Overexpression of Wild-Type <i>tif1γ</i> mRNA or Marrow Transplantation Rescues Embryonic Hematopoiesis in <i>mon</i> Mutants

<div><p>(A) <i>mon^tg234</i> mutants are rescued by injection of mRNA-encoding wild-type Tif1γ protein. At 4 d of development, large numbers of RBCs are visible in the circulation of wild-type zebrafish, shown here by o-dianisidine staining of hemoglobin. Uninjected <i>mon^ttg234</i> homozygous mutants are completely bloodless. Injection of 100 pg of wild-type <i>tif1γ</i> mRNA rescues erythropoiesis in mutant embryos. o-dianisidine-stained larvae are shown in ventral views to highlight blood in vessels.</p> <p>(B) Transplantation of wild-type zebrafish marrow cells carrying a <i>gata1:GFP</i> transgene into 2-d-old embryos reconstitutes erythropoiesis, but not viability, in <i>mon^tg234</i> homozygous mutants. Still frames from movies of live embryos at day 3 posttransplant highlight less than 100 GFP⁺ RBCs in circulation (top). Transplanted cells were observed to proliferate resulting in thousands of donor-derived erythrocytes 7 d later (bottom). Arrows indicate the hearts of control and transplanted zebrafish. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020237#s5" target="_blank">Videos S1–S4</a>.</p></div

The <i>mon/tif1γ</i> Gene Is Highly Expressed in Hematopoietic Mesoderm

<div><p>(A) In situ hybridization of zebrafish embryos demonstrating the embryonic expression of <i>tif1γ. tif1γ</i> is initially expressed as a maternal mRNA. Increased expression of <i>tif1γ</i> in ventral-lateral mesoderm begins between the one- to three-somite stages and increases through early development. By five somites, <i>tif1γ</i> is strongly expressed in lateral stripes of mesoderm that also express <i>scl.</i> At 22 hpf <i>tif1γ</i> is expressed broadly in the brain, spinal cord, trunk, and tail mesenchyme, but is at much higher levels in hematopoietic cells of the blood island. Zebrafish <i>tif1α</i> is also broadly expressed but relatively more uniform in most tissues, in comparison to <i>tif1γ. Tif1α</i> is weakly expressed at early somite stages in hematopoietic mesoderm and uniformly expressed at 22 hpf, including expression in the blood islands. Expression of <i>scl</i> at five somites and 22 hpf highlights the embryonic blood island in comparison to <i>tif1γ</i> expression.</p> <p>(B) In situ hybridization of mouse embryos detects broad expression of <i>Tif1γ</i> at embryonic day 8.5 including the yolk sac blood islands (arrow). AT embryonic day 12.5, there is high level expression in the fetal liver (arrow) and broad expression in the embryonic brain, spinal chord, gut, and muscle.</p></div

Zebrafish <i>mon</i> Mutants Also Have Severe Defects in Definitive Hematopoiesis

<p>Adult phenotype of wild-type and <i>mon</i> mutants. A rare surviving <i>mon^tb222</i> homozygous adult shows significant cardiomegaly in comparison to a wild-type age-matched control. Wright–Giemsa stained marrow of wild-type adult in comparison to a homozygous mutant. Note the dramatic reduction of terminally differentiated erythroid cells and the presence of abnormally large megaloblastic proerythroblasts in the <i>mon^tb222</i> mutant marrow.</p

Positional Cloning Identifies the <i>mon</i> Gene as Zebrafish <i>tif1γ</i>

<div><p>(A) Physical map of the <i>mon</i> locus on zebrafish Chromosome 8. Microsatellite markers z987 and z11001 were used to initially identify recombinants in a panel of 2,200 diploid <i>mon^tg234</i> homozygous mutants. The AFLP marker MA3 was used to initiate a chromosomal walk in PAC libraries. The critical PACS that were isolated to encompass the <i>mon</i> locus are indicated by numbers above bar. The PAC 107N19 defines the critical interval for the <i>mon</i> gene. This PAC was used as a probe to screen cDNA libraries and to identify zebrafish <i>tif1γ</i> cDNAs. Numbers below the bar indicate the number of recombinants identified by SSCP analysis.</p> <p>(B) Clustal-W–generated phylogentic tree of zebrafish (Danio rerio [Dr]) Tif1γ and Tif1α peptide sequences in comparison to TIF1 family members: human (Hs) TIF1α, TIF1β, and TIF1γ; mouse (Mm) Tif1α, Tif1β, and Tif1γ;; and fly (Dm) bonus.</p> <p>(C) Diagrams illustrating the structure of the Tif1γ-predicted peptide and the three identified point mutants. RING finger (RING), B-boxes (B1 and B2), plant homeodomain finger (PHD) and bromodomain (BROMO). Numbers below the first diagram indicate the percent identity shared between each of these domains in zebrafish and human TIF1γ. The predicted truncated proteins are indicated.</p> <p>(D) DNA sequence chromatograms showing the three ENU-induced point mutants in comparison to wild-type control sequences</p></div

Zebrafish <i>mon</i> Mutants Have Severe Defects in Primitive Hematopoiesis

<div><p>(A) Whole-mount TUNEL assays reveal that ventral-posterior mesodermal cells undergo apoptosis in homozygous <i>mon^tg234</i> mutant embryos. Whole-mount in situ hybridization of <i>gata1</i> detected at the 12- and 18-somite stage in genotyped embryos. Posterior views, anterior to the left.</p> <p>(B) Extensive apoptosis is visible in the trunk and tail (arrowhead) and also in hematopoietic cells of the embryonic blood island at 22 h of development (arrow). Whole-mount in situ hybridization at 22 hpf including <i>scl, gata2, gata1, ikaros,</i> and <i>myb</i> in <i>mon^tg234</i> mutants. Expression of <i>myb</i> is greatly reduced in the blood islands because of a loss of erythroid cells, but embryonic macrophages are still present (arrows). The expression of <i>rag1</i> in thymic T-cells appears normal in <i>mon</i> mutants at 5 d postfertilization (arrow heads). Lateral views of 22 hpf and 5-d-old embryos.</p></div