated by CKI and ELISA . To measure Epo concentrations in mouse embryonic parts, mouse embryos FGF. Nat. Cell Biol. 9, 531–540 (2007). were dissected as shown in Supplementary Fig. S2a–c, and the dissected parts from 27. Kulesa, P. M., Bailey, C. M., Kasemeier-Kulesa, J. C. & McLennan, R. Cranial more than five embryos were gathered into a sampling tube. The pooled samples neural crest migration: new rules for an old road. Dev. Biol. 344, 543–554 (2010). were lysed by pipetting after two freeze-thaw cycles and Epo amount was measured 28. Wilson, Y. M., Richards, K. L., Ford-Perriss, M. L., Panthier, J. J. & Murphy, M. using a mouse Epo ELISA kit (R&D Systems). Total protein amount of the lysate Neural crest cell lineage segregation in the mouse neural tube. Development was measured using the Bradford assay (Bio-Rad). 131, 6153–6162 (2004). To measure the Epo secretion, the dissected organs (Supplementary Fig. S2a) from four embryos were pooled into 0.1 ml of RPMI medium (Sigma) that was 29. Engleka, K. A. et al. Insertion of Cre into the Pax3 locus creates a new allele of supplemented with 10% fetal bovine serum (Hyclone). After 24-h incubation under Splotch and identifies unexpected Pax3 derivatives. Dev. Biol. 280, 396–406 (2005). normoxic or hypoxic (1% O2) conditions at 37 °C, the supernatants were measured the Epo concentrations using a mouse Epo ELISA Kit. 30. Yamauchi, Y. et al. A novel transgenic technique that allows specific marking of For ELISA of phosphorylated Stat5, a single-cell suspension of the yolk sac, the neural crest cell lineage in mice. Dev. Biol. 212, 191–203 (1999). which was prepared by pipetting, was fixed by 8% formaldehyde on the 96-well 31. Sauka-Spengler, T. & Bronner-Fraser, M. A gene regulatory network plate under a centrifuging condition (500g) for 20 min at room temperature. The orchestrates neural crest formation. Nat. Rev. Mol. Cell Biol. 9, 557–568 (2008). relative amounts of phospho-Stat5 in each yolk sac were analysed using a rabbit 32. Britsch, S. et al. The transcription factor Sox10 is a key regulator of peripheral anti-Stat5(Y699) antibody from cell-based ELISA system (R&D Systems) following glial development. Genes Dev. 15, 66–78 (2001). the instruction manual. 33. Dottori, M., Gross, M. K., Labosky, P. & Goulding, M. The winged-helix transcription factor Foxd3 suppresses interneuron differentiation and promotes neural crest cell fate. Development 128, 4127–4138 (2001). References 34. Suzuki, N. et al. Identification and characterization of 2 types of erythroid 1. Jelkmann, W. Erythropoietin after a century of research: younger than ever. progenitors that express GATA-1 at distinct levels. Blood 102, 3575–3583 Eur. J. Haematol. 78, 183–205 (2007). (2003). 2. Wojchowski, D. M., Sathyanarayana, P. & Dev, A. Erythropoietin receptor 35. Yokomizo, T. et al. Characterization of GATA-1( þ ) hemangioblastic cells in response circuits. Curr. Opin. Hematol. 17, 169–176 (2010). the mouse embryo. EMBO J. 26, 184–196 (2007). 3. Fried, W. Erythropoietin and erythropoiesis. Exp. Hematol. 37, 1007–1015 36. Mostafa, S. S., Miller, W. M. & Papoutsakis, E. T. Oxygen tension influences the (2009). differentiation, maturation and apoptosis of human megakaryocytes. Br. J. 4. Semenza, G. L., Nejfelt, M. K., Chi, S. M. & Antonarakis, S. E. Hypoxia- Haematol. 111, 879–889 (2000). inducible nuclear factors bind to an enhancer element located 30 to the human 37. Xu, M. J. et al. Evidence for the presence of murine primitive erythropoietin gene. Proc. Natl Acad. Sci. USA 88, 5680–5684 (1991). megakaryocytopoiesis in the early yolk sac. Blood 97, 2016–2022 (2001). 5. Semenza, G. L. Involvement of oxygen-sensing pathways in physiologic and 38. Mikkola, H. K., Fujiwara, Y., Schlaeger, T. M., Traver, D. & Orkin, S. H. pathologic erythropoiesis. Blood 114, 2015–2019 (2009). Expression of CD41 marks the initiation of definitive hematopoiesis in the 6. Suzuki, N. et al. Specific contribution of the erythropoietin gene 30 enhancer mouse embryo. Blood 101, 508–516 (2003). to hepatic erythropoiesis after late embryonic stages. Mol. Cell Biol. 31, 39. Zhu, B. M. et al. Hematopoietic-specific Stat5-null mice display microcytic 3896–3905 (2011). hypochromic anemia associated with reduced transferrin receptor gene 7. Godin, I. & Cumano, A. Of birds and mice: hematopoietic stem cell expression. Blood 112, 2071–2080 (2008). development. Int. J. Dev. Biol. 49, 251–257 (2005). 40. Chu, C. Y. et al. The zebrafish erythropoietin: functional identification and 8. Palis, J. Ontogeny of erythropoiesis. Curr. Opin. Hematol. 15, 155–161 (2008). biochemical characterization. FEBS Lett. 581, 4265–4271 (2007). 9. Kingsley, P. D., Malik, J., Fantauzzo, K. A. & Palis, J. Yolk sac-derived primitive 41. Paffett-Lugassy, N. et al. Functional conservation of erythropoietin signaling in erythroblasts enucleate during mammalian embryogenesis. Blood 104, 19–25 zebrafish. Blood 110, 2718–2726 (2007). (2004). 42. Lux, C. T. et al. All primitive and definitive hematopoietic progenitor cells 10. Fraser, S. T., Isern, J. & Baron, M. H. Maturation and enucleation of primitive emerging before E10 in the mouse embryo are products of the yolk sac. Blood erythroblasts during mouse embryogenesis is accompanied by changes in cell- 111, 3435–3438 (2008). surface antigen expression. Blood 109, 343–352 (2007). 43. Levy, J. E., Jin, O., Fujiwara, Y., Kuo, F. & Andrews, N. C. Transferrin receptor 11. Arcasoy, M. O. The non-haematopoietic biological effects of erythropoietin. Br. is necessary for development of erythrocytes and the nervous system. Nat. J. Haematol. 141, 14–31 (2008). Genet. 21, 396–399 (1999). 12. Noguchi, C. T., Wang, L., Rogers, H. M., Teng, R. & Jia, Y. Survival and 44. Bachmann, S., Le Hir, M. & Eckardt, K. U. Co-localization of erythropoietin proliferative roles of erythropoietin beyond the erythroid lineage. Expert Rev. mRNA and ecto-50-nucleotidase immunoreactivity in peritubular cells of rat Mol. Med. 10, e36 (2008). renal cortex indicates that fibroblasts produce erythropoietin. J. Histochem. 13. Jelkmann, W. Erythropoietin: back to basics. Blood 115, 4151–4152 (2010). Cytochem. 41, 335–341 (1993). 14. Morita, M. et al. HLF/HIF-2alpha is a key factor in retinopathy of prematurity 45. Asada, N. et al. Dysfunction of fibroblasts of extrarenal origin underlies renal in association with erythropoietin. EMBO J. 22, 1134–1146 (2003). fibrosis and renal anemia in mice. J. Clin. Invest. 121, 3981–3990 (2011). 15. Scheerer, N. et al. The anemia of the newborn induces erythropoietin 46. Becker, L., Kulkarni, S., Tiwari, G., Micci, M. A. & Pasricha, P. J. Divergent fate expression in the developing mouse retina. Am. J. Physiol. Regul. Integr. Comp. and origin of neurosphere-like bodies from different layers of the gut. Am. J. Physiol. 299, R111–R118 (2010). Physiol. Gastrointest. Liver Physiol. 302, G958–G965 (2012). 16. Teng, R. et al. Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production. Nat. Commun. 2, 520 (2011). 17. Suzuki, N. et al. Erythroid-specific expression of the erythropoietin receptor Acknowledgements rescued its null mutant mice from lethality. Blood 100, 2279–2288 (2002). We thank Drs Naoshi Obara (University of Tsukuba), Tadashi Nomura (Kyoto 18. Wu, H., Liu, X., Jaenisch, R. & Lodish, H. F. Generation of committed erythroid Prefectural University of Medicine), Takashi Moriguchi (Tohoku University) and BFU-E and CFU-E progenitors does not require erythropoietin or the Doug Engel (University of Michigan) for their help with the transgenic mouse analyses. erythropoietin receptor. Cell 83, 59–67 (1995). We also thank Ms Chisa Sato, Yuko Kikuchi and Atsuko Konuma for their help, and

10 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

Biomedical Research Core and Centre for Laboratory Animal Research of Tohoku Additional information University for supports. This work was supported in part by a research fund from Chugai Supplementary Information accompanies this paper at http://www.nature.com/ Pharmaceutical Co. Ltd. (N.S., M.Y.), Grants-in-Aids from MEXT/JSPS KAKENHI (N.S., naturecommunications M.Y.), the NAITO foundation (M.Y.), the Astellas Foundation for Research on Metabolic Disorders (M.Y.), the Tokyo Biochemistry Society (N.S.) and the Takeda Science Competing financial interests: The authors declare no competing financial interests. Foundation (N.S.). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Author contributions N.S., I.H., N.M. and M.Y. designed the research and wrote the manuscript; N.S., I.H. and How to cite this article: Suzuki, N. et al. Erythropoietin production in neuroepithelial X.P. performed the experiments, generated the gene-modified mouse lines, analysed the and neural crest cells during primitive erythropoiesis. Nat. Commun. 4:2902 data and prepared the figures. doi: 10.1038/ncomms3902 (2013).