Leukemia (2010) 24, 2090–2099 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 www.nature.com/leu ORIGINAL ARTICLE A DEAB-sensitive aldehyde dehydrogenase regulates hematopoietic stem and progenitor cells development during primitive hematopoiesis in zebrafish embryos ACH Ma, MIS Chung, R Liang and AYH Leung Division of Haematology and Bone Marrow Transplantation, Department of Medicine, The University of Hong Kong, Hong Kong, Hong Kong SAR Although aldehyde dehydrogenase (ALDH) activity has become interfering RNA expanded HSPC population, reminiscent of a surrogate of hematopoietic stem and progenitor cells ALDH inhibition with DEAB in human study.6 However, (HSPCs), its function during hematopoiesis was unclear. Here, targeted deletion of aldh1al7 and aldh1a28 had no effects on we examined its role in zebrafish hematopoiesis based on pharmacological inhibition and morpholino (MO) knockdown. murine HSPC development and yolk-sac hematopoiesis, sug- Zebrafish embryos were treated with diethylaminobenzal- gesting that other members in the Aldh family may be involved. dehyde (DEAB, 1 lmol/l) between 0- and 48 hour-post-fertilization The zebrafish embryo has emerged as a model for the study (hpf). MOs targeting aldhs were injected between 1 and 4-cell of embryonic hematopoiesis.9 Like other vertebrates, hemato- stage. The effects on hematopoiesis were evaluated at different poiesis occurs in two successive waves, known as the primitive stages. DEAB treatment between 0 and 18 hpf increased gene and definitive hematopoiesis. In zebrafish, the transitory expression associated with HSPC (scl, lmo2), erythropoiesis (gata1, a-andb-eHb) and myelopoiesis (spi1)aswellasgfpþ primitive hematopoiesis occurs in the intermediate cell mass cells in dissociated Tg(gata1:gfp) embryos. The effects were (ICM), whereas the definitive wave originates from the ventral ameliorated by all-trans retinoic acid (1 nmol/l). Definitive wall of dorsal aorta, thence the kidney, where hematopoiesis hematopoiesis and the erythromyeloid precursors were occurs life-long. The transcription factors involved in HSPC unaffected. In all, 14 out of 15 zebrafish aldhs were detectable formation and differentiation during hematopoiesis are also by reverse transcription PCR in 18 hpf embryos, of which only conserved. The zebrafish genome contains at least 15 aldh aldh1a2 and aldh16a1 were expressed in sites pertinent to genes. Missense mutation of aldh1a2, known as the neckless10 hematopoiesis. Molecular targeting by MOs was demonstrated 11 for 15 aldhs, but none of them, even in combined aldh1a2 and or no-fin mutation, was associated with defects in the aldh1a3 knockdown, recapitulated the hematopoietic expan- hindbrain and brachial cartilage development, absence of sion in DEAB-treated embryos. In conclusion, DEAB expands pectoral fins and more recently expansion of cardiac progenitor HSPC population during primitive hematopoiesis through pool.12 These developmental effects could be explained by inhibition of aldh and retinoic acid synthesis. The specific aldh defective endogenous retinoic acid biosynthesis secondary to isoform(s) remains to be determined. 13 Leukemia (2010) 24, 2090–2099; doi:10.1038/leu.2010.206; aldh1a2 mutation. Interestingly, aldh1a1, the isoform thought published online 7 October 2010 to be associated with mammalian HPSC regulation, is lacking in Keywords: aldehyde dehydrogenase; zebrafish; hematopoiesis; all teleosts.14 It remains unclear if aldh may play a role during hematopoietic stem cells embryonic hematopoiesis. To address this issue, we examined the effect of aldh inhibition in zebrafish using both pharmacological inhibitor and anti-sense morpholino (MO) knockdown. We demonstrated that pharmacological inhibition of aldh with DEAB resulted in Introduction expansion of primitive hematopoiesis. Definitive hematopoiesis was not affected. Molecular targeting of 15 individual aldh Aldehyde dehydrogenase (ALDH) are a group of NAD(P) þ - isoforms did not increase hematopoiesis, implicating a complex dependent enzymes involved in the biosynthesis of retinoic acid mode of regulation by aldh. as well as the metabolism of amino acid, fatty aldehydes, ethanol and cyclophosphamide.1 It was first demonstrated in mouse and human hematopoietic stem and progenitor cells Materials and methods (HSPCs) almost 20 years ago based on their resistance to the cytotoxic effects of cyclophosphamide. In human, inhibition of Zebrafish and MOs ALDH with diethylaminobenzaldehyde (DEAB) in-vitro has Wild-type zebrafish (Danio rerio) were obtained from local been shown to delay differentiation and expand HSPC by 2 aquarium and were maintained and raised under standard inhibiting endogenous retinoic acid biosynthesis. Immuno- conditions at 28 1C. Transgenic Tg(gata1:gfp) embryos was a gift histochemical and gene expression studies have suggested from Dr Anming Meng (Tsinghua University, Beijing, China) ALDH1A1 as the predominant form associated with HSPC 15 3–5 were used to quantify the erythroid cell populations. activity. In mouse, knockdown of Aldh1a1 by small Transgenic Tg(fli1:gfp) embryos bought from ZIRC were used to track vascular development. Anti-sense MOs (Gene-Tools, Correspondence: Dr AYH Leung, Division of Haematology and Bone Philomath, OR, USA) targeting splice junction of scl16 together Marrow Transplantation, Department of Medicine, Queen Mary with 15 MOs newly designed to target the 50-UTR or start codon Hospital, The University of Hong Kong, Pok Fu Lam Road, of zebrafish aldhs and a random sequence MO for setting up Hong Kong, Hong Kong SAR. E-mail: [email protected] control were used (Supplementary Table 1). Procedures for Received 9 July 2010; accepted 30 July 2010; published online micro-injection and whole-mount in-situ hybridization (ISH) 7 October 2010 have been described previously.17,18 ALDH regulates primitive hematopoiesis in zebrafish embryos ACH Ma et al 2091 Treatment with ALDH inhibitor Microscopy and imaging DEAB was used to treat zebrafish embryos at 1 mmol/l. Unless Bight field images were taken under Nikon SMZ800 otherwise specified, the embryos were treated from 0 hour-post- (Nikon Corporation, Kawasaki, Japan) and P-Plan 1X objective fertilization (hpf) until examination. In each experiment, an in 3% methylcellulose with Nikon Coolpix E4500 (Nikon Hong equal volume of dimethyl sulfoxide was added to the control Kong Ltd., Hong Kong), processed with Adobe Photoshop embryos. Version 7.0. Fluorescent images were taken under Olympus IX70 (Olympus Corporation, Tokyo, Japan) and 10X/0.3 NA objective in 3% methylcellulose, with Olympus DP71 (Olympus Cloning of zebrafish aldhs and synthesis of sense and Corporation, Shinjuku-ku, Tokyo, Japan) and Olympus DP-BSW anti-sense RNA probes for ISH basic Software, processed with Adobe Photoshop Version 7.0. Full-length or partial sequences of zebrafish aldhs were PCR amplified with specific primers (Supplementary Table 2) and TA-cloned using pGEM-T Easy Vector System (Promega, Statistical analysis Madison, WI, USA). The sequences and orientation were Results were expressed as mean±s.e.m. unless otherwise stated. confirmed by DNA sequencing and both sense and anti-sense Comparisons between groups of data were evaluated by paired RNA probes were synthesized as described previously.17,18 t-test, Mann–Whitney U-test and Kruskal–Wallis test, where appropriate. P-value of o0.05 was considered statistically significant. Cloning and injection of gfp-fusion expressing vectors for checking MO efficiency To confirm molecular targeting of MOs, we designed primers Results (Supplementary Table 3) to clone the MO targeting region (50-UTR and the start codon) of each aldh isoform in-frame into DEAB treatment induced ICM expansion without pcDNA3.1/CT-GFP-TOPO vectors using the GFP Fusion TOPO perturbing vascular formation TA Expression Kits (Invitrogen, Carlsbad, CA, USA). We injected To examine the effects of aldh inhibition, we treated embryos each GFP vector with or without the respective MO at one-cell with DEAB (1 mmol/l) at 0 hpf. At 18 and 24 hpf (Figures 1a–d), stage. Embryos were examined at 18 hpf under fluorescent there was no morphological abnormality. At 30 hpf, the ICM was microscopy to check for gfp signal. expanded (Figures 1e–j). Thereafter, the ICM expansion gradually decreased and became undetectable at 48 hpf (Figures 1k and l). We also examined the effect of DEAB treatment on vascular Flow cytometry development using Tg(fli1:gfp) embryos. At 30 hpf, both vasculo- Tg(gata1:gfp) embryos at 18 hpf were dechorionated and genesis and angiogenesis were intact (Figures 1m–p) and digested with 0.05% Trypsin/EDTA solution (Invitrogen) for circulation was patent (Supplementary video SV1-4). Therefore, 15 min at 28 1C. The embryos were completely dissociated to the ICM expansion was unlikely due to the accumulation of single-cell suspension by pipetting. Trypsin digestion was blood cells as in embryos with defective circulation. terminated by CaCl2 (2 mmol/l) and the cellular suspension was filtered through a 40 mm cell strainer (BD Falcon, BD Biosciences Discovery Labware, Bedford, MA, USA). Cells were DEAB treatment upregulated genes associated with washed and harvested in phosphate-buffered saline with 2% primitive hematopoiesis fetal bovine serum and the percentage of gfp þ cells were The expansion of ICM in DEAB-treated embryos led us to enumerated by flow cytometry (Cytomics FC500, Beckman examine its hematopoietic gene expression. To focus on Coulter, Brea, CA, USA). For aldehyde dehydrogenase activity primitive