development and disease runx1 is required for zebraﬁsh ... · runx1have been described in aml and...

INTRODUCTION

The mammalian hematopoietic system is established earlyduring embryogenesis and is required for the continuousproduction of blood during fetal and adult life. Duringembryonic development, a transient wave of ‘primitive’hematopoiesis that primarily gives rise to embryonicerythrocytes is followed by a second ‘definitive’ hematopoieticwave that establishes all blood lineages (reviewed by Orkin,2000; Cumano and Godin, 2001). In mammals, hematopoieticstem cells (HSCs) are initially found in the extra-embryonic

yolk sac blood islands and the intra-embryonic para-aorticsplanchnopleura/aorta-gonad-mesonephros region (P-Sp/AGM).In successive waves of migration, HSCs colonize the fetal liver,spleen and bone marrow (reviewed by Dzierzak andMedvinsky, 1995; Robb, 1997). HSCs are mesodermal inorigin, and this tissue also gives rise to angioblasts, theprecursors of the vascular system. Although yet to be directlyidentified in vivo, increasing evidence points towards theexistence of a mesodermally derived bipotential precursortermed the ‘hemangioblast’, from which both HSCs andangioblasts differentiate (Choi et al., 1998; Pardanaud and

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RUNX1/AML1/CBFA2 is essential for definitivehematopoiesis, and chromosomal translocations affectingRUNX1 are frequently involved in human leukemias.Consequently, the normal function of RUNX1and itsinvolvement in leukemogenesis remain subject to intensiveresearch. To further elucidate the role of RUNX1 inhematopoiesis, we cloned the zebrafish ortholog (runx1)and analyzed its function using this model system.Zebrafish runx1 is expressed in hematopoietic andneuronal cells during early embryogenesis. runx1expression in the lateral plate mesoderm co-localizes withthe hematopoietic transcription factor scl, and expressionof runx1 is markedly reduced in the zebrafish mutantsspadetail and cloche. Transient expression of runx1incloche embryos resulted in partial rescue of thehematopoietic defect. Depletion of Runx1 with antisensemorpholino oligonucleotides abrogated the development ofboth blood and vessels, as demonstrated by loss ofcirculation, incomplete development of vasculature and theaccumulation of immature hematopoietic precursors. Theblock in definitive hematopoiesis is similar to that observedin Runx1 knockout mice, implying that zebrafish Runx1has a function equivalent to that in mammals. Our data

suggest that zebrafish Runx1 functions in both blood andvessel development at the hemangioblast level, andcontributes to both primitive and definitive hematopoiesis.Depletion of Runx1 also caused aberrant axonogenesis andabnormal distribution of Rohon-Beard cells, providing thefirst functional evidence of a role for vertebrate Runx1 inneuropoiesis.

To provide a base for examining the role of Runx1 inleukemogenesis, we investigated the effects of transientexpression of a human RUNX1-CBF2T1 transgene[product of the t(8;21) translocation in acute myeloidleukemia] in zebrafish embryos. Expression of RUNX1-CBF2T1 caused disruption of normal hematopoiesis,aberrant circulation, internal hemorrhages and cellulardysplasia. These defects reproduce those observed inRunx1-depleted zebrafish embryos and RUNX1-CBF2T1knock-in mice. The phenotype obtained with transientexpression of RUNX1-CBF2T1validates the zebrafish as amodel system to study t(8;21)-mediated leukemogenesis.

Key words: Runx1, RUNX1-CBF2T1, Zebrafish, Hematopoiesis,Angiogenesis, Hemangioblast, Neuropoiesis, Leukemia

SUMMARY

DEVELOPMENT AND DISEASE

Runx1 is required for zebrafish blood and vessel development and

expression of a human RUNX1-CBF2T1 transgene advances a model for

studies of leukemogenesis

Maggie L. Kalev-Zylinska 1, Julia A. Horsfield 1, Maria Vega C. Flores 1, John H. Postlethwait 2, Maria R. Vitas 1,Andrea M. Baas 1, Philip S. Crosier 1 and Kathryn E. Crosier 1

1Division of Molecular Medicine, The University of Auckland, Auckland, New Zealand2Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA*Author for correspondence (e-mail: [email protected])

Accepted 4 January 2002

2016

Dieterlen-Lievre, 1999). There is also evidence for thedevelopment of HSCs from ‘hemogenic endothelium’ in theventral wall of the aorta (Jaffredo et al., 1998).

Recently, zebrafish (Danio rerio) genetics has contributedtowards the understanding of early blood and vesseldevelopment (Amatruda and Zon, 1999; Paw and Zon, 2000).Hematopoietic programs are largely conserved betweenmammals and zebrafish (Zon, 1995), and the genetic amenity ofthe zebrafish has advanced its cause as a strong model systemfor developmental studies (Knapik, 2000). Several zebrafishmutants with defects in blood development exist. For example,the clochemutant (Stainier et al., 1995) fails to develop matureblood or vessels, providing genetic evidence for the existence ofa hemangioblast. Uncovering the mutation responsible forclocheis expected to provide insight into the molecular eventsthat direct commitment of mesoderm towards blood and/orendothelial fates.

Studies in mammals have identified key transcriptionalregulators that are involved in early commitment to ahematopoietic stem cell fate (Shivdasani and Orkin, 1996;Davidson and Zon, 2000) of which one example is RUNX1(Downing, 1999; Tracey and Speck, 2000). RUNX1 is a memberof the runt family of transcriptional regulators that are involvedin many developmental processes, ranging from segmentationand sex determination in Drosophila to blood and bonedevelopment in mammals (reviewed by Westendorf and Hiebert,1999; Canon and Banerjee, 2000). The RUNX1 gene (alsoknown as AML1/CBFA2/PEBP2αB) was first isolated from thechromosome 21 breakpoint in t(8;21)(q22;q22) (Miyoshi et al.,1991). This translocation is found in approximately 40% ofindividuals with the M2 subtype of acute myeloid leukemia(AML) (Bitter et al., 1987) and results in the formation of achimeric protein, now known as RUNX1-CBF2T1 (formerlyAML1-ETO) (Erickson et al., 1992; Miyoshi et al., 1993).RUNX1is involved in several other chromosomal translocationsin acute leukemias, of which TEL-RUNX1 in t(12;21) andRUNX1-EVI1 in t(3;21) are the most common (reviewed byLutterbach and Hiebert, 2000). In addition, point mutations inRUNX1have been described in AML and myelodysplasia (Osatoet al., 1999; Imai et al., 2000; Preudhomme et al., 2000),and haploinsufficiency at the RUNX1locus causes familialthrombocytopenia with predisposition to AML (Song et al.,1999). Like other runt family members, RUNX1 contains aconserved runt domain (RD) that mediates binding to its targetsequence, TGT/cGGT, and is also necessary for the physicalinteraction with the heterodimeric partner, CBFβ(Meyers et al.,1993; Ogawa et al., 1993b; Crute et al., 1996). Both of thesefunctions are mediated by distinct, non-overlapping RD sites(Nagata and Werner, 2001). The structure of a RD-CBFβcomplex bound to its cognate sequence has recently been solved,providing insight into the molecular basis of human diseaseinvolving RD mutations (Bravo et al., 2001).

Runx1 has been shown to play a critical role in blooddevelopment. Mice that lack Runx1 have no definitivehematopoiesis, display central nervous system (CNS)hemorrhages, and die in midgestation (Okuda et al., 1996; Wanget al., 1996a). P-Sp explant cultures from these mice exhibitdefective vascular formation that can be rescued by HSCs(Takakura et al., 2000). Vascular growth factors were shown toaugment Runx1 expression in an endothelial cell line, andexpression of a CBFβ-MYH11 fusion inhibited the angiogenic

activity of these cells (Namba et al., 2000). These observationssuggest involvement of Runx1in the process of angiogenesis.

Investigations directed at understanding how translocationsinvolving RUNX1contribute to the development of AML haveshown that the RUNX1-CBF2T1 fusion protein is able to repressRUNX1 responsive genes (Okuda et al., 1998; Westendorf et al.,1998). CBF2T1/ETO normally interacts with the nuclearreceptor co-repressor complex [N-CoR/mSin3/histonedeacetylase (HDAC)], and thereby mediates transcriptionalrepression (Melnick et al., 2000; Hildebrand et al., 2001).Therefore, the RUNX1-CBF2T1 fusion protein could recruitHDAC to the promoters of genes that would normally beactivated by RUNX1. Recently, it has been demonstrated thatRUNX1-CBF2T1 influences the regulation of target genes withpotential relevance in myeloid leukemogenesis. The fusionprotein was shown to upregulate TIS11b, which induces myeloidcell proliferation when overexpressed (Shimada et al., 2000),and to downregulate the granulocytic differentiation factorC/EBPα (Pabst et al., 2001).

To investigate the role of RUNX1-CBF2T1 inleukemogenesis, RUNX1-CBF2T1 knock-in mice have beencreated (Yergeau et al., 1997; Okuda et al., 1998). These micedie in midgestation with a phenotype very similar to that seen inRunx1 and Cbfb null mice, providing further evidence thatRUNX1-CBF2T1 abrogates the normal function of RUNX1.The embryonic lethality of RUNX1-CBF2T1 expression isproblematic with respect to the development of a model systemfor the analysis of t(8;21) leukemogenesis. To overcome thisobstacle, mice that conditionally express RUNX1-CBF2T1under control of a tetracycline-responsive element (Rhoades etal., 2000), and mice in which transgene expression is regulatedby the myeloid-specific human MRP8 promoter (Yuan et al.,2001) have been developed. Results show that RUNX1-CBF2T1alone has a restricted capacity to transform cells and suggest thatadditional mutations are necessary to generate a leukemicphenotype. Inducible translocation of the Runx1 and Cbf2t1genes in mice through Cre/loxP-mediated recombination hasbeen described (Buchholz et al., 2000). Together, these miceprovide valuable model systems for the analysis of t(8;21)leukemogenesis; however, it might be helpful to developalternative animal models that are also highly amenable togenetic studies. The zebrafish offers genetic and developmentaladvantages that may facilitate analysis of the role of RUNX1 innormal hematopoiesis and in leukemia.

We consider the viability of the zebrafish as a model for thestudy of Runx1 function, and of RUNX1-CBF2T1-relateddisease. Initially, we cloned the zebrafish runx1 gene andexamined its role in early embryonic development. Zebrafishrunx1 shares a high degree of similarity with its mouse andhuman counterparts, and mapping data reflect conservation ofsynteny between the zebrafish and mammalian Runx1genes.During zebrafish development, runx1 is expressed inhematopoietic and neuronal cells. The abrogation of Runx1function using morpholino antisense oligonucleotidessuggested a role for Runx1 in early hematopoiesis,vasculoangiogenesis and neuropoiesis. Our results confirm thatzebrafish Runx1 functions in a similar manner to its mammalianorthologs. Furthermore, we found additional roles for zebrafishRunx1 in vasculogenesis and neuropoiesis. We theninvestigated the possibility that zebrafish could be used as amodel for the study of RUNX1-CBF2T1-mediated

M. L. Kalev-Zylinska and others

2017Runx1 in zebrafish hematopoiesis

leukemogenesis. Transient expression of a human RUNX1-CBF2T1 cDNA in zebrafish embryos caused a phenotypeanalogous to that observed in the RUNX1-CBF2T1knock-inmice. Therefore, the zebrafish can serve as an alternative modelsystem for examining additional genetic events that contributeto t(8;21)-mediated leukemia.

MATERIALS AND METHODS

Embryo collectionWild-type zebrafish (Danio rerio) were maintained, and embryoscollected and staged as described (Kimmel et al., 1995; Westerfield,1995). Embryos older than 24 hours post-fertilization (hpf) were usuallyincubated in 0.003% 1-phenyl-2-thiourea (PTU; Sigma) to inhibitpigmentation. clochemutant embryos (clom39; obtained from L. Zon),were generated from pairwise crosses between identified heterozygousmales and females.

Isolation of the zebrafish runx1 gene Coding sequence of human AML1a/RUNX1a(X79549) encompassingthe RD was amplified from cDNA derived from the Jurkat T-celllymphoma line using 5′-CCGCTTCACGCCGCCTTCCACC-3′(forward) and 5′-GGGCTGGGTGTGTGGGCTGAC-3′ (reverse)primers. This product was used as a probe to screen a 24 hour zebrafishembryo lambda cDNA library (Stratagene) at low stringency (2×SSC,0.1% SDS at 55°C). One positive clone was isolated and the insertsubcloned into pBluescriptII SK+ (Stratagene). Sequencing was doneusing an ABI 377XL sequencer.

Phylogenetic analysisPhylogenetic analysis was performed using the Phylogenetic InferencePackage, PHYLIP 3.5 (Felsenstein, 1993). The sequences were alignedusing the ClustalW method. Dendrograms were generated from aminoacid sequences of the entire protein or RD and outgrouped toDrosophila runt. Tree reconstruction was done using the neighbor-joining algorithm and percent bootstrap values were derived from 1000replications.

Genetic mappingMapping was performed using SSCP on the MOP haploid mappingpanel (Postlethwait et al., 1998). For display, the position of runx1wasintercalated into the HS panel (Woods et al., 2000). Comparativemapping with human was accomplished using the human chromosome21 database at the Weizmann Institute (http://bioinformatics.weizmann.ac.il/chr21/). The mapping primers were: runx1, 5′-TCTATGCCT-TTTCTATGGTTTCTTTTCTAA-3′ (forward) and 5′-GCTCGCCC-GCTGATTGTG-3′ (reverse); app, 5′-ATGGAGCACCGTCACCCC-TAACC-3′ (forward) and 5′-ACTTTGGCCATTGATTTGAACTGA-3′(reverse); and gart, 5′-GAGCATTCCAGATCCAGACATTCC-3′(forward) and 5′-CCTCTGAGAAGCTCCAGTTTTACTC-3′(reverse).

Generation of expression constructs and microinjection ofzebrafish embryosThe construct pCS2cmv-runx1contained a 1442 bpEcoRI-DraIfragment of zebrafish runx1cloned into the EcoRI and StuI sites of theexpression vector pCS2+ (Rupp et al., 1994). A human RUNX1-CBF2T1cDNA was kindly provided by Dr Scott W. Hiebert (VanderbiltUniversity, Nashville, TN). This was introduced into pCS2+ digestedwith XbaI, to generate pCS2cmv-RUNX1-CBF2T1. For expression inzebrafish embryos, approximately 100 pg of pCS2cmv-runx1and 100-150 pg of pCS2cmv-RUNX1-CBF2T1, both linearized at a 3′ NotI sitewere injected per embryo (Westerfield, 1995). Microinjections werecarried out using a Narishige micromanipulator (type GJ) and an MPPI-2 Milli-Pulse Pressure Injector (Applied Scientific Instrumentation)under a Leica MZ12 dissecting microscope.

Western blot Protein was isolated from pools of embryos injected with pCS2cmv-RUNX1-CBF2T1, after yolk removal as described (Westerfield, 1995).Samples (10 embryos equivalent per lane) were separated on 10% SDS-polyacrylamide gel and transferred to PVDF membrane. The Jurkat cellline that expresses RUNX1(Takahashi et al., 1995), was used as apositive control. The blot was incubated with rabbit anti-humanAML1/RD polyclonal antibody (1:40; Oncogene), and developed withgoat anti-rabbit peroxidase conjugate (1:5000; Amersham) and the ECLdetection system (Amersham).

Morpholino oligonucleotidesTwo morpholino antisense oligonucleotides (MO) targeting the runx1transcript were obtained (Fig. 1A) (Gene-Tools, LLC): runx1-MO1, 5′-TGGCGTCCCAAAGAAAAACCATTT-3′; runx1-MO2: 5′-TTTGGT-ATGTTTTTGTCTCCGTGAG-3′. Sequence complementary to thepredicted start codon is underlined. An antisense oligonucleotide withfour base mismatches when compared with runx1-MO2 was used asa control: 5′-TTTGCTATGATTTTGACTCCCTGAG-3′ (mismatchedbases are underlined). Solutions were prepared and injected asdescribed (Nasevicius and Ekker, 2000).

Preparation of antisense RNA probes and runx1 mRNAFor runx1in situ hybridization with zebrafish embryos, two probes weresynthesized (Fig. 1A). Template 1 (1682 bp) was generated byPCR using 5′-GGTAAGCTTCGGGGAAGATGAGCGAGGGTTT-3′(forward) and 5′-GGGGAATTCTGGGAGGAAACACTAGCTGTGC-3′ (reverse) primers. This template contained 1280 bp of codingsequence (nucleotides 77-1356 including the RD) and 403 bp ofadjacent 3′-UTR. It excluded a further 3′-UTR with homology to azebrafish mermaid repeat. Template 2 (981 bp; generated by ApaI digestof template 1 to exclude RD) contained nucleotides 779-1356 of thecoding sequence and 403 bp of 3′-UTR. For synthesis of RNA probes,template 1 was linearized with HindIII and template 2 with KpnI, andboth were transcribed with T7 polymerase using an RNA labeling kit(Boehringer Mannheim) according to the manufacturer’s instructions.Full-length antisense digoxigenin (DIG)- or fluorescein (FLU)- labeledriboprobes for zebrafish scl, flk-1(kdr – Zebrafish InformationNetwork) myb and βE3-globin (hbbe3 – Zebrafish InformationNetwork) were synthesized using T7 polymerase from templates(provided by L. Zon) that had been linearized with SalI, SmaI, EcoRIand SmaI/KpnI respectively.

To generate synthetic runx1mRNA, a pCS2cmv-runx1constructwas digested with NotI and capped mRNA was synthesized in vitrousing the SP6 mMESSAGE mMACHINE Kit (Ambion). For rescueexperiments, 10 pg of runx1 mRNA was injected, as describedpreviously (Nasevicius and Ekker, 2000).

Whole-mount in situ hybridization and immunostainingIn situ hybridization using DIG- or FLU-labeled antisense riboprobeswas performed as described (Broadbent and Read, 1999) withmodifications. For in situ hybridization with runx1 probes,temperatures of 65-70°C were used for hybridization and wash steps(60-65°C for other probes). Washes were performed as follows:2×SSCT/75%formamide, 2×SSCT/50%formamide, 2×SSCT/25%-formamide, 2×SSCT (15 minutes), 0.2×SSCT (twice, 30 minutes),followed by three washes in PBST (5 minutes) and a rinse in MABT.Embryos were blocked in 2% blocking reagent (BoehringerMannheim) for 3-4 hours. Hybridization was detected with anti-DIGor anti-FLU antibodies (Boehringer Mannheim) coupled to alkalinephosphatase (AP). Excess antibody was removed by eight washeswith PBST (15 minutes). Bound antibody was visualized using theAP substrates BM Purple, Fast Red (Boehringer Mannheim) orNBT/BCIP (Promega). Double in situ hybridization was performed asdescribed (Broadbent and Read, 1999).

After in situ hybridization, immunostaining of zebrafish embryoswas performed as described (Macdonald, 1999). Mouse monoclonal

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anti-HNK-1/N-CAM (1:1000; Sigma) was used as a primaryantibody. This was detected with a goat anti-mouse IgM peroxidaseconjugate (1:300; Sigma).

Histology and cytologyFollowing runx1 in situ hybridization, embryos to be sectioned werere-fixed, mounted in agarose, dehydrated in ethanol and infiltratedwith JB4 resin (Polysciences). Sections (5 µm) were cut using aRM2155 microtome and counterstained with nuclear fast red (Vector).

Other embryos were fixed overnight, embedded in paraffin, sectionedat 3 µm on a Leica RM2135 microtome and stained with Hematoxylinand Eosin (Sigma). Cytological analysis of embryonic blood wasperformed essentially as described (Ransom et al., 1996). Cells wereaspirated with a pulled glass capillary connected to a manual pistonpump (CellTram Oil; Eppendorf).

Imaging and microangiographyMost images were captured on a MZ FLIII stereomicroscope using a


Fig. 1. (A) Sequence analysis (GenBank Accession Number, AF391125). The runt domain (RD) is in red. The region encompassed by twoperpendicular bars indicates the longer template, and the region in green, the shorter template for in situ probes. The runx1-MO1 and runx1-MO2 binding sites are indicated by horizontal lines. (B) Percentage amino acid identity of zebrafish Runx1 when compared with the Xenopus,mouse and human orthologs. Overall identity is indicated in black, and identity within the RD in red. (C) Phylogenetic analysis of runx1. TheGenBank Accession Numbers of the genes included in the analysis are: human RUNX1, L34598; mouse Runx1, D13802; Xenopus runx1,AF035446; human RUNX2, XM_004126; mouse Runx2, AF010284; human RUNX3, X79550; mouse Runx3, AF155880; and zebrafish runx3(runxbtranscript 1), AB043788. Bootstrap support values are given in the nodes. (D) The runx1 locus maps to the upper portion of zebrafishLG1 in a region, showing conserved syntenies with a portion of human chromosome 21. Distances on LG1 are in centiMorgans (cM) (the entirechromosome is 122 cM long), and distances on Hsa21 are in megabases (Mb). Abbreviations: gart (AF257743; D. B. Slavov and K. Gardiner,unpublished), ortholog of GARTin human; app (AF257742; D. B. Slavov and K. Gardiner, unpublished), ortholog of APP in human; mx1(AW202878; Washington University Zebrafish EST Project 1998, unpublished), apparent ortholog of MX1 in human (Woods et al., 2000); othermarkers of the form Z4593 are from Shimoda et al. (Shimoda et al., 1999) and http://zebrafish.mgh.harvard.edu/.

http://zebrafish.mgh.harvard.edu/


Leica DC 200 camera and a PC Pentium III-600 equipped with Leicaimaging software. For two-color runx1in situ imaging and fluorescentphotography, a ProgRes3008 digital camera (Zeiss Jenoptik) withassociated software was used.

Microangiography of zebrafish embryos was performed asdescribed (Weinstein et al., 1995) with modifications.Yellow-green fluoresceinated carboxylated latex beads(Molecular Probes) were used. After dilution, they weresonicated on ice using a Misonix sonicator for 25 minutes(five 5-minute cycles). Injection was performed asdescribed elsewhere (http://mgchd1.nichd.nih.gov:8000/zfatlas/Intro%20Page/angiography.html) (Isogai et al.,2001). Image capture was performed with a Leica MZFLIII stereomicroscope as described above, using astandard FITC filter set.

Accession numberThe cDNA sequence of the zebrafish runx1gene reportedin this manuscript has been deposited in GenBank underthe accession number AF391125.

RESULTS

Isolation of zebrafish runx1A human RUNX1a 620 bp cDNA probeencompassing the RD was used to screen a 24 hourold zebrafish cDNA library at low stringency, and asingle 2.5 kb clone was isolated (Fig. 1A) (GenBankAccession Number, AF391125). Sequence analysisshowed that this clone is homologous to human andmouse Runx1. The cDNA encodes an open readingframe of 451 amino acids that shows an overall 73%and 72% identity with the human and mouse Runx1proteins, respectively. The highest degree ofconservation is present within the RD, which shares96% identity with both human and mouse proteins(Fig. 1B). All amino acid substitutions within theRD are conservative changes. At the DNA level,runx1 is highly similar to the human RUNX1gene(71% overall identity and 84% within RD) and tothe mouse Runx1gene (70% overall and 82% withinRD) in the coding region. Our zebrafish runx1cloneshares 99% sequence identity with a runxacloneisolated by Kataoka et al. (Kataoka et al., 2000).Five of the eight nucleotides that differ are locatedwithin the RD. Despite the nucleotide differences,both clones encode identical proteins. Phylogeneticanalysis, based on amino acid sequences of eitherthe entire protein or RD only, revealed that theclosest relative of zebrafish runx1 is the Xenopusrunx1 (Xaml), and that these two genes formseparate outgroups from their mammalian Runxrelatives (Fig. 1C). Mapping studies showed thatrunx1 is located on the MOP haploid mapping panelat a position equivalent to LG1_7.7 cM on the HSmeiotic mapping panel (Woods et al., 2000) orLG1_3.5cM on the MGH mapping panel (Shimodaet al., 1999) (Fig. 1D). This region in the zebrafishgenome exhibits conserved synteny with a region ofhuman chromosome 21 that includes RUNX1 at21q22.3 and murine Runx1 at 16_62.2 (Miyoshi et

al., 1991; Bae et al., 1994). This conservation of syntenytogether with the phylogenetic analysis of the gene we haveisolated indicates that it is very probably the zebrafishortholog of RUNX1.

Fig. 2.Expression of runx1 in zebrafish wild-type (A-N) and mutant (O,P)embryos. In situ was performed with the longer probe, except D,E, which werecarried out with the shorter probe. (A-E,O,P) Whole embryos; dorsal views,anterior upwards (A-C), lateral views, anterior towards the left (D,E,O,P).(F-H) Posterior halves of embryos; dorsal view, posterior downwards (F), lateralviews, anterior towards the left (G,H). (I,J) Transverse sections of the embryocorresponding to a line in H, with J a higher magnification of the area marked inI. (K-N) Dorsal views of head areas, except K, dorsolateral. Stages in hpf areindicated. (A) Diffuse pre-zygotic expression. (B-F) Expression within the LPM(arrowheads) and in Rohon-Beard cells (arrows). (G-J) Expression in the ICM(arrowheads), Rohon-Beard cells (arrow in G) and ventral wall of the dorsalaorta (arrows in H-J). Endothelial cells are indicated in J. Cells with runx1expression show a different nuclear morphology. (K-N) Expression in theolfactory epithelium (arrowheads) and in putative cranial nerve VIII ganglia(arrows). (O,P) Arrows indicate markedly reduced runx1expression in the ICMin spadetail and cloche respectively. NT, neural tube; NO, notochord; AV, axialvein; e, endothelial cell, ov, otic vesicle. Scale bars: ~25 µm.

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Zebrafish runx1 is expressed in hematopoietic andneuronal cells We used whole-mount in situ hybridization analysis tocharacterize expression of the runx1gene during zebrafishembryogenesis (Fig. 2). In all experiments, the two probesused yielded identical patterns of expression. Diffuse runx1expression at 5 hpf suggests the presence of a maternaltranscript (Fig. 2A). A specific pattern of zygoticrunx1expression can be detected from 12 hpf. Expression was firstdetected as bilateral stripes in the lateral plate mesoderm(LPM), and appeared to be most distinct at the posterior endof the embryo (Fig. 2B-D). At 18 hpf, runx1 expression was

strongest at the end of the yolk extension where the bilateralstripes of runx1expression appeared closer to the midline (Fig.2E,F). From ~18 hpf, cells of the LPM migrate to the midlineto form the intermediate cell mass (ICM) (Al-Adhami andKunz, 1977). Strong runx1 expression was observed in thedeveloping ICM during this time (Fig. 2G). At 24 hpf,expression of runx1in the posterior ICM weakened, while newpatches of runx1expression appeared in a more anteriorposition in the ventral wall of the dorsal aorta (Fig. 2H-J). Asimilar domain of Runx1expression is found during mouseembryogenesis (Simeone et al., 1995). In addition, thetemporal and spatial expression of runx1 is very similar to thatof the hematopoietic transcription factor scl (Gering et al.,1998). Therefore, we investigated in more detail therelationship between scland runx1during the first 24 hours ofdevelopment.

SCL is essential for the development of blood andvasculature (reviewed by Begley and Green, 1999), andzebrafish scl marks the earliest sites of commitment to thesefates (Gering et al., 1998). Double in situ hybridizationrevealed co-expression of scland runx1within the LPM at 12hpf (Fig. 3A). Dual expression of runx1and sclwas observedin individual cells (Fig. 3B,C). At 24 hpf, when runx1expression weakens in the posterior ICM and appears in theventral wall of the dorsal aorta, differences in the domains ofexpression of runx1and sclwithin the posterior ICM wereobserved (Fig. 3D,E).

In addition to expression in hematopoietic tissues, weobserved runx1 expression in neuronal cells within the headand the trunk (Figs 2, 3). Neuronal expression of runx1 firstappeared at 14 hpf in individual cells along the anteroposterioraxis that are located medial to the LPM (Fig. 2C,D).Immunolabeling with an antibody against HNK-1, a neuronalmarker, indicated that these cells are Rohon-Beard neurons(sensory neurons related to the dorsal root ganglia) (Kruse etal., 1984; Artinger et al., 1999) (Fig. 3F,G). Expression ofHNK-1 during early development in the zebrafish has beenpreviously characterized (Metcalfe et al., 1990). The earliestneurons to express this tetrasaccharide are the primary sensoryneurons mediating touch sensitivity, the Rohon-Beard neuronsof the spinal cord and the trigeminal ganglion sensory neuronsin the head. We found that only a subset of the Rohon-Beardcells display runx1expression (Fig. 3F,G). runx1 transcriptswere also detected in other neuronal tissues such as theolfactory placode (Fig. 2K,L). At 18 hpf, HNK-1 and runx1mRNA co-localize in clusters of cells located against theanterior-medial surface of the otic vesicle (Fig. 3H,I). Thesecells may represent acoustico-vestibulo (VIII) cranial nerveganglia.

runx1 acts downstream of spadetail and clocheTo determine whether runx1is implicated in molecularpathways leading to the commitment of mesoderm to a bloodor vascular fate, we analyzed runx1expression in spadetail(sptb104) and cloche(clom39) zebrafish mutant embryos (Fig.2O,P). The sptmutation abrogates the zebrafish tbx16 gene(Griffin et al., 1998), and results in defective differentiation ofmesodermal cells such that they fail to converge duringgastrulation (Ho and Kane, 1990). Consequently, sptembryosexhibit aberrant patterning of somites and the accumulation ofmesodermal cells in the tail. Additional data shows that spt


Fig. 3.Characterization of runx1expression. (A-E) Two-color in situhybridization using runx1(purple; NBT/BCIP) and scl(red; FastRed) riboprobes (purple and red arrowheads, respectively).(C,E) Fluorescence of sclsignal using rhodamine filter. (F-I) Co-labeling of embryos hybridized with runx1riboprobe (purple) withanti-HNK-1 (brown) (purple and brown arrows respectively).(A) Dorsal view of whole embryo. (B,C) Higher magnifications of anarea boxed in A. (D,E) Lateral view of posterior portion of embryo,anterior towards the left. (F-I) Dorsal views of mid-trunk embryoregions, anterior towards the left. (G,I) Higher magnifications ofareas boxed in F,H, respectively. Stages in hpf are indicated.(A) runx1and scloverlap in the LPM (arrowheads). (B,C) Overlapof runx1and sclin individual cells (arrowheads). (D,E) runx1expression is weaker than sclin the posterior ICM. (F,G) Rohon-Beard cells with no runx1expression (brown arrowheads), and a cellwith dual expression (purple arrowhead). (H,I) Putative cranial nerveVIII nuclei with overlapping runx1and HNK-1 expression (purplearrowheads). Otic vesicle (ov) is indicated.


embryos fail to generate differentiated blood (Thompson et al.,1998). At 18 hpf, runx1 expression in the ICM region of sptembryos is maintained, although at significantly reduced levels(Fig. 2O). cloembryos are defective in the production of bothblood and endothelial cells (Stainier et al., 1995), therefore theclo gene product is probably required for mesodermalcommitment to both of these fates. In 24 hpf clo embryos, norunx1 expression was detected in the region that wouldnormally form the dorsal aorta, and runx1expression inthe ICM region was markedly reduced compared withwild-type embryos (Fig. 2P). Furthermore, transientexpression of runx1 partially rescued the clohematopoietic defect (Fig. 4). 683 embryos obtained fromclo heterozygote mating pairs were injected with 100 pgof pCS2cmv-runx1. These were scored for the mutantphenotype on the basis of the enlarged heart at 48 hpf. In22% (36/163) of cloembryos, red blood cells were seenin the trunk (Fig. 4A). This was confirmed by a hbbe3in

situ (Fig. 4D). Overexpression of the same doseof pCS2cmv-runx1 in wild-type zebrafishembryos produced ectopic blood in up to 10%of injected embryos (Fig. 4C).

These findings are consistent with a role forrunx1 downstream of spt and cloin pathwaysleading to the differentiation of blood andvasculature.

runx1 is required for the development ofblood and vasculature and forneuropoiesis Antisense, morpholino-modified oligonucleotides(morpholinos) have recently proved to beeffective and specific translational inhibitors inzebrafish (Ekker, 2000; Nasevicius and Ekker,2000). We used this targeted gene knock-downtechnique to generate runx1loss-of-functionembryos. Morpholinos were injected into theyolk of zebrafish embryos at one to eightcell stages. Two morpholino oligonucleotidestargeting runx1 (Fig. 1A) were used to providea control for specificity. Amounts of injectedmorpholino ranged from 0.5 to 16 ng. Injection

of amounts higher than 4 ng were associated with nonspecificeffects in addition to a specific phenotype. For runx1-MO1,nonspecific effects consisted of gastrulation defects that causedembryo death by 18 hpf. High levels of runx1-MO2 led tononspecific necrosis within the CNS. To generate the datashown, we injected 2 ng of runx1-MO1. Eighty percent ofembryos presented a specific phenotype, in comparison with

Fig. 4. Effects of runx1expression in cloche(A,B,D-F) and wild-type (C) zebrafishembryos at 48 hpf. Lateral views of anterior regions (A-C) and whole embryos (D-F),anterior towards the left. (A-C) Morphology and (D-F) expression of hbbe3(globin).(A,B) Blood in the trunk of cloembryo injected with runx1(arrowhead) comparedwith uninjected clo. Arrows indicate dilated heart. (C) Ectopic blood (arrowhead). (D-F) hbbe3(globin) expression on the yolk (arrowhead) and in the trunk (arrow) incloinjected with runx1compared with uninjected cloand wild type.

Fig. 5. Phenotypic effects of runx1-MO injections. (A-H)Morphological changes at 24 hpf (A-C), 48 hpf (D-H) andcirculation defect visualized by microangiography at 52 hpf(I,J). All are lateral views of whole embryos (A-E), posteriorhalves of embryos (F-H) and mid-trunk regions (I,J), anteriortowards the left. Control embryos are indicated. (A,B) Bloodcells accumulated in the anterior (arrows) and posterior(arrowheads) ICM in comparison with control in C. (D) Lackof normal circulation. Blood cells accumulated in the aorta(region encompassed by two black arrows). Empty heart andedematous vitelline vessels (red arrow). Underdeveloped head(red arrowhead). Otic vesicle containing three otoliths (blackarrowhead). (E) Normal circulating blood cells (arrow). (F,G)Collections of blood cells in ventral tail ICM (arrowheads). (H)Normal tail circulation with caudal artery (arrowhead) and vein(arrow). (I) Interrupted aortic blood flow (red arrow) with lackof flow in cardinal vein and intersegmental vessels. (J) Normalcirculation; dorsal aorta (red arrow), posterior cardinal vein(green arrow) and intersegmental vessel (red arrowhead).

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50% for the same dose of runx1-MO2. In both cases there wereminimal nonspecific consequences, and the remaining embryoswere normal. Injection of 2-6 ng of a runx1control morpholinowith four mismatched bases did not produce a phenotype. Theeffects of both active morpholinos (2 ng) were fully rescuedwith 10 pg runx1mRNA, an amount that was insufficient toinduce a phenotype on its own (data not shown).

We found that runx1-MO injected embryos (runx1-moembryos) displayed dramatic defects in hematopoiesis,vasculogenesis and neuropoiesis in early embryonicdevelopment (Figs 5-8). Later embryos became severelyedematous, and death occurred after 6-7 days. This edematousphenotype was similar to that seen in vegfamorphant embryos(Nasevicius et al., 2000).

Embryos injected with runx1-morpholino showdisrupted vasculature and lack normal circulationThe most striking abnormality observed in the runx1-MOinjected embryos was the lack of normal circulation at 48 hpf,with accumulation of red blood cells in the aorta and ventraltail (Fig. 5A-H). This phenotype occurred despite thepresence of a beating heart. A small amount of blood wasoccasionally seen in the vitelline vessels, but was found to bestationary (data not shown). Consistent with the observedaccumulation of blood cells in the dorsal aorta,microangiography performed at 52 hpf demonstrated a lack

of circulation through the trunk and tail, with arrest of flowat the mid-trunk level (Fig. 5I,J).

Erythrocyte expression of hbbe3was used to examine thefunctional integrity of the circulatory system of runx1-moembryos (Fig. 6A,B). hbbe3 mRNA was limited to theposterior half of the injected embryos, with a marked reductionof blood cells seen in the vitelline and cranial vessels whencompared with normal. The observed distribution of hbbe3mRNA suggests that blood cells are unable to circulate andhave become trapped in the dorsal aorta. To investigate thenature of the vascular defects observed in runx1-moembryos,we used the expression of flk-1 (kdr – Zebrafish InformationNetwork) (Yamaguchi et al., 1993; Liao et al., 1997) to markdeveloping vascular endothelial cells (Fig. 6E,F,I,J). In normalembryos, two stripes of flk-1-positive cells, corresponding tothe developing aorta and axial vein were distinguishable in thetrunk region. flk-1 expression was also present in theintersomitic vessels. By contrast, flk-1expression in runx1-moembryos was grossly perturbed and indicated missing segmentsof vasculature, abnormal axial vessels, formation of atypical orectopic structures and deficient formation of intersomiticvessels. Furthermore, transverse sections through the trunk andtail revealed dilated and disorganized vascular channels in 48hpf runx1-moembryos (Fig. 6C,D,G,H,K,L). These findingsimply that the lack of normal circulation can be at leastpartially attributed to defective development of the blood


Fig. 6. Abnormal vasculature in the runx1-moembryos demonstrated by molecular (A,B,E,F,I,J) and histological (C,D,G,H,K,L) analyses at 48hpf. Expression of hbbe3(globin) (A,B) and flk-1(E,F,I,J). Lateral views of whole embryos (A,B) and posterior embryo region (E,F,I,J);anterior towards the left. Cross sections of the mid-trunk (C,D) and tail (G,H,K,L) regions. (K,L) Higher magnifications of G,H, respectively.Controls are indicated. (A)hbbe3(globin) expression is limited to the posterior embryo (arrow) with lack of expression in the circulation(arrowhead). (B) Normal hbbe3(globin) expression within vitelline vessels (arrowhead) and aorta (arrow). (E) ‘Wavy’ pattern of flk-1expression in axial vessels (arrowhead). (F) Ectopic (arrowhead) and missing (arrow) flk-1expression. (I) Interrupted expression in the axialvein (arrow), expansion of expression in the tail (black arrowhead) and a loss of expression in intersegmental vessels (blue arrowhead).(J) Normal flk-1expression in the axial vessels (arrow) and intersegmental vessels (blue arrowhead). (C) Multiple and disorganized vascularchannels replacing normal trunk vessels (arrow). (D) Normal dorsal aorta (arrow) and axial vein (arrowhead). (G,K) Expansion of tail regionwith multiple dilated capillaries (arrow). (H,L) Normal caudal vessels (arrows). Scale bars: ~50 µm.


vessels in the trunk. Our results suggest that normal Runx1function is essential for vasculoangiogenesis.

runx1-mo embryos accumulate immaturehematopoietic progenitorsA characteristic feature observed in runx1-mo embryos wasenlargement of the ICM region (Fig. 5A,B). This was firstobserved at 24 hpf and persisted until at least 48 hpf. Weexamined the developmental status of this cell population. Thehematopoietic transcription factor sclis expressed in blood-generating regions and later in circulating cells during normalembryonic development, but declines markedly before 50 hpf(Gering et al., 1998). Inrunx1-mo embryos, in situhybridization revealed that cells that accumulated in the ICMregion from 24 hpf were scl positive and that this scl-positivecell population remained dominant at 48 hpf (Fig.7A,B,I,J,M,N). Consistent with the vascular phenotype ofrunx1-MO injected embryos, there was no evidence of sclexpression in circulation (Fig. 7E,F). Because scl expressionmarks early differentiation of hematopoietic cells, scl-positivecells that accumulate in the ventral tail are likely to representimmature hematopoietic progenitors. This is consistent withthe observed immature morphology of these cells. Microscopicanalysis revealed their blast-like features characterized by ahigh nuclear to cytoplasmic ratio, open chromatin and scantybasophilic cytoplasm (Fig. 7C,D). Immature erythroblastswere also seen (Fig. 7G,H). Together, these results support theidea that maturation of blood progenitors is delayed or arrested

in runx1-mo embryos, resulting in decreased numbers ofprimitive erythrocytes.

runx1-mo embryos have a block in definitivehematopoiesisThe mybgene encodes a transcription factor that promotes thedifferentiation of definitive hematopoietic cell types (Mucenskiet al., 1991). To investigate whether runx1 is upstream of mybin the regulatory cascade, we carried out in situ hybridizationon runx1-moembryos using a mybriboprobe (Fig. 7O,P). Innormal embryos at 48 hpf, myb was expressed in clusters ofcells in the ventral wall of the dorsal aorta, a region equivalentto the mammalian AGM that is likely to contain definitivehematopoietic stem cells (Thompson et al., 1998). In 48 hpfrunx1-moembryos, expression of mybin the dorsal aorta wasmarkedly reduced by comparison. Consistent with the role ofRunx1 in mammals, our results imply that runx1 function isrequired for definitive hematopoiesis in zebrafish.

runx1-mo embryos exhibit neurologicalabnormalitiesIn addition to the vascular and hematopoietic phenotypesdescribed above, runx1-moembryos also exhibit a number ofneurological defects (Fig. 8).

Normally, otic vesicles of developing zebrafish embryoscontain two otoliths. Strikingly, we observed otic vesicles inrunx1-mo embryos that had irregular numbers of otoliths(commonly three) (Fig. 8A,B), indicating that the expression

Fig. 7. Abnormal hematopoiesis inrunx1-moembryos. Two-color insitu hybridization for flk-1 (purple)and scl(red) expression(A,B,E,F,I,J,M,N) and singlehybridization with the mybriboprobe(O,P). Lateral views of the posterior(A,B,I,J,M,N) and anterior (E,F)embryo regions, and whole embryos(O,P); anterior towards the left.(M,N) Higher magnification of theICM regions. Histological cross-sections of the tail (C) and trunk(G,K). Cytology of cells collectedfrom ICM collections (D) andvitelline vessels (H,L). All are 48hpf, except A,B, which are 24 hpf.Controls are indicated. (A,B)scl-positive cells (red arrow) accumulatein the ICM of runx1-moembryo.(E,F) Lack of sclexpression incirculation (red arrowhead) in runx1-moembryo. (I) Cells that accumulatein the tail maintain sclexpression(red arrowhead). In A,E,I, axialvessels (purple arrows) andintersegmental vessels (purplearrowheads) are poorly formed whencompared with controls (B,F,J).(M,N) scl-positive cells (red arrow)predominate in runx1-moembryowhen compared with thepredominantly flk-1-positive population in control (purple arrows). (C) Large immature cells (arrow) mixed with necrotic cells (arrowhead).(D) Blast-like morphology of accumulated cells with a mitotic figure (arrow). (G, arrow; H) Erythroid cells with delayed maturation compared withnormal in K,L. (O,P) Reduction in mybexpression in the aorta of runx1-moembryo compared with control (arrowheads). Scale bars: ~10 µm.

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of runx1 adjacent to the forming otic vesicle is probablyrequired for its development. Some embryos also exhibitedhead deformities such as enlargement of the ventricles in thebrain, and, more rarely, head underdevelopment (Fig. 8A,B andFig. 5D,E respectively). In addition, notochord abnormalitieswere observed, including missing cells (Fig. 8C,D), waviness,angulation and shortening of the anteroposterior body axis(data not shown).

To further investigate the role of runx1 in neuropoiesis, wedetermined the distribution of the neuronal marker HNK-1 inrunx1-moembryos. HNK-1 marks primary sensory neurons andthe axons of these neurons become strongly positive for HNK-1 as they develop (Metcalfe et al., 1990). Immunostaining withanti-HNK-1 revealed abnormal neurogenesis inrunx1-moembryos. These embryos commonly displayed disruptedaxonogenesis and abnormal localization of Rohon-Beard cells(Fig. 8E,F). These results provide the first functional evidenceto our knowledge that Runx1 is necessary for development ofthe CNS.

Overexpression of human RUNX1-CBF2T1 cDNA inzebrafish embryos reproduces abnormalities seen inrunx1-mo embryos and RUNX1-CBF2T1 transgenicmiceStudies in mice have shown that expression of a RUNX1-

CBF2T1fusion gene during embryogenesis causes embryoniclethality, probably by dominant interference with normalRUNX1 function (Yergeau et al., 1997; Okuda et al., 1998).To examine the validity of zebrafish as a model forleukemogenesis, we sought to determine whether the RUNX1-CBF2T1 fusion protein could produce similar effects inzebrafish embryos. To this end, a pCS2cmv-RUNX1-CBF2T1construct was used to drive transient expression of humanRUNX1-CBF2T1during zebrafish embryogenesis (Figs 9-11).Embryos were injected with 100-150 pg of pCS2cmv-RUNX1-CBF2T1, and the presence of human RUNX1-CBF2T1 proteinwas confirmed by western analysis (Fig. 9A). Abnormalitieswere observed in 32-41% of injected embryos at 48 hpf andcomprised defective development of blood and circulation andinternal hemorrhaging (Fig. 9B). Depending on the amountof pCS2cmv-RUNX1-CBF2T1injected, 13-41% of embryoslacked normal circulation and accumulated blood cells in theaorta and ventral tail (Fig. 9E,F). Some embryos alsoexhibited perturbed and/or reduced circulation (Fig. 9G,H).These defects were similar to those observed in runx1-moembryos. Hemorrhages were found in the CNS andpericardium in a subset of embryos with establishedcirculation (Fig. 9H-N). Within the CNS, both intracerebraland intraventricular areas of bleeding were observed.Extravasation of blood in the brain was confirmed byhistological analysis (Fig. 10).

To investigate the molecular effects of RUNX1-CBF2T1on hematopoiesis, in situ hybridization using selectedhematopoietic and vascular markers were performed onpCS2cmv-RUNX1-CBF2T1-injected embryos with thephenotypes described above (Fig. 11A,B,E,F,I,J). Consistentwith the diminished blood circulation, the expression of hbbe3was significantly reduced in a subset of embryos (Fig. 11A,B).In embryos displaying CNS hemorrhages, ectopic hbbe3expression was observed in the head (data not shown). Theexpression of flk-1was perturbed in embryos that lackedcirculation, indicating aberrant development of the trunkvasculature (Fig. 11E,F). A marked reduction in mybexpression indicates defective definitive hematopoiesis (Fig.11I,J).

To determine the effect of RUNX1-CBF2T1 onhematopoiesis at a cellular level, we examined the cytologyof blood cells collected from 48 hpf embryos injected withpCS2cmv-RUNX1-CBF2T1(Fig. 11C,D,G,H,K,L). In theseembryos, immature blood cell precursors were seen toaccumulate in the ventral tail region. The immature cellshad a blast morphology similar to that described forrunx1-mo embryos (Fig. 11C). In addition, some earlycells showed atypical features of nuclear cleavage andperinuclear cytoplasmic clearing, more characteristic ofatypical myeloid than erythroid precursors (Fig. 11D). Inthe injected embryos, we also observed circulatingerythroid cells with dysplastic features which includednuclear bridges, doughnut-shaped and irregular nuclei,binucleation and delayed maturation (Fig. 11G,H,K,L).Our results demonstrate that the expression of RUNX1-CBF2T1 in developing zebrafish embryos leads to aphenotype that shares similarities with the runx1-moembryos, and also aligns closely with the data obtained inRUNX1-CBF2T1 knock-in mice (Yergeau et al., 1997; Okudaet al., 1998).


Fig. 8. Neurological effects of Runx1 depletion. Morphologicalphenotype (A-D) and effect on HNK-1 expression (E,F) at 48 hpf.(A,B) Lateral and (E,F) dorsal views of head regions. (C,D) Lateralviews of tail regions; anterior towards the left. Controls are indicated.(A,B) Enlarged ventricular space (arrowhead) and supernumeraryotoliths (arrow) in runx1-moembryo compared with normal.(C,D) Interrupted notochord (arrow indicates its anterior andarrowhead indicates posterior region in runx1-moembryo). The redarrow in C indicates an accumulation of blood cells. In control,notochord (black arrow) and caudal vessels (red arrow) are shown.(E,F) Reduction in trigeminal descending (arrow) and Rohon-Beardascending (arrowhead) central axons and abnormal localization ofRohon-Beard cells (brown arrow).

DISCUSSION

We have isolated a zebrafish ortholog of the runx1 gene. In thisstudy, we describe the expression of zebrafish runx1 duringearly embryogenesis, and show that it is involved inhematopoiesis, vasculogenesis and neuropoiesis. Furthermore,we provide evidence that the zebrafish may be used as analternative model for studies of t(8;21)-mediatedleukemogenesis.

The zebrafish runx1gene we isolated shares 99% nucleotidesequence identity with a previously described gene, runxa

(Kataoka et al., 2000). Although Kataoka et al. obtained asimilar pattern of neuronal expression, they did not observerunx1 expression in hematopoietic tissues. The probe used intheir studies encompassed 483 bp of sequence 3′ of the RD andcontained 248 bp that overlapped with our shorter probe.Within this region of overlap, one nucleotide differed (H.Kataoka, personal communication). When we reproduced theirexperimental conditions, the results were unchanged fromthose shown in Fig. 2. The reasons for the differenthybridization patterns are unclear. However, it is possible thattheir probe detects an alternatively spliced exon of runx1 that


Fig. 9. Effects of RUNX1-CBF2T1expression in zebrafish embryos. (A) Western analysis of Jurkat cell lysate (lane 1) and extracts from 18 hpfembryos injected with pCS2cmv-RUNX1-CBF2T1(lane 2), pCS2 vector alone (lane 3) and uninjected embryos (lane 4). (B) Tabulatedsummary of phenotypes generated by transient expression of RUNX1-CBF2T1following injection of construct at two doses, compared withpCS2 vector alone and uninjected embryos. (C-N) Morphological changes at 24 hpf (C,D) and 48 hpf (E-N). (C-J) Whole embryos and (K-N)head regions; lateral views except L, which is ventral; anterior towards the left for all. Controls are indicated. (C,D) Blood accumulated in theICM region compared with normal (arrowheads). (E,F) Embryos with no circulation and blood accumulated in the proximal aorta (arrow) andICM (arrowhead). (G,H) Aberrant circulation with blood pooling in the ICM (arrows) and associated CNS bleed (arrowhead). (I) Normalcirculation with CNS hemorrhage (arrowhead). (K-M) Arrowheads indicate intraventricular, intracerebral and pericardial hemorrhagesrespectively.

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is only found in neuronal but not in hematopoietic tissues, andthat this probe is too short to detect hematopoietic runx1expression. Intron/exon boundaries are present in this region inthe human RUNX1gene (Levanon et al., 2001).

The hematopoietic transcription factor SCL has previouslybeen shown to be required for the correct differentiation ofblood and vascular tissue in the zebrafish (Gering et al., 1998;Liao et al., 2000). Recent evidence suggests that SCL is likelyto be directly involved in hemangioblast specification inmammalian cells (Robertson et al., 2000). Therefore, the co-expression of SCL and runx1at 12 hpf observed in our studiessuggests a role also for Runx1 at the hemangioblast level ofstem cell differentiation. In addition, we found that runx1expression was lost in the zebrafish mutants spadetail andcloche, and the forced expression of runx1 in clo resulted inpartial rescue of hematopoiesis. These findings indicate thatrunx1 acts downstream of spadetailand clochegenes andsupport an involvement of runx1 in the development of bloodand vasculature. This is strengthened by the morpholino data,showing that the depletion of Runx1 leads to aberrantvasculoangiogenesis as well as the accumulation of immaturehematopoietic progenitors. Together, these results support theexistence of a hemangioblast and argue that runx1 is involvedin its development in the zebrafish.

Previously, Runx1 function was shown to be required fordefinitive, but not primitive hematopoiesis in mice (Okudaet al., 1996; Wang et al., 1996a). It is therefore surprisingthat zebrafish runx1 expression was detected so early inmesodermal cells, as they give rise to the primitive blood.Nevertheless, consistent with its presence in differentiatingmesoderm, analysis of Runx1-depleted embryos supports arole for Runx1 in zebrafish primitive hematopoiesis. Anexpanded population of blast-like, scl-positive cellsaccumulated in the ventral tail of runx1-moembryos. Thesecells failed to differentiate and maintained sclexpression foran extended period of time (Fig. 7). Because runx1-moembryos also displayed aberrant vasculature, it is likely that

the accumulation of progenitors resulted from aninability of these cells to circulate and/ordifferentiate, rather than from increasedproliferation. To support this, an increase in thescl-positive population was not observed inrunx1-mo embryos at 14 hpf, prior tovascularization (M. K., unpublished). Therefore,we conclude that there is a delay or arrest in thematuration of primitive erythrocytes in Runx1-depleted embryos.

There is evidence that Runx1 may be involvedin primitive hematopoiesis in other vertebrates.The Xaml is expressed in sites of primitivehematopoiesis and functional studies indicatedits requirement for the formation of primitiveblood (Tracey et al., 1998). The involvement ofRunx1 in zebrafish and Xenopus primitivehematopoiesis may therefore represent anancestral condition from which the mammalianstate was derived. In the mouse, all newlyemerging primitive erythrocytes transientlyexpress Runx1until around 10.5 dpc (North etal., 1999), but it does not appear to be requiredfor their development. However, circumstantial

evidence hints that Runx proteins may be involved in primitivehematopoiesis in mammals. CBFβis absolutely required forRunx1 function (Wang et al., 1996b) and can heterodimerizewith all three Runx proteins (Ogawa et al., 1993a; Wang et al.,1993). A dominant negative form of CBFβ resulting from theCBFβ-MYH11fusion impaired primitive hematopoiesis in vivo(Castilla et al., 1996), implying involvement of a CBFβ-Runxcomplex in primitive hematopoiesis. It has been proposed thatthere is functional redundancy amongst runt family members,which would reconcile the absence of a defect in primitivehematopoiesis in Runx1-null mice with Runx1expression inprimitive lineages (Tracey et al., 1998).

Consistent with its role in mammals, Runx1 is required fordefinitive hematopoiesis in zebrafish. Zebrafish runx1 isexpressed in the ventral wall of the dorsal aorta, a siteassociated with the development of early definitive progenitorsin mice (Dzierzak and Medvinsky, 1995; North et al., 1999;Mukouyama et al., 2000). Furthermore, Runx1-depletedembryos showed a marked reduction in the expression of myb(Fig. 7).

Sites of runx1expression coincide with the expression ofzebrafish cbfb (Blake et al., 2000). Like runx1, zebrafish cbfbexpression was also detected in the LPM and ICM regions.Interestingly, the neural expression of cbfb is more extensivethan runx1, implying that it may interact with other membersof the runt family. The neural expression of runx1 was verysimilar to that previously described for runxa (Kataoka et al.,2000). We observed expression of runx1 in a subset of Rohon-Beard cells, olfactory placode and the presumptive cranialnerve VIII ganglia. The RUNX1homolog lozengeis requiredfor olfactory sense organ and eye development in Drosophila(Flores et al., 1998; Gupta et al., 1998), and also forhematopoiesis (Lebestky et al., 2000). In addition, both mouseRunx1and Xaml are expressed in neural structures, includingneural crest and Rohon-Beard cells, respectively (Simeone etal., 1995; Tracey et al., 1998). However, a neuronal functionfor Runx1 had not previously been demonstrated in a


Fig. 10. Histology of RUNX1-CBF2T1-related intracranial hemorrhages.Hematoxylin and Eosin staining of sagittal head sections at 48 hpf. Control isindicated. Arrows in A and B indicate intraventricular and intracerebral hemorrhages,respectively. (D,E) Higher magnifications of areas boxed in A,B respectively, witharrows indicating areas of bleeding. In D, an associated small intracerebralhemorrhage is shown (arrowhead). (F) At higher magnification, immature erythroidcells were present within hemorrhages (arrow). Scale bars: ~50 µm.


vertebrate. Strikingly, Runx1-depleted zebrafish embryosshowed gross abnormalities in neuropoiesis as revealed by adisorganized distribution of Rohon-Beard cells and perturbedaxonogenesis (Fig. 8). In addition, Runx1 was found to berequired for specification of the number of otoliths (two) in thedeveloping otic vesicle. Embryos compromised for Runx1function were found to have supernumerary otoliths. This isthe first functional evidence that runx genes contribute toneuronal and sensory organ development in vertebrates. TheCNS hemorrhages that occurred in Runx1null mice primarilyaffected cranial nerves (especially VII-VIII complex) anddorsal root ganglia. Moreover, necrosis was observed in neuralcrest and endothelial cells (Wang et al., 1996a). As Rohon-Beard cells are developmentally related to neural crest cells(Artinger et al., 1999), the CNS hemorrhages may reflect a rolefor Runx1 in neurogenesis.

A zebrafish model for RUNX1-CBF2T1-mediatedleukemogenesisThe RUNX1-CBF2T1 fusion gene product that results fromt(8;21) in humans has been studied in mice as a model fort(8;21)-derived leukemia (Yergeau et al., 1997; Okuda et al.,1998; Rhoades et al., 2000; Yuan et al., 2001). Because thezebrafish is highly amenable to genetic and developmentalstudies (Amatruda and Zon, 1999), we sought to create thebasis for a RUNX1-CBF2T1 leukemogenic model usingzebrafish. As a starting point, we overexpressed a humanRUNX1-CBF2T1 cDNA construct in zebrafish embryos.

This transient overexpression caused disorganized, reducedor absent circulation, along with internal hemorrhages (CNSand pericardial). Abnormal vascular development anddefective hematopoiesis contributed to the observed defects, asrevealed by aberrant flk-1and reduced mybexpression, and thepresence of dysplastic and immature blood cells. Although

misexpression of a heterologous fusion protein might induceartefacts that affect the observed phenotype, theseabnormalities were also observed in the zebrafish runx1-moembryos, consistent with the idea that the RUNX1-CBF2T1fusion protein dominantly inhibits endogenous runx1function.The phenotype generated by RUNX1-CBF2T1in zebrafishvaried with the amount of the construct injected, consistentwith the observation that RUNX1 functions in a dose-dependent manner (Wang et al., 1996a; Song et al., 1999; Caiet al., 2000). Significantly, this phenotype aligns with thechanges observed in RUNX1-CBF2T1 knock-in mice (Yergeauet al., 1997; Okuda et al., 1998). Furthermore, fetal liver cellsfrom RUNX1-CBF2T1 knock-in mice form dysplastichematopoietic progenitors with a high self-renewal capacity invitro (Okuda et al., 1998). Consequently, it was suggested thatRUNX1-CBF2T1 mediates inappropriate cell proliferation inaddition to repression of normal Runx1 function. The RUNX1-CBF2T1 protein was also shown to block myeloiddifferentiation in vitro (Rhoades et al., 2000). Consistentwith these data, we observed accumulation of immaturehematopoietic precursors in the ventral tail ICM and dysplasticerythroid cells.

Because the expression of RUNX1-CBF2T1 in zebrafishgenerates a phenotype that mimics the equivalent situation inmice, we conclude that the zebrafish presents a valid modelsystem for the evaluation of t(8;21)-mediatedleukemogenesis. There are precedents for the use of zebrafishas a model for human hematopoietic disease with the alas-emutation in sauternes, urodin yquem, ferrochelataseindracula and sptb in riesling offering models for congenitalsideroblastic anemia, hepatoerythropoietic porphyria,erythropoietic protoporphyria and hereditary spherocytosis,respectively (Brownlie et al., 1998; Wang et al., 1998; Childset al., 2000; Liao et al., 2000). We believe that the zebrafish

Fig. 11. Molecular andcellular effects of RUNX1-CBF2T1expression at 48hpf. Whole-mount in situhybridization for hbbe3(globin) (A,B), flk-1 (E,F)and myb(I,J) expression.Lateral views, anteriortowards the left. Cellsaspirated from the ICMregion (C,D) and circulation(G,H,K,L). Controls areindicated. (A,B)hbbe3(globin) expression isreduced in the circulation(arrowhead) and tail (arrow)compared with control.(E,F)flk-1expression in thetail (arrows) is abnormal.(I,J) mybexpression ismarkedly reduced in thedorsal aorta (arrows).(C) Immature, blast-likecells accumulated in theICM. (D) Cluster of atypicalearly cells (arrow).(G) Nuclear bridge (arrowhead), doughnut-shape nucleus (arrow). (H) Binuclear cell (arrowhead). (K) Cleaved nuclei (black arrowheads),mitotic figure (arrow) and a megaloblast (purple arrowhead). Scale bars: ~10 µm.

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offers a promising, genetically amenable alternative to themouse for examining the role of t(8;21) in leukemogenesis.The phenotype generated by expressing human RUNX1-CBF2T1 cDNA in zebrafish embryos represents the firstdemonstration of a human oncogenic protein modeled inzebrafish. To further explore this exciting possibility, we arecurrently generating a zebrafish transgenic line with inducibleRUNX1-CBF2T1 expression. We intend to use this line in agenetic screen for molecules that modulate the effects ofRUNX1-CBF2T1.

We thank Anne Bardsley for help in screening the zebrafish cDNAlibrary, Ross Bland for advice on the in situ work, Chris Hall forfacilitating microangiography and Latifa Khan for technicalassistance. This study would not have been possible without theexcellent fish facility run by Dr Peter Cattin, who also helped withembryo sectioning. We appreciate the help of Natalie Potter and IsobelEarly with histology, Michelle Petrasich with cytology and JeffGreenwood with Western blotting. Selected digital imaging wascarried out at The University of Auckland Biomedical ImagingResearch Unit with the help of Hilary Holloway. We are grateful toDr Len Zon for providing fixed clocheand spadetailembryos, clom39

line and plasmids for the scl, flk-1, myband hbbe3in situ probes. Oursincere thanks go to Dr Scott Hiebert for providing the humanRUNX1-CBF2T1cDNA, Dr Hiroko Kataoka for helpful discussionand to Dr Gordon Keller for comments on the manuscript. M. K. holdsa Ruth Spencer Medical Research Fellowship from the AucklandMedical Research Foundation and the New Zealand Guardian Trust.This work was also supported by the Health Research Council of NewZealand, the Leukaemia and Blood Foundation of New Zealand andgrant RR10715 from NIH to J. H. P.

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