We demonstrate that in zebrafish, the microRNA miR-451 plays a crucial role in promoting erythroid maturation, in part via its target transcript gata2. Zebrafish miR-144 and miR-451 are processed from a single precursor transcript selectively expressed in erythrocytes. In contrast to other hematopoietic mutants, the ze-brafish mutant meunier (mnr) showed intact erythroid specification but diminished miR-144/451 expression. Although erythropoiesis initiated normally in mnr, erythrocyte maturation was morphologically retarded. Morpholino knockdown of miR-451 increased erythrocyte immatu-rity in wild-type embryos, and miR-451 RNA duplexes partially rescued erythroid maturation in mnr, demonstrating a requirement and role for miR-451 in erythro-cyte maturation. mnr provided a selectively miR-144/451-deficient background, facilitating studies to discern miRNA function and validate candidate targets. Among computer-predicted miR-451 targets potentially mediating these biologic effects, the pro-stem cell transcription factor gata2 was an attractive candidate. In vivo reporter assays validated the predicted miR-451/gata2-3UTR interaction, gata2 down-regulation was delayed in miR-451-knockdown and mnr embryos, and gata2 knockdown partially restored erythroid maturation in mnr, collectively confirming gata2 down-regulation as piv-otal for miR-451-driven erythroid matura-tion. These studies define a new genetic pathway promoting erythroid maturation (mnr/miR-451/gata2) and provide a rare example of partial rescue of a mutant phenotype solely by miRNA overexpres-sion. (Blood. 2009;113:1794-1804)

IntroductionZebrafish (Danio rerio) have contributed to hematologic research for more than 50 years. Interest in zebrafish embryology dates to the 1930s, 1 and they have long been used for zoology and toxicology research. 2 Their developmental hematology entered the literature in 1963 with the second appearance of zebrafish in the journal Nature, as a representative teleost fish with an intraembryonic origin for blood. 3 The first descriptions of zebrafish blood cell morphology appeared in the 1970s. [4][5][6] The "modern" phase of zebrafish hematology research, driven by genetic experimental approaches, started just over 10 years ago with the collection of zebrafish mutants with hematopoietic defects, mostly recognized for their anemia. [7][8][9] Genetic approaches to studying hematopoiesis were pioneered in the mouse. Originally, spontaneous mutants provided the majority of models, 10 but functional studies of specific genes, using transgenic and gene targeting methods in vivo, have dominated murine research for the last 25 years. However, the genes selected for such reverse genetic functional analyses are by definition biased by prior knowledge, and a reverse genetic gene-by-gene approach is unlikely to reveal the full set of genes contributing to hematopoiesis. Indeed, the genetic basis of many congenital human blood diseases remains unknown.Unbiased forward genetic approaches, particularly saturation mutagenesis and phenotype-based screening for mutants, sample the genome for all genetically interruptible, functionally mandatory steps in complex biologic pathways and have been of proven value in nonmammalian models (eg, Caenorhabditis elegans, Drosophila). 11 Unfortunately, the hematologic system of Drosophila is different from that of vertebrates such as mammals. 12 At a genetic level, hematologically important transcription factor families and signaling pathways are represented, 12 but at a cellular level, Drosophila hematology is primitive compared with mammals and adaptive immunity is entirely lacking.Outcomes from forward genetic screens in mice looking for hematopoietic mutants are now being reported, with mutants in genes both expected (Ikaros,14 Gata-1 15 ) and unexpected (C1galt1, 16 Bcl-x(L) 17 ). Although these examples show that murine genetic screens are feasible, they are logistically difficult because of their high cost and resource requirements and hence are typically scaled well below saturation screening and are currently limited to a few large projects.Zebrafish combine an affordable, genetically tractable vertebrate model with biology particularly suited to studying early development (eg, rapid ex vivo development, high fecundity, and optical transparency). Key reverse genetic techniques elsewhere reviewed 18,19 for functionally studying genes of interest in zebrafish include transient gene overexpression and knockdown, stable transgenesis, 20 and recovering stable mutated alleles by TILLING (Targeting Induced Local Lesions In Genomes), 21 or from catalogued libraries of insertional muta...