Iron is required to produce haem and iron-sulphur (Fe-S) clusters, processes thought to occur independently. Here we show that the hypochromic anaemia in shiraz (sir) zebrafish mutants is caused by deficiency of glutaredoxin 5 (grx5), a gene required in yeast for Fe-S cluster assembly. We found that grx5 was expressed in erythroid cells of zebrafish and mice. Zebrafish grx5 rescued the assembly of grx5 yeast Fe-S, showing that the biochemical function of grx5 is evolutionarily conserved. In contrast to yeast, vertebrates use iron regulatory protein 1 (IRP1) to sense intracellular iron and regulate mRNA stability or the translation of iron metabolism genes. We found that loss of Fe-S cluster assembly in sir animals activated IRP1 and blocked haem biosynthesis catalysed by aminolaevulinate synthase 2 (ALAS2). Overexpression of ALAS2 RNA without the 5' iron response element that binds IRP1 rescued sir embryos, whereas overexpression of ALAS2 including the iron response element did not. Further, antisense knockdown of IRP1 restored sir embryo haemoglobin synthesis. These findings uncover a connection between haem biosynthesis and Fe-S clusters, indicating that haemoglobin production in the differentiating red cell is regulated through Fe-S cluster assembly.

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...