Hematopoiesis is precisely orchestrated by lineage-specific DNA-binding proteins that regulate transcription in concert with coactivators and corepressors. Mutations in the zebrafish moonshine (mon) gene specifically disrupt both embryonic and adult hematopoiesis, resulting in severe red blood cell aplasia. We report that mon encodes the zebrafish ortholog of mammalian transcriptional intermediary factor 1γ (TIF1γ) (or TRIM33), a member of the TIF1 family of coactivators and corepressors. During development, hematopoietic progenitor cells in mon mutants fail to express normal levels of hematopoietic transcription factors, including gata1, and undergo apoptosis. Three different mon mutant alleles each encode premature stop codons, and enforced expression of wild-type tif1γ mRNA rescues embryonic hematopoiesis in homozygous mon mutants. Surprisingly, a high level of zygotic tif1γ mRNA expression delineates ventral mesoderm during hematopoietic stem cell and progenitor formation prior to gata1 expression. Transplantation studies reveal that tif1γ functions in a cell-autonomous manner during the differentiation of erythroid precursors. Studies in murine erythroid cell lines demonstrate that Tif1γ protein is localized within novel nuclear foci, and expression decreases during erythroid cell maturation. Our results establish a major role for this transcriptional intermediary factor in the differentiation of hematopoietic cells in vertebrates.

The pharyngeal arch arteries (PAAs) are transient embryonic blood vessels that make indispensable contributions to the carotid arteries and great vessels of the heart, including the aorta and pulmonary artery1, 2. During embryogenesis, the PAAs appear in a craniocaudal sequence to connect pre-existing segments of the primitive circulation after de novo vasculogenic assembly from angioblast precursors3, 4. Despite the unique spatiotemporal characteristics of PAA development, the embryonic origins of PAA angioblasts and the genetic factors regulating their emergence remain unknown. Here, we identify the embryonic source of PAA endothelium as nkx2.5+ progenitors in lateral plate mesoderm long considered to adopt cell fates within the heart exclusively5, 6. Further, we report that PAA endothelial differentiation relies on Nkx2.5, a canonical cardiac transcription factor not previously implicated in blood vessel formation. Together, these studies reveal the heart field origin of PAA endothelium and attribute a novel vasculogenic function to the cardiac transcription factor nkx2.5 during great vessel precursor development.

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...