The stem-cell leukemia (SCL, also known as TAL1) gene encodes a basic helix-loop-helix transcription factor that is essential for the initiation of primitive and definitive hematopoiesis, erythrocyte and megakarocyte differentiation, angiogenesis, and astrocyte development. Here we report that the zebrafish produces, through an alternative promoter site, a novel truncated scl (tal1) isoform, scl-β, which manifests a temporal and spatial expression distinct from the previously described full-length scl-α. Functional analysis reveals that while scl-α and -β are redundant for the initiation of primitive hematopoiesis, these two isoforms exert distinct functions in the regulation of primitive erythroid differentiation and definitive hematopoietic stem cell specification. We further demonstrate that differences in the protein expression levels of scl-α and -β, by regulating their protein stability, are likely to give rise to their distinct functions. Our findings suggest that hematopoietic cells at different levels of hierarchy are likely governed by a gradient of the Scl protein established through temporal and spatial patterns of expression of the different isoforms.

Proper cell fate choice in myelopoiesis is essential for generating correct numbers of distinct myeloid subsets manifesting a wide spectrum of subset-specific activities during development and adulthood. Studies have suggested that myeloid fate choice is primarily regulated by transcription factors; however, new intrinsic regulators and their underlying mechanisms remain to be elucidated. Zebrafish embryonic myelopoiesis gives rise to neutrophils and macrophages and represents a promising system to derive new regulatory mechanisms for myeloid fate decision in vertebrates. Here we present an in vivo study of cell fate specification during zebrafish embryonic myelopoiesis through characterization of the embryos with altered Pu.1, Runx1 activity alone, or their combinations. Genetic analysis shows that low and high Pu.1 activities determine embryonic neutrophilic granulocyte and macrophage fate, respectively. Inactivation and overexpression of Runx1 in zebrafish uncover Runx1 as a key embryonic myeloid fate determinant that favors neutrophil over macrophage fate. Runx1 is induced by high Pu.1 level and in turn transrepresses pu.1 expression, thus constituting a negative feedback loop that fashions a favorable Pu.1 level required for balanced fate commitment to neutrophils versus macrophages. Our findings define a Pu.1-Runx1 regulatory loop that governs the equilibrium be- IntroductionMyeloid cells are a collection of morphologically, phenotypically, and functionally distinct blood cells that conventionally consist of 2 separated lineages, granulocytes (neutrophils, basophils, and eosinophils), and monocytes/macrophages. The importance of this class of cells is exemplified by their wide engagement in diverse physiologic processes, including organogenesis, tissue homeostasis, and immune defense. Perturbation in the formation, differentiation, and function of these cells will incur devastating consequences, such as leukemia. Hence, a comprehensive understanding of how functional myeloid system is established will render novel therapeutic opportunities for curing myeloid malformationassociated diseases. During ontogeny, there exist multiple waves of myeloid cell production, termed myelopoiesis. In the adult myelopoiesis, all granulocytes and monocytes/macrophages are derived from the hierarchical transformation of multipotential hematopoietic stem cells (HSCs) into lineage-restricted progenitors and subsequent maturation of unilineage-restricted progenitors. 1 In the developing embryos where HSCs are not formed, myeloid cells are transitorily supplied by unipotent or bipotent progenitors, which appear to arise directly from mesoderm without transiting through HSC-like multipotent progenitors.A key indispensable step shared by different waves of myelopoiesis is the specification of granulocyte or monocyte/macrophage fate from initially equivalent pool of myeloid progenitors. Resolving granulocyte versus monocyte/macrophage fate is thought to be primarily dictated by the interplay among various transcription factors and...

Hematopoiesis is a complex process which gives rise to all blood lineages in the course of an organism's lifespan. However, the underlying molecular mechanism governing this process is not fully understood. Here we report the isolation and detailed study of a newly identified zebrafish ugly duckling (Udu) mutant allele, Udu sq1 . We show that loss-of-function mutation in the udu gene disrupts primitive erythroid cell proliferation and differentiation in a cell-autonomous manner, resulting in red blood cell (RBC) hypoplasia. Positional cloning reveals that the Udu gene encodes a novel factor that contains 2 paired amphipathic ␣-helix-like IntroductionHematopoiesis in vertebrate occurs in 2 waves, primitive and definitive. [1][2][3] In the mouse, the primitive or embryonic wave of hematopoiesis occurs around embryonic day 7.5 in the yolk-sac blood island, and produces primitive erythrocytes and macrophages. 4,5 This primitive wave of hematopoiesis lasts for a transient period of a few days and is subsequently replaced by the definitive program. Murine definitive hematopoiesis is believed to originate from a distinctive region known as the aorta-gonad-mesonephros at embryonic days 7.5 to 8. 6,7 These definitive hematopoietic precursors, presumably the definitive hematopoietic stem cells, then migrate to the fetal liver, where they undergo rapid proliferation and differentiation, and finally colonize the bone marrow for adult hematopoiesis. In contrast to primitive hematopoiesis, the definitive hematopoietic program gives rise to all the mature blood cell types and remains active throughout the lifetime of the organism.In zebrafish, hematopoiesis also comprises both primitive and definitive programs and produces mature blood cell types similar to those found in mammals. 8,9 Zebrafish primitive erythropoiesis begins at the 4-somite stage as a pair of bilateral stripes in the posterior lateral mesoderm. 10 These stripes extend anteriorly and posteriorly, and then converge in the midline at the 20-somite stage to form the intermediate cell mass (ICM), where erythroid precursors further develop and enter the circulation by 24 to 26 hours after fertilization (hpf). 10,11 On the other hand, primitive myelopoiesis originates from the rostral blood island in the anterior lateral mesoderm at around the 10-somite stage and produces mainly macrophages and possibly some neutrophils. [12][13][14][15] Recent studies demonstrate that zebrafish definitive hematopoiesis initiates in the ventral wall of dorsal aorta between 26 and 48 hpf, 16,17 and then establishes the adult hematopoietic organ in the kidney by 5 days after fertilization (dpf). 11 The zebrafish mutant, ugly duckling (Udu tu24 ), was first isolated from the 1996 Tuebingen large-scale screen as a mutant defective in morphogenesis during gastrulation and tail formation. 18 In this article, we report the isolation and detailed study of a new udu allele, udu sq1 , as a mutant with a defect in blood cell development. Cell-cycle, cytologic, and transplantation analyses sho...

A processing error occurred when the full text HTML for Development 136, 647-654 was produced.Throughout the full text version the alpha symbol in αe1-globin was converted to β, thus in all cases 'βe1-globin' should read 'αe1-globin'.The full text has now been corrected. The PDF and print copy are correct.We apologise to authors and readers for this mistake.

SUMMARYRecent studies have shown that nascent hematopoietic stem cells (HSCs) derive directly from the ventral aortic endothelium (VAE) via endothelial to hematopoietic transition (EHT). However, whether EHT initiates from a random or predetermined subpopulation of VAE, as well as the molecular mechanism underlying this process, remain unclear. We previously reported that different zebrafish stem cell leukemia (scl) isoforms are differentially required for HSC formation in the ventral wall of the dorsal aorta. However, the exact stage at which these isoforms impact HSC development was not defined. Here, using in vivo time-lapse imaging of scl isoformspecific reporter transgenic zebrafish lines, we show that prior to EHT scl-β is selectively expressed in hemogenic endothelial cells, a unique subset of VAE cells possessing hemogenic potential, whereas scl-α is expressed later in nascent HSCs as they egress from VAE cells. In accordance with their expression, loss-of-function studies coupled with in vivo imaging analysis reveal that scl-β acts earlier to specify hemogenic endothelium, which is later transformed by runx1 into HSCs. Our results also reveal a previously unexpected role of scl-α in maintaining newly born HSCs in the aorta-gonads-mesonephros. Thus, our data suggest that a defined hemogenic endothelial population preset by scl-β supports the deterministic emergence of HSCs, and unravel the cellular mechanisms by which scl isoforms regulate HSC development.

Zebrafish is an excellent model organism for studying vertebrate development and human disease. With the availability of increased numbers of zebrafish mutants and microarray chips, gene expression profiling has become a powerful tool for identification of downstream target genes perturbed by a specific mutation. One of the obstacles often encountered, however, is to isolate large numbers of zebrafish mutant embryos that are indistinguishable in morphology from the wild-type siblings for microarray analysis. Here, we report a method using amplified cDNA derived from five embryos for gene expression profiling of the 18-somite zebrafish cloche (clo) mutant, in which development of hematopoietic and endothelial lineages is severely impaired. In total, 31 differentially expressed target genes are identified, of which 13 have not been reported previously. We further determine that of these 13 new targets, 8 genes, including coproporphyrinogen oxidase (cpo), carbonic anhydrase (cahz), claudin g (cldn g), zinc-finger-like gene 2 (znfl2), neutrophil cytosol factor 1 (ncf1), matrix metalloproteinase 13 (mmp13), dual specificity phosphatase 5 (dusp5), and a novel gene referred as zebrafish vessel-specific gene 1 (zvsg1) are predominantly expressed in hematopoietic and endothelial cells. Comparative analysis demonstrates that this method is comparable and complementary to that of the conventional approach using unamplified sample. Our study provides valuable information for studying hematopoiesis and vessel formation. The method described here offers a powerful tool for gene expression profiling of zebrafish mutants in general.

A processing error occurred when the full text HTML for Development 136, 647-654 was produced.Throughout the full text version the alpha symbol in αe1-globin was converted to β, thus in all cases 'βe1-globin' should read 'αe1-globin'.The full text has now been corrected. The PDF and print copy are correct.We apologise to authors and readers for this mistake.

Key Points c-Myb is essential for neutrophil terminal differentiation by targeting granule gene expression. c-Myb and Cebp1 act cooperatively to regulate neutrophil maturation in zebrafish.