Date Published: August 17, 2004
Publisher: Public Library of Science
Author(s): David G Ransom, Nathan Bahary, Knut Niss, David Traver, Caroline Burns, Nikolaus S Trede, Noelle Paffett-Lugassy, Walter J Saganic, C. Anthoney Lim, Candace Hersey, Yi Zhou, Bruce A Barut, Shuo Lin, Paul D Kingsley, James Palis, Stuart H Orkin, Leonard I Zon
Abstract: 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.
Partial Text: Hematopoiesis involves the coordinated processes of cell proliferation and differentiation of a relatively small number of progenitor cells into billions of circulating red and white blood cells (Thisse and Zon 2002). Hematopoiesis in vertebrates, from zebrafish to humans, is an evolutionarily conserved program that produces two waves of stem or progenitor cells that differ both in their embryonic origins and in the lineages of differentiated blood cells produced (Palis and Yoder 2001; Orkin and Zon 2002; Galloway and Zon 2003). The first, or primitive, wave of hematopoiesis originates from ventral mesoderm and gives rise to progenitor cells that differentiate in embryonic blood islands. The primitive wave of hematopoiesis produces a burst of embryonic erythrocytes and macrophages. The second, or definitive, wave of hematopoiesis arises from self-renewing stem cells that develop primarily in the intraembryonic aorta–gonad–mesonephros region. These definitive hematopoietic stem cells seed the later developing marrow spaces, to produce all lineages of adult blood cells, including definitive erythrocytes, myeloid cells, and lymphocytes.
The zebrafish is an excellent model system to elucidate the molecular machinery controlling gene expression during hematopoiesis (Thisse and Zon 2002; Galloway and Zon 2003). As part of a large-scale forward genetic screen, we originally identified a complementation group of independent mutant alleles in the zebrafish gene that we named moonshine (Ransom et al. 1996). Positional cloning was used to identify the mon gene, establishing a critical role for a transcriptional intermediary factor, Tif1γ, during hematopoietic development.
Transplantation of wild-type zebrafish marrow cells carrying a gata1:GFP transgene into 2-d-old embryos reconstitutes erythropoiesis, but not viability, in montg234 homozygous mutants. Movies of live embryos at day 3 posttransplant highlight less than 100 GFP+ RBCs in circulation. Transplanted cells were observed to proliferate, resulting in thousands of donor-derived erythrocytes 7 d later. Movies present GFP-fluorescent images of live zebrafish larvae.