Functional conservation of erythropoietin signaling in zebrafish

Abstract

Erythropoietin (Epo) and its cognate receptor (EpoR) are required for maintaining adequate levels of circulating erythrocytes during embryogenesis and adulthood. Here, we report the functional characterization of the zebrafish epo and epor genes. The expression of epo and epor was evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization, revealing marked parallels between zebrafish and mammalian gene expression patterns. Examination of the hypochromic mutant, weissherbst, and adult hypoxia-treated hearts indicate that zebrafish epo expression is induced by anemia and hypoxia. Overexpression of epo mRNA resulted in severe polycythemia, characterized by a striking increase in the number of cells expressing scl, c-myb, gata1, ikaros, epor, and ␤e1-globin, suggesting that both the erythroid progenitor and mature erythrocyte compartments respond to epo. Morpholino-mediated knockdown of the epor caused a slight decrease in primitive and complete block of definitive erythropoiesis. Abrogation of STAT5 blocked the erythropoietic expansion by epo mRNA, consistent with a requirement for STAT5 in epo signaling. Together, the characterization of zebrafish epo and epor demonstrates the conservation of an ancient program that ensures proper red blood cell numbers during normal homeostasis and under hypoxic conditions. (Blood. 2007;110:2718-2726) © 2007 by The American Society of Hematology IntroductionThe glycoprotein erythropoietin (Epo) is essential for definitive erythropoiesis during ontogeny and for maintaining appropriate numbers of circulating erythrocytes in the adult. 1,2 Epo binds the erythropoietin receptor (EpoR) on erythroid progenitors and stimulates an intracellular signaling cascade initiated by autophosphorylation of the receptor-associated Janus kinase 2 (Jak2) and subsequent tyrosine phosphorylation of EpoR (reviewed in Richmond et al 3 ). Proteins with Src homology 2 (SH2) domains, such as STAT5 (signal transducer and activator of transcription factor 5) and phosphoinositide 3-kinase (PI3K), associate with the EpoR and are activated by Jak2 phosphorylation. 4 One primary action of Epo is to inhibit apoptosis, 5 which is mediated by STAT5 induction of the antiapoptotic B-cell lymphoma-X L (bcl-X L ) response pathway and activation of Akt by PI3K. 6 The Epo-EpoR interaction also activates the Ras-mitogen-activated protein kinase (MAPK) pathways 7 and nuclear factor-B (NFB)-dependent transcription. 8 In mammals, Epo is produced by hepatocytes during development and by interstitial peritubular cells in the adult kidney. [9][10][11] In response to chronic hypoxia, extrarenal Epo is expressed by the adult liver and spleen. In contrast, EpoR is expressed by primitive and definitive erythroid progenitors, endothelial cells, neural cells, and at low levels in cardiomyocytes. [12][13][14][15][16] Mice lacking Epo or EpoR have fewer primitive erythrocytes in the yolk sac blood islands and die between embryonic day 13 and embryonic day 15 due to severe an...

Erythropoietin production in neuroepithelial and neural crest cells during primitive erythropoiesis

“…We further have discovered that a major source of Epo for primitive erythropoiesis might be a subpopulation of neural and neural crest cells, which we refer to as NEP cells. Consistent with our observation, the principal Epo-producing tissue in zebrafish larvae is neural tissue, and Epo production moves to the heart and liver during adulthood 40,41 . We also observed that a subpopulation of NEP cells migrate along the blood vessels in the intersomitic space.…”

“…We have also observed that Epo-KO embryos exhibit no apparent abnormality until E11.5 in non-haematopoietic organs. In zebrafish, the overexpression and knockdown of Epo dramatically alter erythropoiesis in the embryonic intermediate cell mass and the adult kidney, but these alterations in Epo levels do not affect the development of non-haematopoietic systems 41 . Moreover, NEP cells express low EpoR mRNA levels compared with haematopoietic cells.…”

Erythropoietin Gene Expression: Developmental-Stage Specificity, Cell-Type Specificity, and Hypoxia Inducibility

“…This evidence suggests that NEP-derived cells in organs other than the kidney and liver may retain their ability to produce Epo under pathological conditions. Interestingly, in Zebrafish, one of the major Epo-producing sites is the brain (Paffett-Lugassy et al 2007). Therefore, transient Epo production by NEP cells in mouse embryos may support the so-called "recapitulation theory", which argues that ontogeny recapitulates phylogeny (Sander 2002).…”

“…At the transcriptional level, Epo gene expression is tightly regulated by hypoxia-inducible factors (HIFs), which are the master transcription factors for oxygen-dependent gene regulation (Suzuki et al 2007;Miyata et al 2013;Franke et al 2013). Because non-mammalian Epo genes also respond to hypoxic stimuli (Paffett-Lugassy et al 2007), Epo is considered to be evolutionarily indispensable for maintaining oxygen homeostasis in animals with hemoglobins encapsulated by erythrocytes.…”

“…Epo binding to the receptors causes conformation changes in the intracellular domains of an EpoR homodimer, bringing it into close proximity to initiate Epo-EpoR signal transduction (Suzuki et al 2015). Because EpoR is highly expressed only in committed erythroid progenitors and erythroblastic cells, the principal function of Epo is fundamentally restricted to erythropoiesis; it is not involved in the growth of other cell lineages (Suzuki et al 2002;Paffett-Lugassy et al 2007). Additionally, Epo signaling is important for the growth and terminal maturation of erythroid cells and not for the commitment of hematopoietic stem cells to the erythroid lineage (Wu et al 1995).…”