Development of multilineage adult hematopoiesis in the zebrafish with a runx1 truncation mutation

Abstract

Runx1 is required for the emergence of hematopoietic stem cells (HSCs) from hemogenic endothelium during embryogenesis. However, its role in the generation and maintenance of HSCs during adult hematopoiesis remains uncertain.Here, we present analysis of a zebrafish mutant line carrying a truncation mutation, W84X, in runx1. The runx1 W84X/W84X embryos showed blockage in the initiation of definitive hematopoiesis, but some embryos were able to recover from a larval "bloodless" phase and develop to fertile adults with multilineage hematopoiesis. Using cd41-green fluorescent protein transgenic zebrafish and lineage tracing, we demonstrated that the runx1 W84X/W84X embryos developed cd41 ؉ HSCs in the aorta-gonad-mesonephros region, which later migrated to the kidney, the site of adult hematopoiesis. Overall, our data suggest that in zebrafish adult HSCs can be formed without an intact runx1. (Blood. 2010;115(14): 2806-2809) IntroductionRunx1 plays a critical role in the emergence of hematopoietic stem cells (HSCs) in the aorta-gonad-mesonephros (AGM) at the beginning of definitive hematopoiesis. 1 The function of Runx1 in adult HSCs, however, has not been fully resolved. Some studies suggest that Runx1 is required for the maintenance of HSCs, whereas others indicate that it negatively regulates the number of HSCs. 2,3 Overall, though, it has been difficult to explore the role of Runx1 in initiation of adult hematopoiesis because of limitations of conditional knockout mouse models. We recently reported on the generation of a zebrafish line, W84X, with a premature truncation in the runt domain of the runx1 protein. 4 This truncation removes most of the residues important in runx1 activity, such as CBF␤ and DNA binding, and nuclear localization signal. 5,6 Consistent with the Runx1 knockout mice, 7 the mutant embryos had normal primitive hematopoiesis but blockage of definitive hematopoiesis. 4 In this study, we analyzed the role of runx1 in adult hematopoiesis using the W84X mutant line. MethodsZebrafish were maintained under an approved National Institutes of Health animal use protocol. All zebrafish handling and breedings were performed as described. 8 Peripheral blood smears were prepared from tail clips of killed fish. Whole kidney marrow preparation and flow cytometric analysis were performed as described. 9 Killed adult fish were fixed in 10% formalin for histology. Lineage tracing by uncaging was performed as described. 10 Protocols for genotyping, microscopy and imaging, morpholino design, microinjections, reverse-transcription polymerase chain reaction (RT-PCR), and time lapse are described in the supplemental Methods (available on the Blood website; see the Supplemental Materials link at the top of the online article). Results and discussionAll runx1 W84X/W84X embryos lack definitive hematopoiesis, as demonstrated by lack of expression of HSC and lineage-specific markers between 36 hours after fertilization and 5 days after fertilization 4 (supplemental Figure 1A-G). Despite lack of runx1-expressing cells b...

Ddx46 Is Required for Multi-Lineage Differentiation of Hematopoietic Stem Cells in Zebrafish

“…Consistent with observations that have been made in mice, zebrafish cmyb mutants have defects in definitive hematopoiesis, suggesting that cMyb function is evolutionarily conserved [9,10]. On the other hand, runx1 is expressed in the VDA of mouse and zebrafish embryos [11,12], and analyses of Runx1 knockout mice and zebrafish mutants have revealed that Runx1 is required for the emergence of HSCs at the beginning of definitive hematopoiesis [5,13,14]. In contrast to these 2 factors, analyses of mutant animals have shown that the T-cell acute lymphocytic leukemia 1 (Tal1; also known as Scl) gene, encoding a basic-helix-loop-helix transcription factor, is required for both hematopoietic and endothelial development [1,2,5].…”

“…Several lines of genetic evidence have revealed that at least two transcription factors, cMyb and Runt-related transcription factor 1 (Runx1), have been implicated in the initiation, maintenance, and/or differentiation of HSCs during vertebrate development [5][6][7][8][9][10][11][12][13][14]. Studies on murine and human hematopoietic cell lines have shown that the cmyb protooncogene, encoding a transcription factor, is expressed mainly in HSCs, and its expression is downregulated in differentiated hematopoietic cells [5].…”

Hemogenic endothelium specification and hematopoietic stem cell maintenance employ distinct Scl isoforms

“…Zebrafish strains AB, Tg , , Tg(scl-α:DsRed), Tg(kdrl:eGFP) (Jin et al, 2005), Tg(kdrl:Ras-mCherry) s896 (Lee et al, 2009), Tg(cmyb:eGFP) (North et al, 2007), Tg(CD41:eGFP) (Lin et al, 2005) and runx1 w84x Sood et al, 2010) were used in this study.…”

CBFβ and RUNX1 are required at 2 different steps during the development of hematopoietic stem cells in zebrafish

“…20,21 Further studies from other groups demonstrated that, in zebrafish, runx1 is also required for the emergence of HSCs from the hemogenic endothelium in the AGM. 6 On the other hand, relatively little is known about the exact role of Cbfb during the early stages of HSC development in the mouse, 22 although it is assumed that Cbfb plays a similar role as Runx1 does.…”