The model organism Danio rerio, also known as the zebrafish, is an excellent system for studying the developmental process of hematopoiesis. It is an ideal model for in vivo imaging, and it is useful for large-scale genetic screens. These have led to the discovery of previously unknown players in hematopoiesis, as well as helped our understanding of hematopoietic development. In this review, we will summarize hematopoiesis in the zebrafish and discuss how genetic approaches using the zebrafish system have helped to build our current knowledge in the field of hematopoiesis. KEY WORDS: hematopoietic stem cell, HSC, hemangioblast, primitive hematopoiesis, definitive hematopoiesisIn the last decade, zebrafish rose as a new genetic system to analyze hematopoietic development. The zebrafish system has a number of unique advantages compared to other vertebrate model organisms. Its embryos are externally fertilized and transparent, enabling in vivo visualization of early embryonic processes ranging from birth of hematopoietic stem cells (HSCs) in the mesoderm to migration of blood cells. In addition, large production of embryos makes phenotype-based forward genetics feasible (de Jong and Zon, 2005). For example, 26 complementation groups with blood defects were identified from two seminal large-scale mutagenesis projects performed in the 1990s (Ransom et al., 1996; Weinstein et al., 1996). Cloning and characterization of these mutants helped us analyze hematopoietic ontogeny and blood-related disease mechanisms. Most importantly, even though sites of hematopoiesis are very different in fish and mammals, the genetic program governing hematopoiesis was found to be highly conserved, which made new knowledge gained from the zebrafish field applicable to mammalian hematopoiesis (Davidson and Zon, 2004). Overview of vertebrate hematopoiesisAll vertebrate organisms experience waves of hematopoiesis in their lifetime (Galloway and Zon, 2003). During mammalian and avian development, the first HSCs appear from the blood islands in the extraembryonic yolk sac, giving rise to erythrocytes and macrophages that are required for growing tissues of the embryos (Palis and Yoder, 2001). This primitive wave is only transient, and the successive definitive wave starts intraembryonically in the Int. J. Dev. Biol. 54: 1127-1137 (2010) doi: 10.1387/ijdb.093042ep aorta-gonad-mesonephros (AGM) region. In contrast to primitive HSCs, the definitive HSCs are multipotent, giving rise to all different lineages of blood. Subsequently, HSCs born from the AGM then migrate to the fetal liver where they will proliferate and ultimately seed the bone marrow, which is the adult hematopoietic organ (Cumano and Godin, 2007).Zebrafish also have waves of hematopoiesis, which occur in a spatially unique manner compared to other vertebrate model organisms (Fig. 1). Its primitive HSCs are born intraembryonically in ventral mesoderm derived tissue called the intermediate cell mass (ICM) . During this wave, the anterior part of the embryo generates myelo...
Defining the genetic pathways essential for hematopoietic stem cell (HSC) development remains a fundamental goal impacting stem cell biology and regenerative medicine. To genetically dissect HSC emergence in the aorta-gonadmesonephros (AGM) region, we screened a collection of insertional zebrafish mutant lines for expression of the HSC marker, c-myb. Nine essential genes were identified, which were subsequently binned into categories representing their proximity to HSC induction. Using overexpression and loss-of-function studies in zebrafish, we ordered these signaling pathways with respect to each other and to the Vegf, Notch, and Runx programs. Overexpression of vegf and notch is sufficient to induce HSCs in the tbx16 mutant, despite a lack of axial vascular organization. Although embryos deficient for artery specification, such as the phospholipase C gamma-1 (plc␥1) mutant, fail to specify HSCs, overexpression of notch or IntroductionSpecification of definitive hematopoietic stem cells (HSCs) capable of generating the blood cell lineages is a vertebrate-specific process that occurs in the aorta-gonad-mesonephros (AGM) region of the developing embryo. 1 In the mouse, HSCs are located near the ventral endothelium of the dorsal aorta on embryonic day (E) 10, approximately the same time that HSC activity is present in the AGM region. [2][3][4] The proto-oncogene c-myb and the transcription factor runx1 are both excellent markers of these emerging HSCs and are essential for mammalian HSC development. 5 Runx1 is thought to operate very early in HSC specification as the mouse knockout lacks intraaortic hematopoietic clusters. 4 Based on analysis of a point mutant, c-Myb is thought to act in concert with p300 to regulate the proliferation and differentiation of HSCs. 6 Similarly, zebrafish have an AGM-like region in the ventral wall of the dorsal aorta also marked by c-myb and runx1 expression. 5,[7][8][9][10][11] Lineage tracing experiments have shown cells exiting this region and ultimately seeding the kidney and thymus, the sites of definitive hematopoiesis in the zebrafish. 12-14 Consistent with the mouse knockout data, morpholino knockdown of runx1 translation in zebrafish results in the loss of c-myb cells in the aortic region as well as a loss of definitive marker expression in the thymus. 8 Although many studies have focused on the molecular events important for HSC emergence from the AGM, very little is understood about the signals essential for specifying these cells. 15 Some progress has been made through the analysis of several zebrafish mutants. In zebrafish, definitive HSCs are located between and in the axial vasculature, 14 which is developmentally derived from intermediate mesoderm. Mutants with defects in vascular formation and patterning show deficiencies in HSC specification. For example, the cloche (clo) mutant lacks endothelium and thus a vasculature, has no AGM HSCs and does not undergo definitive hematopoiesis. 11 Loss of vasculature organization, such as that in the spadetail (spt) mutant ha...
SummaryDeletion of caudal/cdx genes alters hox gene expression and causes defects in posterior tissues and hematopoiesis. Yet, the defects in hox gene expression only partially explain these phenotypes. To gain deeper insight into Cdx4 function, we performed chromatin immunoprecipitation sequencing (ChIP-seq) combined with gene-expression profiling in zebrafish, and identified the transcription factor spalt-like 4 (sall4) as a Cdx4 target. ChIP-seq revealed that Sall4 bound to its own gene locus and the cdx4 locus. Expression profiling showed that Cdx4 and Sall4 coregulate genes that initiate hematopoiesis, such as hox, scl, and lmo2. Combined cdx4/sall4 gene knockdown impaired erythropoiesis, and overexpression of the Cdx4 and Sall4 target genes scl and lmo2 together rescued the erythroid program. These findings suggest that auto- and cross-regulation of Cdx4 and Sall4 establish a stable molecular circuit in the mesoderm that facilitates the activation of the blood-specific program as development proceeds.
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