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A Nature Research Journal. 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.
These results thus suggest that NEP cells secrete Epo and might support the development of primitive erythropoiesis. Access provided by. Erythropoietin Epo is one of the best-characterized cytokines that stimulates the proliferation and differentiation of erythroid cells 1.https://lentireenling.tk
Diverse of Erythropoiesis Responding to Hypoxia and Low Environmental Temperature in Vertebrates
Epo binds to the Epo receptor EpoR on the surface of erythroid progenitor EP cells and stimulates the proliferation and maturation of progenitor cells via the phosphorylation of EpoR and associated signalling molecules 2. Tissue hypoxia that is caused by anaemia or altitude sickness induces Epo gene expression in the kidney and liver of adult humans and mice 1 , 3. This hypoxia-induced Epo gene expression is transcriptionally regulated by hypoxia-inducible transcription factors HIFs. The first wave of murine erythropoiesis begins in the blood islands of the visceral yolk sac at embryonic day 7.
This erythropoiesis is referred to as primitive erythropoiesis 7 , 8. The fetal liver subsequently becomes the principal erythropoietic tissue in late-stage embryos. Around birth, erythropoiesis moves to the bone marrow and spleen 6. The latter type of erythropoiesis is referred to as definitive erythropoiesis 7 , 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 yolk sac and complete their maturation enucleation while circulating 9 , 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 clarified 11 , 12 , 13 , 14 , 15 , In contrast, transgenic complementation rescue analyses of EpoR gene revealed that EpoR expression in non-haematopoietic tissues is dispensable for mouse survival and fertility Thus, while Epo also has non-haematopoietic functions under pathological or stress conditions, the principal role of Epo is to support erythropoiesis.
We have identified renal Epo-producing REP cells in the interstitial space of the kidney 19 , 20 , REP cells secrete Epo into the bloodstream where it acts as an endocrine factor for bone marrow EPs 6 , While adult mouse hepatocytes also produce low levels of Epo, this hepatic Epo production is dispensable for erythropoiesis in adult mice 6 , In contrast, fetal liver-derived Epo stimulates liver erythropoiesis in a paracrine manner 6. Importantly, Epo or EpoR gene knockout mice also exhibit an inefficient production of primitive erythrocytes, suggesting that Epo plays important roles in primitive erythropoiesis However, Epo-producing cells in the early stages of mouse development have not been identified.
Therefore, in the present study we address Epo production in early-stage embryos. We observe Epo production in neural and neural crest cells of E8. We also determine that this Epo production is able to induce the proliferation and differentiation of yolk sac-derived erythroid cells under ex vivo co-culture conditions. The major source of Epo for primitive erythropoiesis might be a subpopulation of neural and neural crest cells, which we refer to as neural Epo-producing NEP cells. S1 20 , No apparent abnormality was observed in Epo-KO embryos at E8.
Nonetheless, the yolk sacs of Epo-KO mice were moderately and severely anaemic at E We found that Epo-KO embryos died by E Scale bars, 1. FACS data from more than three embryos in each group are presented with s. To characterize the yolk sac cells of Epo-KO embryos, we conducted flow cytometry analyses. These results support our contention that Epo contributes to primitive erythropoiesis and is required for the differentiation of primitive erythroid cells towards the CDpositive stage in yolk sacs after E9. To our current knowledge, the earliest time of Epo expression is at E9. However, the impaired primitive erythropoiesis in Epo-KO mice implies that Epo is produced in embryos around E9.
To identify Epo-producing tissues or cells in mid-gestational stage embryos, we dissected embryos Supplementary Fig. S2a—c at E8.
Epo mRNA expression was detected in the head region and in the fetal liver in E9. The relative Epo mRNA expression level in the head region gradually decreased during development and was undetectable at E The data indicate the averages of the relative expression levels with s. 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. GFP-positive cells are observed in or around the neural folds arrowheads in b — d at E8.
Dotted circle, fetal liver; orange arrow, optic vesicle; E, otic vesicle; NT, neural tube. Scale bars, 0. To identify which cell types express Epo in the embryonic head region, we next examined transgenic mouse lines that express green fluorescent protein GFP under the control of the Epo regulatory region. To this end, we used transgenic mouse lines that carried various bacterial artificial chromosome-based GFP reporter transgenes that we previously established Table 1 ; Supplementary Fig. Importantly, it has been shown that GFP fluorescence in these mice faithfully recapitulates the endogenous Epo gene expression profile in adult livers and kidneys.
In E8. 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 region at E8. Thus, these results demonstrate that cells in neural tissues of E8. We refer to these cells as NEP cells. We also conclude that the transactivation event that induces Epo gene expression in NEP cells resides within a kb region that spans the kb upstream and kb downstream of the Epo gene Supplementary Fig.
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These findings were drawn by using mouse lines that carry the wt-Epo-GFP and its mutant transgene constructs. Taking advantage of these transgenic mouse lines, we examined the contribution of these cis -acting elements to Epo gene expression in NEP cells. Since GFP expression was observed in the neuroepithelium around hindbrain Fig. Orange arrow, optic vesicle; E, otic vesicle; dotted circle, fetal liver.
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. A dorsal view of the posterior P —anterior A axis was obtained using a laser confocal microscope with Amira software. Serial sections are exhibited in a rostral-caudal order upper-lower. 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. Epo-GFP expression was also observed in neuroepithelia of the otic and optic vesicles Figs 2e,f and 3a,b , and the expression was detectable in the retinal neuroepithelium until E These results thus demonstrate that Epo is expressed transiently in a subpopulation of developing neural cells. 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 cells 27 , and the cells seemed to be derived from the initially emerged NEP cells in E8.
At E9. It seems plausible that the close proximity between NEP cells and the vessels Fig. Since trunk neural crest cells migrate into the intersomitic space from dorsal to ventral side of embryos 27 , we conclude that NEP cells consist of a subpopulation of neural crest cells and a subpopulation of neural lineage cells. S3c The results demonstrated that GFP-positive cells were partially overlapped with tdTomato-positive cells in these tracer mouse lines Supplementary Fig. DAPI blue was used for immunofluorescent counterstaining.
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.
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. The GFP-positive cells in E9.
In the head region, the expression levels of the neural crest markers Wnt1, Foxd3 and Sox10 31 , 32 , 33 were enriched in the GFP-positive cell fraction compared with the GFP-negative cell fraction Fig. This indicates that a subpopulation of neural 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 marker Fig. This result conclusively demonstrates that the Epo-expressing head cells are a different type of cells from Epo-expressing fetal hepatocytes 6 , Moreover, EpoR expression was strong in the GFP-negative liver cell fraction; the majority of these cells were definitive haematopoietic cells Fig. When E8. However, no Epo was observed in the supernatant from the heart or yolk sac regions Fig.
Epo production was not altered by hypoxic exposure of the neural tissue culture Fig. In this assay, we collected haematopoietic cells from yolk sacs of E8. The yolk sac cells were incubated with the dissected head region that contained NEP cells or heart region from E8. The whole embryo with the yolk sac left was dissected into the yolk sac YS , the head region Hd and the heart region Ht. Scale bar, 1.
The morphology of the cells that were incubated for 5 days c and of E8. Note that whereas most cells from the culture with Epo or Hd are erythroblastic cells that stain dark purple, many megakaryoblastic cells arrowheads with multiple nuclei are observed in the cells that were incubated with Ht or without supplementation No.
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