MiR-144 regulates hematopoiesis and vascular development by targeting meis1 during zebrafish development

The International Journal of Biochemistry & Cell Biology 49 (2014) 53–63

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MiR-144 regulates hematopoiesis and vascular development by targeting meis1 during zebrafish development Zhenhong Su a,b , Wenxia Si a , Lei Li a , Bisheng Zhou a , Xiuchun Li a , Yan Xu a , Chengqi Xu a , Haibo Jia a , Qing K. Wang a,c,∗ a Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Center for Human Genome Research, Cardio-X Institute, Huazhong University of Science and Technology, Wuhan, PR China b Key Laboratory of Kidney Disease Pathogenesis and Intervention of Hubei Province, Key Discipline of Pharmacy of Hubei Department of Education, Medical College, Hubei Polytechnic University, Huangshi, Hubei, PR China c Center for Cardiovascular Genetics, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA

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Article history: Received 20 August 2013 Received in revised form 24 December 2013 Accepted 7 January 2014 Available online 18 January 2014 Keywords: MiR-144 Meis1 Zebrafish (Danio rerio) Primitive and definitive hematopoiesis

a b s t r a c t Hematopoiesis is a dynamic process by which peripheral blood lineages are developed. It is a process tightly regulated by many intrinsic and extrinsic factors, including transcriptional factors and signaling molecules. However, the epigenetic regulation of hematopoiesis, for example, regulation via microRNAs (miRNAs), remains incompletely understood. Here we show that miR-144 regulates hematopoiesis and vascular development in zebrafish. Overexpression of miR-144 inhibited primitive hematopoiesis as demonstrated by a reduced number of circulating blood cells, reduced o-dianisidine staining of hemoglobin, and reduced expression of hb˛e1, hbˇe1, gata1 and pu.1. Overexpression of miR-144 also inhibited definitive hematopoiesis as shown by reduced expression of runx1 and c-myb. Mechanistically, miR-144 regulates hematopoiesis by repressing expression of meis1 involved in hematopoiesis. Both real-time RT-PCR and Western blot analyses showed that overexpression of miR-144 repressed expression of meis1. Bioinformatic analysis predicts a target binding sequence for miR-144 at the 3 -UTR of meis1. Deletion of the miR-144 target sequence eliminated the repression of meis1 expression mediated by miR-144. The miR-144-mediated abnormal phenotypes were partially rescued by co-injection of meis1 mRNA and could be almost completely rescued by injection of both meis1 and gata1 mRNA. Finally, because meis1 is involved in vascular development, we tested the effect of miR-144 on vascular development. Overexpression of miR-144 resulted in abnormal vascular development of intersegmental vessels in transgenic zebrafish with Flk1p-EGFP, and the defect was rescued by co-injection of meis1 mRNA. These findings establish miR-144 as a novel miRNA that regulates hematopoiesis and vascular development by repressing expression of meis1. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Hematopoiesis is a life-long, highly regulated, multi-stage process by which blood cell lineages are generated (Hogan et al., 2006; Rhodes et al., 2005; Yamaguchi et al., 1998; Yeh et al., 2008). Hematopoiesis in vertebrates occurs in two successive waves, including primitive and definitive hematopoiesis (Du et al., 2011; Warga et al., 2009; Yuan et al., 2011). Understanding genetic regulation of hematopoiesis is crucial because perturbations in hematopoiesis result in diseases such as anemia,

∗ Corresponding author at: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Center for Human Genome Research, Cardio-X Institute, Huazhong University of Science and Technology, Wuhan, PR China. E-mail addresses: [email protected], [email protected] (Q.K. Wang). 1357-2725/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2014.01.005

thrombocytopenia, leukemia, and lymphoma (Ellett and Lieschke, 2010; Sood and Liu, 2012). Similar to most vertebrates, zebrafish embryonic hematopoiesis originates from the lateral plate mesoderm (LPM), which gives rise to hemangioblasts, cardiac and other tissues (Ellett and Lieschke, 2010). Hemangioblasts are multipotential progenitors that generate pluripotent self-renewing hematopoietic stem cells (HSCs) and vascular endothelial cells. HSCs can subsequently produce erythrocytes, myelocytes, and other blood cells (Ma et al., 2011; Palis and Segel, 1998). Primitive hematopoiesis, which produces erythrocytes and macrophages, begins in the blood island of the yolk sac at the embryonic stage of about 12 h post-fertilization (hpf) in zebrafish. However, definitive hematopoiesis, which produces HSCs that give rise to all blood lineages, begins in the aorta-gonadmesonephros (AGM) region at about 36 hpf in zebrafish (Ellett and Lieschke, 2010; Gardiner et al., 2007). Prior to the differential expression of either myeloid or erythroid genes in these

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distinct precursor pools, both regions express the transcription factor gene scl. The stem cell leukemia (scl) gene acts upstream of gata1 and pu.1, which control the common myelo-erythroid progenitors (MEPs) to determine erythroid versus myeloid fate (Amali et al., 2013; Juarez et al., 2005; Liang et al., 2012). In addition to a variety of protein-coding mRNAs, accumulating evidence suggests that microRNAs (miRs, i.e. short, ∼22-nucleotide non-coding RNAs) regulate hematopoiesis by acting as post-transcriptional modulators of gene expression and exerting significant effects on cell growth, development, and differentiation (Ambros, 2004; Bartel, 2004; Bushati and Cohen, 2007). Several miRs have been reported to regulate hematopoiesis. Deregulation of miR-155, miR-451, and miR-320 has been shown to result in blood diseases (Bruchova et al., 2007, 2008; Chen et al., 2008; Felli et al., 2005, 2009; Masaki et al., 2007; Yang et al., 2009). Zebrafish miR-144 is specifically expressed in the hematopoietic tissue, cotranscribed with miR-451, and shares the same nucleotide sequence with miR transcripts from other species. MiR-144 has been reported to selectively regulate ␣-hemoglobin synthesis through modulating the expression of the klfd gene (Fu et al., 2009; Lalwani et al., 2012; Pase et al., 2009; Rasmussen et al., 2010). In this study, through ectopic expression of miR-144 in zebrafish embryos, we show that miR-144 regulates both primitive and definitive hematopoiesis as well as vascular development by targeting meis1, which provides significant insights into a novel miR-based molecular signal pathway for hematopoiesis and vascular development.

(http://www.targetscan.org/), EIMMO (http://www.mirz.unibas. ch/ElMMo2/), MicroCosm Targets Version 5 (http://www.ebi. ac. uk/enright-srv/microcosm/htdocs/targets/v5/), and PicTar (http:// pictar.mdc-berlin. de/). We analyzed the interaction between miR-144 and its target mRNA based on the minimum free energy hybridization of the site 1 and site 2 using the RNAhybrid tool (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/submission. html) (Grabher et al., 2011; Zhou et al., 2013a,b). Meis1 was predicted to be the direct downstream target gene of miR-144 by all the programs. 2.4. In vitro target validation by dual luciferase assays HCT116 cells were cultured in 24-well plates and co-transfected with 100 ng of pMIR-meis1-3 UTR(wt) or pMIR-meis1-3 UTR(mut) together with 10 ng of pRL-TK vector containing Renilla luciferase (Promega) and 100 nM of miR-144 mimics or negative control miRNA mimics using lipofectamine 2000 and Opti-MEM I reduced serum media (Gibco) according to the manufacturer’s instruction. Forty-eight to sixty hours after transfection, cells were harvested, lysed and used for luciferase assays. Firefly and renilla luciferase activities were measured using the Dual-Glo luciferase assay kit (Promega, USA) and analyzed as described (Ambros, 2004; Fan et al., 2009; Fu et al., 2009; Zhou et al., 2013a,b). Each experiment was performed in triplicate and repeated at least three times. 2.5. MiRNA mimics and preparation of mRNA

2. Materials and methods 2.1. Fish maintenance and cell culture

MiR-144 mimics and scrambled control miRNA mimics were purchased from RioboBio (RiboBio, China). The mRNA samples of meis1 and gata1 used in rescue experiments were prepared by in vitro transcription using the Sp6 mMessage mMachine Kit (Ambion) following the manufacturer’s instruction.

Wild-type zebrafish (Danio rerio) (AB type) and a line of transgenic zebrafish, Tg(flk1:EGFP), were maintained at 28.5 ◦ C under standard conditions as described previously (Chen et al., 2013; Li et al., 2014; Westerfield, 2000). To prevent pigmentation, embryos were raised and maintained at 28.5 ◦ C in “egg water” consisting of 0.003% 1-phenyl-2-thiourea (PTU, Sigma) after 12 hpf. Studies using zebrafish were approved by the Animal Care and Use Committee of Huazhong University of Science and Technology. Cell line HCT116 was purchased from ATCC (American Type Culture Collection, Rockville, MD, USA), and maintained in the Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) in a humidified incubator with 5% CO2 at 37 ◦ C (Zhou et al., 2013a,b).

One to two cell stage fertilized embryos were microinjected with 1–2 nl of synthetic miR-144 duplexes (10 ␮M) and control mimics (10 ␮M) as described (Pase et al., 2009). For rescue experiments, 100 pg of synthetic meis1 mRNA without 3 -UTR, gata1 mRNA without 3 -UTR or both was co-injected into one to two-cell stage embryos with 1–2 nL of synthetic miR-144 duplexes (10 ␮M) (Chen et al., 2013; Li et al., 2014).

2.2. Plasmid construction

2.7. In vivo target validation by EGFP reporter assays

The 3 -UTR of meis1 was amplified by PCR from a cDNA library prepared from 24 hpf zebrafish embryos, and cloned between the Spe I-Hind III sites of the pMIR -REPORT Luciferase vector (Zhou et al., 2013a,b), resulting in pMIR-meis1-3 UTR(wt). The ORF of EGFP was cloned between the BamH I-EcoR I sites of the pCS2+vector (Du et al., 2009; Grabher et al., 2011), whereas the meis1-3 -UTR was cloned between the EcoR I-Xbal I sites of the PCS2 vector. In addition, the miR-144 targeting sites in the 3 -UTR of meis1 in reporter plasmids were mutated by sequential inverse PCR and ligation via an acquired restriction enzyme site, resulting in pMIR-meis1-3 UTR (mut) and EGFP-meis1-3 UTR (mut). The primers used in this study are listed in Table S1. All constructs were validated by restriction enzyme digestion and direct DNA sequencing analysis.

Zebrafish embryos at the 1–2 cell stage were injected with 1–2 nl of a mixed solution including approximately 100 pg of mRNA for a wild type EGFP reporter with duplex miR-144 or negative control mimics. The other set of embryos were injected with mRNA for a mutant EGFP reporter with miR-144 targeting sequence mutated. At 30 hpf, developed embryos were collected for examining the intensity of green fluorescence under a fluorescent microscope (Olympus SZX16). The level of EGFP protein signal was also assessed using Western blot analysis (Grabher et al., 2011; Pase et al., 2009).

2.3. Bioinformatic analysis The direct targets and binding sites for miR-144 were predicted using several online programs, including TargetScan

2.6. Microinjection of synthetic miR-144 duplexes and mRNA

2.8. Quantitative real time PCR analysis Embryos were collected at indicated time points (12, 32 hpf). Total RNA samples were extracted with TRIzol reagent (TaKaRa, Japan) and genomic DNA was removed with DNA-freeTM DNase treatment and removal reagents (ABI, Foster City, CA, USA). Extracted RNA samples were quantified by spectrometry. Two

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micrograms of RNA samples were transcribed into cDNA by reverse transcription using the a cDNA Synthesis kit (Invitrogen) with random primers or a specific miR-144 RT primer. The cDNA products generated using random primers were used for real time PCR analyses of meis1, Hb˛e1, and Hbˇe1 using a FastStart Universal SYBR Green Master kit (Roche, USA). For expression analysis of miR144, Bulge-loopTM miRNA qRT-PCR Primer Sets specific for miR-144 were purchased from RiboBio (Guangzhou, China). Real time PCR analysis was carried out using a FastStart Universal SYBR Green Master kit (Roche, USA) in a 10 ␮l reaction volume on an ABI 7900 Genome Analyzer System. GAPDH mRNA and U6 small RNA were used as internal controls. Data analysis was performed using the 2−Ct (RQ value) method (Wang et al., 2011; Yuan and Sun, 2009; Zhou et al., 2013a,b). The sequences of primers used for qRT-PCR are listed in Table S2. 2.9. Western blot analysis Approximately 40 embryos at indicated time points (32, 36 hpf) were collected and transferred into a 1.5 ml micro eppendoff tube. Embryo sacs were dissociated by rinsing with deyolk buffer (150 mM NaCl, 50 mM Tris–HCl pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 1 ␮g/ml leupeptin, 1 mM deoxycholic acid and 1 mM EDTA) for three times. The embryos were then mixed with 10 ␮l of lysates buffer (55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO3 ) containing a cocktail of protease inhibitors and phosphatase inhibitors (Calbiochem, Darmstadt, Germany) per embryo, and scrambled with supersonic wave 3 times for 3 s. About 50 ␮g of protein extracts were separated by 12% SDSPAGE and transferred to a PVDF membrane (Millipore, Bedford, MA, USA) using a Bio-Rad electroblotting apparatus. Protein signals were detected with a mouse anti-tublin antibody (1:20,000) and a rabbit anti-human MEIS1(1:25,000) for 2 h at room temperature or overnight at 4 ◦ C, followed by incubation with an horseradish peroxidase conjugated secondary antibody (1:10,000) and enhanced chemiluminescence kit (Pierce). Protein bands were imaged using a ChemiDoc XRS (Bio-Rad), and quantified using Quantity One software (Bio-Rad) (Ro and Dawid, 2011).

Fig. 1. Bioinformatic analysis identified miR-144 targeting sequences in the 3 -UTR of meis1 from various vertebrate species. (A) 3 -UTR sequences of meis1 containing the miR-144 targeting sites (‘seeds’) were aligned among humans (Hsa), chimpanzee (Ptr), rat (Rno), mouse (Mmu), horse (Eca), and zebrafish (Dre). Numbers indicate nucleotide positions targeted by the seed sequence of miR-144. The zebrafish meis1 3 UTR contained a pair of target sequences (sites 1 and 2) for miR-144. (B) The identity of the sequence of mature miR-144 among different species. (C) The RNAhybrid tool (http://bibiserv.techfak. uni-bielefeld.de/rnahybrid/submission. html) found a minimum free energy hybridization between sites 1 and 2 for the meis1 3 -UTR and miR-144. (D) Interaction between the mutated meis1 3 -UTR and miR-144 in zebrafish based on the minimum free energy hybridization.

2.10. O-dianisdine staining Staining of hemoglobin of zebrafish by o-dianisdine was carried out as described (Fu et al., 2009; Pase et al., 2009). First, we prepared a staining solution containing 0.2% benzidine (Sigma), sodium acetate (0.01 M, pH 4.5) and ethanol (40%). Prior to use, 1/10 V of 3% hydrogen peroxide was added. Embryos were collected in 2 ml micro eppendoff tubes with 200 ␮l of staining solution that covers all embryos. The embryos were stained for 15–20 min at room temperature to avoid light, and inspected at a 3 min interval. The embryos were then imaged under a microscope, and analyzed by measuring the corresponding stained areas of the embryos with Image J (version 2.1.4.7). 2.11. Whole-mount in situ hybridization (WISH) WISH was carried out using Dig-labeled mRNA antisense probes as described previously (Chen et al., 2013; Thisse and Thisse, 2008). Embryos at 12 hpf, 24 hpf, 36 hpf or 48 hpf were dechorionated, fixed in 4% paraformaldehyde (PFA)/phosphate buffer saline (PBS) overnight at 4 ◦ C, washed twice in PBS at room temperature, and then maintained in absolute methanol at −20 ◦ C until use. Embryos were rehydrated by successive dilutions using methanol/PBS (75, 50 and 25%). Then, the embryos were treated with proteinase K (10 ␮g/ml) at room temperature, refixed with 4% PFA/PBS for 20 min, washed several times

with PBST (PBS + 0.1% Tween-20), and prehybridized for 2 h with the hybridization solution without the probe. Then, the digoxinlabeled probe was added (1 ng/␮l) and incubated overnight at 65 ◦ C. Hybridization signals were detected using anti-digoxigeninAP (Roche, Mannheim, Germany) and by staining with BCIP/NBT (Promega). The probes used for WISH were described by us previously (Chen et al., 2013; Li et al., 2014).

2.12. Image acquisition Embryos images were captured under an Olympus SZX16 microscope with an Olympus DP72 digital camera with cellSens version1.6 software, and processed with Adobe Photoshop CS4 software. Identical modifications and adjustments were applied to all the images in the same experiment (Azzouzi et al., 2012).

2.13. Statistical analysis All experiments were repeated at least three times. The data were presented as mean ± SD. Statistical analysis was performed using the Student’s t-test with Spass13.0 software. A P value of <0.05 was considered to be statistically significant.

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Fig. 2. In vitro and in vivo reporter assays revealed that miR-144 directly targeted meis1. (A) A schematic diagram showing construction of dual luciferase reporter plasmids for wild-type and mutant 3 -UTR of meis1. The zebrafish meis1 3 -UTR contains 2 predicted miR-144 binding sites (sites 1 and 2). A mutation of the meis1 3 -UTR was introduced to destroy the sites bound by the seed sequence. (B) Effects of the miR-144 mimics on meis1 3 -UTR luciferase reporters in HCT116 cells. The negative control mimics was co-transfected as a control. Luciferase activities were calculated as the ratio of firefly/renilla activities and normalized to the negative control. Results were obtained from 3 independent experiments with each experiment performed in triplicate. Data are shown as means ± SD. (C) A schematic diagram showing the construction of EGFP sensor reporter plasmids (similar to A). (D) EGFP sensor reporter assays were used to validate the interaction between the meis1 3 -UTR and miR-144. Single-cell embryos were microinjected with EGFP sensor mRNA fused to wild-type or mutated meis1 3 -UTRs and miR-144 or control duplex, and the EGFP fluorescence intensity was measured at 28–30 hpf. (E) MiR-144 directly targets the meis1 3 -UTR. Top panel: embryos injected with mRNA of the EGFP sensor and negative control mimics; Middle panel: Embryos injected with mRNA of the EGFP sensor and miR-144 mimics (EGFP fluorescence intensity was reduced); Bottom panel: Embryos injected with mRNA of the EGFP sensor harboring the mutated meis1 3 -UTR and miR-144 mimics (EGFP fluorescence intensity was similar to that of the negative control). Scale bar, 150 ␮m. (F and G) Western blot analysis was performed to quantify the expression of EGFP. ␤-Tubulin was used as an internal control.

3. Results 3.1. Bioinformatic analysis identifies meis1 as a potential downstream target of miR-144 Bioinformatic analysis with TargetScan, EIMMo, MicroCosm Targets Version 5, and PicTar software programs was used to identify potential downstream targets regulated by miR-144. We analyzed the 3 -untranslated region (3 -UTR) of meis1 for potential target sites for miRNAs. We found that the meis1 3 -UTR of zebrafish contained two adjoining target sites for miR-144 (Fig. 1A). The miR-144-binding site 2 was highly conserved during evolution among the human (Hsa), chimpanzee (Ptr), rat (Rno), mouse (Mmu), horse (Eca), and zebrafish (Dre) (Fig. 1A). The corresponding miR-144 seed sequences were also conserved among these species (Fig. 1B). The RNAhybrid tool based on analysis of the minimum free energy suggests that both the miR-144-binding site 1 and site 2

show Watson–Crick matches to the seed region of miR-144 (Fig. 1C). To analyze the potential function of the miR-144-binding sites, we created a mutation in the binding sites. As shown in Fig. 1D, the mutation disrupted the matches between the miR-144-binding sites and the miR-144 seed sequence. 3.2. MiR-144 represses the expression of meis1 To assess whether miR-144 directly regulates expression of meis1, we cloned the meis1 3 -UTR downstream of the firefly luciferase coding region in the pMIR-REPORT luciferase vector. The two putative miR-144 binding sites were also mutated to other sequences to generate a mutant reporter gene (Fig. 2A). Luciferase assays were carried out to determine the effect of miR-144 on expression of meis1 in HCT116 cells co-transfected with miR-144 mimics and a reporter gene. Transfection with the miR-144 mimics significantly reduced the ratio of firefly luciferase/renilla luciferase

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Fig. 3. MiR-144 down-regulates the expression of meis1 and inhibits primitive hematopoiesis in zebrafish embryos. (A) Successful overexpression of miR-144 through microinjection of miR-144 mimics as determined by real-time qPCR analysis. The negative control mimics served as a control. (B) MiR-144 mimics significantly reduced the level of meis1 mRNA in zebrafish embryos as demonstrated by real-time qPCR analysis. Gapdh was used as an internal control. Data are shown as means ± SD from 3 independent experiments performed in triplicate. (C and D) Western blot analysis was performed to quantify the expression of the MEIS1 protein. ␤-Tubulin was used as an internal control. (E) Overexpression of miR-144 significantly reduced the expression of hemoglobin by o-dianisidine staining. Scale bar, 300 ␮m. (F) Overexpression of miR-144 significantly reduced the expression of gata1, a marker for erythroid progenitors. Scale bar, 100 ␮m.

by 2.38-fold (P < 0.001; Fig. 2B) as compared to the control (cotransfected with negative control mimics and a reporter gene). In contrast, the mutation in the miR-144 binding sites abolished the reduced luciferase reporter activity by miR-144 (Fig. 2B). These results suggest that miR-144 represses the expression of meis1 by targeting the miR-144 binding sites at the 3 -UTR. To confirm that miR-144 represses the expression of meis1, we carried out an in vivo reporter assay in zebrafish embryos. The open reading frame (ORF) of EGFP was cloned upstream of the wild type or mutant 3 -UTR of meis1 to create an EGFP reporter (Fig. 2C). A reporter was co-injected with the miR-144 mimics into 1–2 cell stage embryos and the intensity of the EGFP signal was analyzed. Co-injection of miR-144 resulted in a statistically significant decrease in EGFP fluorescence compared to co-injection with a negative control miRNA mimics, indicating a negative regulatory function of miR-144 on meis1. This effect was abolished in embryos injected with the mutant reporter (Fig. 2D and E), suggesting that the miR-144 binding sites are important in its regulation of meis1. The expression level of EGFP was also assessed using Western blotting analysis (Fig. 2F). MiR-144 significantly reduced the expression of EGFP from the reporter with the wild type 3 -UTR of meis1 (P < 0.05; Fig. 2G), but not that from the mutant 3 -UTR. These data suggest that miR-144 interacted with its predicted target sites at the meis1 3 UTR to repress translation of meis1. The regulatory role of miR-144 on meis1 expression was also characterized by real-time RT-PCR analysis and Western blotting analysis in zebrafish embryos injected with miR-144 mimics or a negative control mimics (Fig. 3A). Real-time RT-PCR analysis showed that miR-144 significantly decreased the expression of meis1 mRNA by 1.5-fold (P = 0.0423; Fig. 3B). Similarly, miR-144 significantly reduced the expression level of the MEIS1 protein compared with the negative control (P < 0.001; Fig. 3C and D). MiRs regulate gene expression through either cleavage or translational repression of targeted mRNA (Rosen et al., 2009). The effect of

miR-144 on meis1 was more pronounced at the protein level than at the mRNA level, suggesting that miR-144 may reduce the stability of meis1 mRNA and inhibit translation of the MESI1 protein. Taken together, these results indicate that miR-144 represses the expression of meis1 by directly targeting the meis1 3 UTR. 3.3. MiR-144 regulates primitive hematopoiesis through repression of meis1 during embryogenesis in zebrafish To demonstrate the physiological function of miR-144-mediated repression of meis1 expression, we analyzed the effects of miR144 on hematopoiesis. Previous reports have shown that meis1 is required for primitive and definitive hematopoiesis in zebrafish embryos (Cvejic et al., 2011; Hu et al., 2009; Pillay et al., 2010). Therefore, we hypothesized that miR-144 was also involved in hematopoiesis by repressing meis1. We analyzed erythropoiesis by o-dianisidine staining of hemoglobin (Fig. 3E). Ectopic expression of miR-144 dramatically reduced the number of circulating blood cells in 32 hpf embryos (Fig. 3E) and 2 dpf embryos (Supplementary Videos). To further assess the role of miR-144 in primitive hematopoiesis, we performed whole-mount in situ hybridization (WISH) analysis for the expression of gata1, a marker for erythroid progenitors (Pase et al., 2009). Injection of miR-144 mimics significantly reduced the expression of gata1 compared to embryos injected with a negative control mimics at 12 hpf and 26 hpf (Fig. 3F). These results suggest that miR-144 is involved in the initiation of primitive hematopoiesis during zebrafish embryogenesis. 3.4. Overexpression of meis1 and gata1 rescued the effect of miR-144 To further demonstrate the inhibitory effect of miR-144 on hematopoiesis, we performed rescue experiments by co-injection

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Fig. 4. Rescue of deficiencies in primitive hematopoiesis caused by miR-144 by overexpression of meis1, gata1, or combination of meis1 and gata1. (A) Analysis of the expression of miR-144 by real-time qPCR analysis. GAPDH was used as an internal control. Data are shown as means ± SD from 3 independent experiments performed in triplicate. (B) MiR-144 mimics significantly reduced the level of meis1 mRNA in zebrafish embryos as measured by real-time RT-PCR analysis. Meis1 mRNA was successfully increased by injection with meis1 mRNA. GAPDH was used as an internal control. Data are shown as means ± SD from 3 independent experiments performed in triplicate. (C and D) Western blot analysis was performed to quantify the expression of the MEIS1 protein in zebrafish embryos. MiR-144 significantly reduced the expression of MEIS1 (P < 0.05). Co-injection of meis1 mRNA significantly increased the expression of MEIS1 (P < 0.001). ␤-Tubulin was used as an internal control. (E) Overexpression of miR-144 significantly reduced the expression of hemoglobin by o-dianisidine staining. Decreased hemoglobin expression was partially restored by co-injection with meis1 or gata1 mRNA and almost fully restored by co-injection with both meis1 and gata1 mRNA. Scale bar, 150 ␮m. (F) Analysis of the effects of miR-144 on erythropoiesis using Image J software. (G and H) Overexpression of miR-144 significantly reduced the level of hb˛e1 and hbˇe1 mRNA in zebrafish embryos as measured by real-time RT-PCR analysis. The expression levels of these targets were almost fully restored by co-injection with both meis1 and gata1 mRNA. Gapdh was used as an internal control. Data are the means ± SD from 3 independent experiments performed in triplicate.

of miR-144 mimics with meis1 mRNA, gata1 mRNA or both into 1–2 cell stage embryos. The meis1 mRNA used for injection lacks the miR-144 binding sites. Injection of miR-144 mimics increased expression of miR-144 by more than 16-fold (P < 0.001) as expected (Fig. 4A). Injection of miR-144 significantly reduced meis1 expression (P < 0.05) (Fig. 4B–D). Injection of meis1 mRNA significantly increased the expression level of the MEIS 1 protein by more than 2-fold (P < 0.01; Fig. 4B–D). O-dianisidine staining showed that co-injection of miR-144 together with meis1 or gata1 mRNA partially restored the reduced erythropoiesis by miR-144 compared to injection with miR-144

alone (Fig. 4E and F). Triple co-injection of miR-144 with meis1 mRNA and gata1 mRNA almost fully restored the reduced erythropoiesis by miR-144 (Fig. 4E and F). To confirm the effect of miR-144-mediated meis1 regulation on erythropoiesis, we carried out real-time RT-PCR analysis for ˛- and ˇ-hemoglobin genes. Ectopic expression of miR-144 significantly reduced the mRNA levels of both hb˛e1 and hbˇe1 by 2-fold, respectively (P < 0.001) (Fig. 4G and H). The reduced expression of the two hemoglobin genes by miR-144 was partially rescued by 50% by co-injection of meis1 mRNA (P < 0.001). These results suggest that miR-144 acts upstream of meis1 to regulate initiation of primitive

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Fig. 5. MiR-144 regulates primitive hematopoiesis in zebrafish embryos as analyzed by whole-mount in situ hybridization. (A) Overexpression of miR-144 significantly reduced the expression of gata1 and pu.1 at 12 hpf. The decreased expression of pu.1 by miR-144 was partially restored by co-injection with meis1 mRNA at 12 hpf. The decreased expression of gata1 by miR-144 was partially restored by injection with meis1 mRNA. Scale bar, 100 ␮m. (B) Overexpression of miR-144 significantly reduced the expression of gata1 and pu.1 at 26 hpf. The decreased expression of pu.1 was partially restored by co-injection of meis1 mRNA at 26 hpf. The decreased expression of gata1 by miR-144 was restored by injection of meis1 mRNA. Scale bar, 300 ␮m.

hematopoiesis. Co-injection of the gata1 mRNA slightly, but significantly, rescued the reduced expression of the hb˛e1 gene by miR-144 by 25% (P = 0.009), but not that of hbˇe1 (P = 0.18). Interestingly, co-injection of both meis1 and gata1 mRNA fully rescued the reduced expression of the two hemoglobin genes by miR-144 (P < 0.001) (Fig. 4G and H). Co-injection of miR-144 with meis1 mRNA rescued the reduced expression of pu.1 by miR-144, whereas overexpression of gata1 failed to rescue miR-144-mediated pu.1 reduction (Fig. 5). These results suggest that miR-144 regulate the initiation of primitive hematopoiesis by targeting meis1. The reason for the failed rescue by gata1 may be that overexpression of gata1 will expand erythroid lineage at the expense of myeloid lineage (Nerlov et al., 2000; Semerad et al., 2009), presumably due to the decrease of pu.1 expression. 3.5. MiR-144 is required for definitive hematopoiesis through repression of meis1 in zebrafish embryos Definitive hematopoiesis generates hematopoietic stem cells (HSCs) (Sood and Liu, 2012). To assess the role of miR-144 in definitive hematopoiesis, we analyzed the expression of runx1 and c-myb (markers of HSCs) in zebrafish embryos injected with miR-144 mimics by WISH. Injection of miR-144 significantly reduced the

expression of runx1 and c-myb, which suggests that miR-144 is required for definitive hematopoiesis (Fig. 6A and B). The reduced expression of c-myb and runx1 phenotype by miR-144 was partially rescued by co-injection with meis1 mRNA or a mixture of meis1 and gata1 mRNA, but not by gata1 mRNA (Fig. 6A and B). These results suggest that miR-144 is required for definitive hematopoiesis through repression of meis1 in zebrafish embryos. Scl is critical to both primitive and definitive hematopoiesis (Dooley et al., 2005; Qian et al., 2007). Therefore, we used WISH to analyze the effect of miR-144 on expression of scl. Overexpression of miR-144 dramatically reduced the signal for scl, and this effect was partially rescued by injection of meis1 mRNA (Fig. 6C). These results further suggest that miR-144 regulates both primitive and definitive hematopoiesis by repressing meis1 expression. 3.6. MiR-144 regulates vascular development by repressing expression of meis1 during embryogenesis in zebrafish Previous studies suggest that meis1 plays an important role in the development of the vasculature (Azcoitia et al., 2005; Minehata et al., 2008) and that scl is also involved in endothelial development (Dooley et al., 2005; Patterson et al., 2005; Van Handel et al., 2012). Therefore, we hypothesized that miR-144, which regulates expression of meis1 and acts upstream of scl, is also involved in vascular

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Fig. 6. MiR-144 regulates definitive hematopoiesis. (A) The expression of c-myb, a marker for definitive hematopoiesis and generation of HSCs, was significantly decreased when miR-144 was overexpressed in zebrafish embryos at 36 and 48 hpf. The decreased expression of c-myb by miR-144 was restored by co-injection of meis1 mRNA. Scale bar, 300 ␮m. (B) The expression of runx1, another marker for definitive hematopoiesis and generation of HSCs, was significantly decreased when miR-144 was overexpressed in zebrafish embryos. The decreased expression of runx1 by miR-144 was restored by co-injection with meis1 mRNA. Scale bar, 300 ␮m. (C) WISH showed that overexpression of miR-144 reduced the expression of scl. The decreased expression of scl by miR-144 was rescued by co-injection of meis1 mRNA. Scale bar, 100 ␮m.

development. We utilized a transgenic zebrafish line, Tg(flk1:EGFP) with an EGFP transgene under the control of an endothelial specific flk1 promoter to test the hypothesis. Overexpression of miR-144 by injection of miR-144 mRNA into 1–2 cell stage Tg(flk1:EGFP) embryos resulted in defective development of intersegment vessels (ISVs) (Fig. 7A). The ISV defect was rescued by injection of meis1 mRNA (Fig. 7A). Moreover, the intensity of endothelial signal in the dorsal aorta and cardinal vein areas was decreased in embryos with injection of miR144 compared to those with injection of control miRNA and the effect was rescued by overexpression of meis1 (Fig. 7A). To furthermore assess the role of miR-144 in vascular development, we analyzed the effect of overexpression of miR-144 on development of arterial and venous vessels. WISH analysis showed that compared to zebrafish embryos injected with a control miRNA mimics, those with injection of a miR-144 mimics displayed a reduced signal for arterial marker ephrinB2, which was partially rescued by co-injection of meis1 mRNA (Fig. 7B). No effect was observed for venous marker flt4 by miR-144 overexpression (Fig. 7C).

4. Discussion In this study, we demonstrated that miR-144 regulated both primitive and definitive hematopoiesis by targeting meis1. Bioinformatic analysis showed that miR-144 regulated meis1 by binding to the 3 -UTR of meis1 (Fig. 1). Reporter assays confirmed that miR-144 repressed the expression of meis1 in vitro and in vivo (Fig. 2). Overexpression of miR-144 significantly reduced the number of erythrocytes and the expression of gata1 (the marker for erythroid progenitors) and the effect on erythropoiesis was partially rescued by injection of either meis1 or gata1 mRNA and fully rescued by co-injection of both meis1 and gata1 mRNA (Figs. 3–5). Overexpression of miR-144 also significantly reduced the number of myeloid and the expression of pu.1 (the marker for myeloid cells) in zebrafish embryos and the effect was partially rescued by injection of meis1 (Figs. 3–5). Similarly, overexpression of miR-144 significantly reduced the expression of runx1 and c-myb (markers for HSCs), and the effect was partially rescued by injection of mRNA of meis1 (Fig. 6). Interestingly, as shown in Fig. 6C, overexpression of miR-144 decreased expression of scl, which is an early

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Fig. 7. MiR-144 regulates vascular development. (A) Development of vessels was inhibited by overexpression of miR-144 with injection of miR-144 mimics in a transgenic zebrafish line, Tg(flk1:EGFP) as shown by the reduced EGFP fluorescence signal for endothelial cells. Development of ISVs was inhibited by overexpression of miR-144 but the defect was rescued by co-injection with meis1 mRNA. Scale bar, 200 ␮m. (B) Overexpression of miR-144 reduced the expression of ephrinB2 at 26 hpf. Scale bar, 300 ␮m. (C) Overexpression of miR-144 had no apparent effects on the expression of flt4 at 26 hpf. Scale bar, 300 ␮m.

upstream regulator of both primitive and definitive hematopoiesis. Overexpresison of meis1 partially rescued the miR-144-mediated inhibition of scl expression (Fig. 6C). Together, these results indicate that miR-144 is a new molecular determinant required for both primitive and definitive hematopoiesis. Mechanistically, the regulation of hematopoiesis by miR-144 is through repression of meis1. This conclusion is consistent with the spatial and temporal expression profile of miR-144. WISH revealed strong expression of miR-144 in the ICM in 20 hpf embryos and in the posterior blood island at 72 hpf and 120 hpf (Fu et al., 2009), supporting its role in hematopoiesis. Here we also show that miR-144 is involved in regulation of vascular development. Overexpression of miR-144 led to abnormal development of ISVs and mild perturbation of WISH staining for arterial marker ephrinB2 and the defects were partially rescued by overexpression of meis1 (Fig. 7). Therefore, miR-144 is not only a new factor required for hematopoiesis, but also a new regulator of vascular development. The regulation may be through miR-144-mediated inhibition of meis1 expression, which may lead to inhibition of scl expression. The MEIS1 protein is a 3-amino acid loop extension (TALE) homeodomain protein that was previously identified as playing an important role in primitive and definitive hematopoiesis during zebrafish development (Cvejic et al., 2011). Meis1 was shown to interact with Pbx1 to regulate hematopoiesis (Thisse and Thisse, 2008). Previous studies suggested that meis1 played an essential role in the development of hematopoiesis and leukemiagenesis, however, the role of meis1 in the hematopoietic signal pathway during zebrafish development is controversial. Cvejic et al. demonstrated that the production of erythroid was repressed in zebrafish morphants independent of gata1, whereas Pillay et al.

suggested that meis1 acted upstream of gata1 to regulate primitive hematopoiesis (Cvejic et al., 2011; Pillay et al., 2010; Sangokoya et al., 2010). Overexpression of meis1 and meis2 greatly enhanced the formation of hematopoietic colonies from embryonic stem cells by maintaining proliferation of hematopoietic progenitor cells and skewing the megakaryocyte-erythroid progenitor to erythroid and toward megakaryocyte development (Cai et al., 2012; Moskow et al., 1995). Knockdown of meis1 in zebrafish resulted in a dramatic decrease in the number of erythrocytes (Rosen et al., 2009). The results above support our conclusion that miR-144 represses the expression of meis1 and therefore plays an important role in both primitive and definitive hematopoiesis. Our data showed that overexpression of meis1 partially rescued abnormal phenotypes caused by miR-144. The reason why meis1 cannot fully rescue abnormal phenotypes caused by miR-144 is unknown. One possibility may be that there are other important target genes regulated by miR-144, all of which are involved in hematopoiesis. Overexpression of both meis1 and gata1 (encoding an erythroid-specific transcription factor) fully rescued the abnormal erythropoiesis caused by miR-144, but only partial rescue was observed with meis1 mRNA alone. Bioinformatic analysis did not identify any potential miR-144 binding site on the gata1 mRNA (data not shown). It is interesting to note that meis1 was shown to act upstream of gata1 to regulate primitive hematopoiesis (Pillay et al., 2010). Thus, miR-144 may indirectly regulate gata1 expression through regulating expression of meis1. This may explain why overexpression of gata1 partially rescued the abnormal hematopoietic phenotypes associated with miR-144 overexpression. Moreover, miR-144 may indirectly regulate gata1 expression through regulating expression of other regulatory genes. This may explain why overexpression of both meis1 and gata1 fully rescue the defects by

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miR-144. Interestingly, gata1 was shown to directly activate the transcription of miR-144 (Dore et al., 2008), which forms a looped signaling pathway involved in hematopoiesis. Pillay et al. found that a meis1 morpholino increased WISH signals for pu.1 and l-plastin (downstream of pu.1) at 24 hpf (Pillay et al., 2010). In contrast, Cvejic et al. reported that two different meis1 morpholinos dramatically decreased l-plastin signal at 28 hpf by 3.3-fold (Cvejic et al., 2011). The reason for the different observations is unknown. However, our data are consistent with the finding by Cvejic et al., i.e. overexpression of miR-144 led to decreased expression of meis1 and decreased WISH signal for pu.1. Moreover, we found that reduced expression of pu.1 by miR-144 was rescued by injection of meis1 mRNA. Therefore, our data support the notion that miR-144-mediated pu.1 reduction is through regulation of meis1. On the other hand, because miR-144 may regulate expression of other downstream target genes, we cannot exclude the possibility that miR-144-mediated pu.1 reduction may also involve other target genes of miR-144. In conclusion, we found that miR-144 is required for both hematopoiesis and vascular development by down-regulating the expression level of meis1. This study therefore identifies a new regulator for concerted hematopoietic and vascular development. Funding This study was supported by Chinese National Basic Research Programs (973 Programs 2013CB531101 and 2012CB517801), the Hubei Province’s Outstanding Medical Academic Leader Program, Specialized Research Fund for the Doctoral Program of Higher Education from the Ministry of Education, the “Innovative Development of New Drugs” Key Scientific Project (2011ZX09307-001-09), Hubei Province Natural Science Key Program (2008CDA047), and State Key Laboratory of Freshwater Ecology and Biotechnology (2011FB16). Conflict of interest The authors declare no competing financial interests. Author contributions Most experiments were performed by Zhenhong Su. Wenxia Si made some plasmid constructs and assisted with real time PCR analysis. Bisheng Zhou, Lei Li, Chengqi Xu, Yan Xu, and Xiuchun Li provided help and insights into the project. Zhenhong Su drafted the manuscript. Haibo Jia and Lei Li participated in revision of the manuscript. Qing Wang designed and supervised the overall project, and critically revised the manuscript. Acknowledgement We thank all the members of the Cardio-X team for helpful discussions and fish husbandry. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biocel. 2014.01.005. References Amali AA, Sie L, Winkler C, Featherstone M. Zebrafish hoxd4a acts upstream of meis1.1 to direct vasculogenesis, angiogenesis and hematopoiesis. PLoS ONE 2013;8:e58857. Ambros V. The functions of animal microRNAs. Nature 2004;431:350–5.

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