Genome-Wide Analysis of the Zebrafish ETS Family Identifies Three Genes Required for Hemangioblast Differentiation Or Angiogenesis

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Genome-Wide Analysis of the Zebrafish ETS Family Identifies Three Genes Required for Hemangioblast Differentiation or Angiogenesis

Feng Liu, Roger Patient

Abstract—ETS domain transcription factors have been linked to hematopoiesis, vasculogenesis, and angiogenesis. However, their biological functions and the mechanisms of action, remain incompletely understood. Here, we have performed a systematic analysis of zebrafish ETS domain genes and identified 31 in the genome. Detailed gene expression profiling revealed that 12 of them are expressed in blood and endothelial precursors during embryonic development. Combined with a phylogenetic tree assay, this suggests that some of the coexpressed genes may have redundant or additive functions in these cells. Loss-of-function analysis of 3 of them, erg, fli1, and etsrp, demonstrated that erg and fli1 act cooperatively and are required for angiogenesis possibly via direct regulation of an endothelial cell junction molecule, VE-cadherin, whereas etsrp is essential for primitive myeloid/endothelial progenitors (hemangioblasts) in zebrafish. Taken together, these results provide a global view of the ETS genes in the zebrafish genome during embryogenesis and provide new insights on the functions and biology of erg, fli1, and etsrp, which could be applicable to higher vertebrates, including mice and humans. (Circ Res. 2008;103:1147-1154.) Key Words: zebrafish Ⅲ gene duplication Ⅲ ETS transcription factors Ⅲ hemangioblast Ⅲ angiogenesis

ebrafish has been recognized as an excellent genetic and during embryonic development and adulthood and have been Zdevelopmental biology model to study hematopoiesis linked with diverse biological processes, from hematopoiesis, and vessel development. Large numbers of genetic mutants vasculogenesis, and angiogenesis to neurogenesis. Many and transgenic lines in both blood and endothelial lineages important blood and endothelial regulators have well- have become available. More importantly, developmental conserved and functional ETS binding sites in their promoter mechanisms, including the functions of many known impor- and/or enhancer regions.12–15 For instance, the stem cell tant regulators involved in both blood and vessel develop- leukemia gene, Scl, a key regulator during hemangioblast ment, are conserved between higher vertebrates and ze- formation and hematopoiesis, is regulated by a complex that brafish.1 It has been proposed that hematopoietic and consists of ETS transcription factors (Fli1 and Elf1) and other endothelial cells share a common progenitor, termed the partners.12 Moreover, many ETS genes are coexpressed in hemangioblast.2 This idea was initially conceived from the hematopoietic and endothelial progenitor cells, which sug- observation that these 2 cell types develop in close proximity gests they may have redundant roles or are synergistically within the embryo.3 Coexpression of many important regula- required during hematopoiesis and vasculogenesis.11 Defects tors of hematopoiesis and vasculogenesis in early embryos in human ETS counterpart genes, either by inappropriate further supports the existence of a common progenitor for expression or by fusions with other proteins, cause carcino- hematopoietic and endothelial cells. Furthermore, loss of genesis, such as leukemia.16 function of several of these regulators, including the zebrafish To comprehensively explore the functional roles of ETS cloche (clo) mutant, affects both cell types.4–7 Recent work factors during hematopoiesis and vasculogenesis in zebrafish, by us and others subsequently showed that several important we have used bioinformatics to systematically analyze the hematopoietic and endothelial regulators in mammals, such ETS family from the zebrafish genome (Table I in the online as Scl/Tal1 and Lmo2, also play similar roles in zebrafish.8–10 data supplement, available at http://circres.ahajournals.org). ETS domain genes encode a super family of transcription Interestingly, 12 of the 31 ETS genes identified showed factors organized into 11 subfamilies based on domain expression in blood and endothelial precursors during embry- structures, which are conserved from worm and fly to onic development, which, together with phylogenetic analy- mammals.11 These transcription factors are involved in cell sis, suggested that some of them may have redundant or fate specification, proliferation, migration, and differentiation additive functions. Functional analysis of 3 of them, etsrp,

Original received May 19, 2008; revision received September 17, 2008; accepted September 18, 2008. From the Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, United Kingdom. Correspondence to Prof Roger Patient, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, United Kingdom. E-mail [email protected] © 2008 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.108.179713

Downloaded from http://circres.ahajournals.org/1147 by guest on November 25, 2015 1148 Circulation Research November 7, 2008 erg and fli1, which are strongly expressed in blood and endothelial precursors/hemangioblasts, demonstrated that they are required for hemangioblast differentiation and angiogenesis. Materials and Methods Fish Strains and Embryos Zebrafish embryos were obtained by natural spawning of adult AB strain zebrafish. Embryos were raised and maintained at 28.5°C in system water and staged as described.17 flk1:gfp/gata1:dsred transgenic line and etsrpy11 mutant were kindly provided by Stefan Schulte-Merker (Utrecht, The Netherlands) and Brant Weinstein (NIH), respectively. Bioinformatics To search for ETS genes, the sequences of the human and mouse ETS domains were used to TBLASTN the zebrafish genome in NCBI (http://www.ncbi.nlm.nih.gov). The predicted zebrafish ETS genes and proteins were named based on the alignment of their coding regions to known entries in public databases. To find the conserved domain in the protein sequences, NCBI conserved domain database was used (http://www.ncbi.nlm.nih.gov/Structure/cdd/ wrpsb.cgi). To generate a phylogenetic tree, the full-length amino acid sequences of ETS proteins were aligned using the CLUSTALW program. Chromosome locations of ETS genes and their surrounding genes were obtained from the Ensembl zebrafish genome database as above. Morpholinos Antisense morpholinos (MOs) were obtained from GeneTools (Philomath, Ore). The MOs used in this work were erg atgMO (5Ј-AGTGACACTCACTCTCTCTGAGGTA-3Ј), erg spliceMO (5Ј-AGACGCCGTCATCTGCACGCTCAGA-3Ј), and flil utrMO (5Ј-TTTCCGCAATTTTCAGTGGAGCCCG-3Ј). etsrp atgMO was 18 as reported previously. Stock solutions (1 mmol/L) in dH2O were Figure 1. Phylogenetic analysis of zebraﬁsh ETS genes. The prepared, and 2 to 10 ng amounts were injected into 1- to 2-cell stage tree was generated from the alignment of the amino acid embryos at the yolk/blastomere boundary. Both erg atgMO and sequence of the whole protein including ETS DNA binding spliceMO gave similar phenotype and coinjection of both MOs did domain and Pointed domain (PNT) using the CLUSTAL W not give a more severe phenotype than single injection. method. Zebraﬁsh genes with incomplete or highly divergent ETS domains were not included in this analysis. Groups are Reverse Transcription–Polymerase Chain Reaction named according to.16 RT-PCR analysis to determine efficacy of MO splicing inhibition was carried out with primers 5Ј-AGGCGCTGTCGGTGGTGA- GTGAG-3Ј (erg forward) and 5Ј-TCTGCTGGCGTCTGGAGG- systematically search the whole zebrafish genome (National GTAG-3Ј (erg reverse). cDNA template was produced from mRNA Center for Biotechnology Information [NCBI]; Ensembl, isolated from 24 hpf wild-type (wt) and erg spliceMO-injected zv7/Vega) for the potentially responsible ETS factors regu- embryos. PCR products were analyzed on 1% agarose gel. lating hemangioblast development. Computational searching Whole-Mount In Situ Hybridization of sequence databases revealed 31 ETS domain-containing Whole mount in situ hybridization was performed as described.19 genes in the zebrafish genome sequence (supplemental Table The following probes were used: fli1, scl,gata1, flk, pu.1,9,18 etsrp,18 I and data not shown), and their putative chromosomal 20 pax2.1. erg, ets2, fev, elk4, gabpa, elf1, and elf2 were first reported locations were mapped according to the latest zebrafish in this work and their EST clones were obtained from ImaGenes (Berlin, Germany). genome mapping information (supplemental Table I). Phylogenetic analysis with the complete open reading Photography frames of the ETS transcription factors, identified 11 subfam- Images of live embryos were captured using a Nikon SMZ 1500 ilies within the ETS gene family (supplemental Table I and dissecting microscope (ϫ1 HR Plan Apo objective; numeric aper- ture, 0.13) with a Nikon DXM1200 digital camera driven by Nikon Figure 1), which is consistent with the classification of Act-1 version 2.12 software (Nikon). Confocal images were acquired mammalian counterparts based on their structural composi- with a Zeiss LSM 510 META confocal laser microscope and 3D tion and their similarities in the DNA-binding ETS domains. projections were generated using Zeiss LSM software (Carl Zeiss Inc). Besides the conserved ETS domain, a subset of ETS family proteins has another evolutionarily conserved domain called Results the POINTED (PNT) domain at their N-terminal regions, Identification and Phylogenetic Analysis of which is required for protein–protein interactions. Notably, Zebrafish ETS Domain Genes because of genome duplication, there are several genes that The presence of ETS binding sites in the regulatory elements are closely related in this family (supplemental Table I). For of key hematopoietic and/or endothelial genes led us to example, there are 3 erf-like genes, erfl1, erfl2, and erfl3,

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Figure 2. etsrp, fli1, fli1b, and erg are expressed in hematopoietic and/or endothelial cells during zebrafish embryogenesis. etsrp (A through D), fli1 (E through H), fli1b (I through L), and erg (M through P). Flat-mount view: A through C, E through G, I through K, and M through O; lateral view, D, H, L, and P. Anterior to the left in all images. similar to erf itself, which are the orthologs of the mammalian Expression Analysis in clo Mutants Confirms That erf gene. There are another 3 pairs of ETS genes, ie, elk4 and Twelve ETS Genes Are Specifically Expressed in elk4l1, elf2 and elf2l1, spic and spicl1, which show high Hematopoietic or Endothelial Cells homology to a single mammalian gene. To confirm the expression of the 12 ETS genes in hematopoietic and endothelial cells, we tested whether their expres- Developmental Expression Patterns of Twelve sion was affected in clo mutants, which lack both blood and Hematopoietic or Endothelial ETS Genes endothelial cells. So far, clo mutants exhibit the earliest To systemically characterize expression of these ETS genes hematopoietic and endothelial lineage defects in zebrafish.7 in wt embryos, we performed a detailed expression pattern As shown in Figure 3 and supplemental Figure II, endothelial analysis at various developmental stages (Figure 2, supple- expression of 11 ETS genes, ie, ets1, ets2, etsrp, fli1, fli1b, mental Figure I, and data not shown). We obtained cDNA erg, fev, gabpa, elf1, elf2, and elk4, and myeloid expression clones for 27 of the 31 ETS genes (the other 4 clones are of pu.1 were reduced or absent in clo embryos at 26 hpf, duplicate genes, ie, erfl2, elk4l1, elf2, and spicl1, which suggesting that they are indeed expressed in hematopoietic or should give similar expression patterns because of high similarity of the sequences) (supplemental Table I) and subjected zebrafish embryos to in situ hybridization at 1-somite (1s), 6s, 10s, and 26-hpf stages when hemangioblasts and erythroid/myeloid and endothelial lineages are clearly identifiable (Figure 2, supplemental Figure I, and data not shown). Compared to the known hemangioblast marker, scl (supplemental Figure I, A through D), we found that 12 ETS genes are expressed in the right place and early enough to be involved in hemangioblast formation (Figure 2 and supplemental Figure I). These are ets1, ets2, etsrp, fli1, fli1b, erg, fev, gabpa, elf1, elf2, pu.1, and elk4. Importantly, among them, ets2, erg, fev, gabpa, elf1, elf2, and elk4 are reported for the first time in this study. Of note, etsrp is the earliest ETS gene strongly expressed in the anterior lateral plate mesoderm (ALM) (Figure 2A), which will give rise to myeloid and endothelial cells in zebrafish. In the posterior lateral plate mesoderm (PLM), fli1 is the earliest and strongest expressed at 1s among these 12 genes (Figure 2E). erg is also expressed in the PLM at the 1s stage (Figure 2M). Later on, all of them Figure 3. Expression of etsrp, fli1, fli1b, and erg was reduced or except pu.1 are expressed in endothelial cells at 26 hpf lost in clo mutant embryos at 26 hpf. A, etsrp.B,fli1.C,erg.D, fli1b. Note that endothelial-specific expression was reduced or (Figure 2 and supplemental Figure I), suggesting that they absent in the clo embryos (arrows). Anterior to the left and dor- might play roles in angiogenesis. sal to the top.

Downloaded from http://circres.ahajournals.org/ by guest on November 25, 2015 1150 Circulation Research November 7, 2008 endothelial cells. Of note, neural crest expression of fli1, ets1, a truncated protein containing only a small portion of the gabpa, elf1, and elf2 were relatively normal in clo embryos PNT domain and, because of a frameshift, no downstream (Figure 3B and supplemental Figure II, A, D, E, and F), ETS DNA binding domain (Figure 4A). To test MO effi- compared to wt siblings. ciency, RT-PCR analysis of erg spliceMO-injected embryos was carried out, revealing the formation of aberrantly spliced etsrp Is Required for Anterior Hemangioblast RNAs (Figure 4B, lower arrow in spliceMO-injected em- (Endothelial/Myeloid Progenitor) Differentiation bryos), instead of the wt product, which was virtually Gene expression profiling showed that fli1, erg, and etsrp are abolished (Figure 4B, upper arrow in control embryos). Thus, strongly expressed in the putative hemangioblast population endogenous erg activity was substantially knocked down in 10s stage embryos and later on are restricted to endothelial with the splice MO. spliceMO-injected embryos developed cells (Figure 2). We reasoned that they might be involved in normally without any obvious morphological defects, apart blood/endothelial progenitor specification and differentiation, from a hemorrhage in the head area between 2.5 and 3 days as well as in angiogenesis. erg has previously been implicated postfertilization (see below). The atgMO gave a similar in angiogenesis and in some leukemia cases in mammalian hemorrhage phenotype (data not shown), indicating that the cell lines.21 Because no knockout mice for erg had been hemorrhage is a specific phenotype caused by erg reported (but see Discussion), we decided to use the zebrafish knockdown. as a model to study the function of erg in vivo. To knock- To determine the consequences of loss-of-function of fli1, down erg expression in zebrafish, we designed 2 antisense we designed an antisense MO against the 5Ј untranslated MOs (Figure 4A). The erg atgMO was designed to target the region immediately upstream of the fli1 translation initiation erg ATG and inhibit translation. The erg spliceMO was codon. This fli1 utrMO completely abolished the green directed against the boundary of exon 4 and intron 4, fluorescent protein (GFP) expression in fli1:gfp embryos, spanning part of the PNT domain, to skip exon 4 and generate suggesting that this antisense MO knockdown strategy was efficient (Figure 4D). The fli1 morphants also developed a hemorrhage in the head around 2.5 days postfertilization (supplemental Figure IV). etsrp has been demonstrated to be involved in endothelial differentiation but is dispensable for erythroid development in zebrafish embryos.18 However, its role in the earliest precursor, the hemangioblast, has not yet been tested. Knock- down experiments showed that whereas fli1MO and ergMO had no effects on the early hemangioblast program in both ALM and PLM, etsrpMO caused significant reduction of scl and an absence of pu.1 and flk1 expression in the ALM, which gives rise to myeloid and endothelial lineages in wt embryos (supplemental Figure III). In contrast, the erythroid marker gata1 was not affected in the PLM of all 3 morphants (supplemental Figure III, U through X). To confirm the etsrpMO phenotype, we used the available etsrp mutant, y11, in zebrafish.22 The predicted Etsrp polypeptides synthesized in the homozygous mutant contain only a quarter of the wt Etsrp protein and do not have the well-conserved ETS DNA binding domain essential for the function of ETS genes. Therefore, it is very likely to be a null allele, which should phenocopy the loss-of-function phenotype induced by etsrp atgMO injection. Whole-mount in situ hybridization shows that the hemangioblast marker, scl, was significantly reduced or absent in the ALM of y11 embryos (Figure 5A). The myeloid marker pu.1 was completely Figure 4. MO efficiency test. A, Partial erg gene structure show- absent, and the endothelial marker flk1 was also absent in ing the targets of erg atgMO and spliceMO and the positions of forward (F) and reverse (R) primers for RT-PCR to verify the both the ALM and the PLM (Figure 5B, 5C, and 5E). In the splicing defects. The ETS DNA binding domain is located in PLM, scl expression was downregulated in y11 embryos exon 8/9. B, RT-PCR analysis revealed formation of an alterna- (Figure 5D), suggesting that etsrp is also involved in posterior tive splice product following spliceMO injection (446 bp, lower black arrow), indicating knockdown of wt transcrpit (656 bp, hemangioblast differentiation into the endothelial lineage. upper black arrow) caused by exon 4 skipping. erg plasmid and The erythroid marker gatal was not affected at either the 10s efla primers were used as controls to verify primers and stage in the PLM (Figure 5F) or at 24 hpf in the intermediate RT-PCR, respectively. C and D, 3 ng of fli1 utrMO can effi- ciently knockdown GFP expression in fli1:gfp transgenic cell mass (data not shown), confirming that etsrp is not embryos (D). required for erythroid differentiation.18

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Figure 5. etsrp is essential for anterior hemangioblast formation in zebrafish embryos. ALM expression of scl was nearly absent in the etsrp mutant (A, 6/6). pu.1 expression in ALM was completely lost in etsrp mutants (B, 6/6). flk1 expression in ALM was absent in etsrp mutants (C, 5/5). PLM expression of scl was reduced in etsrp homozygous mutants (D, 5/5), whereas flk1 expression in PLM was completely lost in etsrp mutants (E, 6/6). gata1 expression in PLM was normal in etsrp mutants (F, 5/5). Dorsal view and anterior to the top; wt and y11 embryos were at 6s (A through F).

Knockdown of erg and fli1 Causes Angiogenesis 6H). In the controls, ISVs were arranged in very regular Defects and Downregulation of VE-Cadherin chevron shapes (Figure 6G). However, in the erg morphants, To further characterize the erg morphant phenotype, we ISVs often did not follow this chevron shape and grew either injected ergMOs into flk1:gfp-gata1:dsRed transgenic em- straight or reversely (Figure 6H). bryos to visualize the whole vasculature and blood circula- Fli1 and Erg belong to the same subfamily in the ETS tion. The hemorrhage phenotype was reproduced in Ϸ40% of family (supplemental Table I and Figure 1), and interestingly injected embryos. Confocal microscopy clearly showed that fli1 and erg MOs gave similar hemorrhage phenotypes in the the hemorrhage occurred in the head vasculature from 60 hpf head at around 60 to 72 hpf (supplemental Figure IV, A onward in erg spliceMO-injected embryos (Figure 6A through C). Moreover, fli1 knockout mice also showed a through 6F, arrows). In addition, we also found that inter- hemorrhage phenotype in the head.23,24 These data suggest somitic vessel (ISV) patterning was slightly affected (Figure that erg and fli1 are involved in establishing and/or maintain-

Figure 6. erg and fli1 are additively required for angiogenesis. A through I, Confocal microscopy at 72 hpf showed that hemorrhage occurred specifically in the head vasculature by using flk1:GFP/ gata1:dsRed (A through F, arrows) and slightly disorganized ISVs (H, red arrows), compared to control embryos (A through C and G). Double knockdown by ergMO and fli1MO gave more severely disorganized ISV patterning (K, 22/32, arrows). The dorsal longitudinal anastomotic vessel is indicated by dotted boxes. Lateral views with anterior toward left (A through I).

Downloaded from http://circres.ahajournals.org/ by guest on November 25, 2015 1152 Circulation Research November 7, 2008 ing vessel integrity. Because the 2 genes have similar expres- ysis further confirmed the likelihood of a genome-wide sion patterns in mice and also in zebrafish, these 2 ETS duplication. For example, the ets1-fli1 pair on chromosome transcription factors could either be equally or redundantly 18 has been duplicated and their duplicates, etsrp and fli1b required for angiogenesis and vessel remodeling after the are located on chromosome 16. In the most extreme case, erf endothelial cells have been specified (vasculogenesis). How- has 3 additional duplicates. These duplicated ETS genes, ever, double knockdown of both fli1 and erg gave a more together with their surrounding genes, often form duplicated severe phenotype than the individual knockdowns, namely a chromosome segments located on either the same chromo- more severe ISV patterning defect and higher penetrance of some or a different chromosome, further suggesting genome the hemorrhage (71% versus 41%, Figure 6I versus 6H and duplication occurred. Of note, their close neighboring genes data not shown). In the double knockdown, ISVs were more also have putative mammalian orthologs located near the disorganized and they did not grow properly toward the human counterpart genes, highlighting highly conserved syn- dorsal side of the embryo (Figure 6I, arrows). In addition, the tenies between zebrafish and mammals such as human and dorsal longitudinal anastomotic vessel did not develop prop- mouse. Taken together, our data and published data strongly erly in the double knockdown morphants (Figure 6I, dotted support the view that the zebrafish-specific ETS genes arose box), which contrasts with the individual knockdowns (Fig- from genome-wide duplication.26–29 ure 6H, dotted box). Thus, these data demonstrate that erg and fli1 are additively required for vessel integrity and A Role for Etsrp in Hemangioblast Formation angiogenic remodeling. The list of genes involved in both blood and endothelial VE-cadherin, a key regulator of endothelial intercellular lineages is expanding, although the existence of a bipotential junctions, has recently been shown, by chromatin immuno- progenitor, the hemangioblast, is still contentious. Scl, Lmo2 precipitation and functional assays, to be a direct target of erg and Gata2 have all been demonstrated to be critical for the in human umbilical vein endothelial cells.25 We, therefore, hemangioblast program.8,9,20,30–32 Using binding and transac- looked at VE-cadherin mRNA expression in the erg, fli1, and tivation assays, Pimanda et al have implicated Fli1 in the double morphants. At 24 hpf, VE-cadherin was equally control of scl and gata2 expression; however, no perturba- expressed in both controls and the morphants (data not tions were performed to support such a role in hemangio- shown). However, there was a reduction of VE-cadherin blasts.32 We show here that, although erg and fli1 are expression in both the trunk and the head at 50 hpf in the expressed in the hemangioblast population, loss of their morphants (supplemental Figure IV, D through G, arrows), functions in zebrafish has no effect on hemangioblast devel- just before the hemorrhage occurred, suggesting that down- opment, suggesting that they are either not required or regulation of VE-cadherin by erg and fli1 knockdown might possibly redundantly required. We have recently found that be responsible for the hemorrhage and ISV patterning defects fli1 is indeed essential for blood and endothelial development observed in the morphants. in Xenopus embryos and that gain-of-function of fli1 can induce many hemangioblast markers in zebrafish, consistent Discussion with a role for this family at the top of gene regulatory To initiate a comprehensive evaluation of ETS gene function hierarchy.33 However, even a quadruple knockdown of erg, in hematopoiesis and endothelial cells, we have systemati- fli1, fli1b, and fev (belonging to the same subfamily) did not cally characterized the ETS family in an organism ideally give a hemangioblast phenotype (supplemental Figure V), suited to functional testing, the zebrafish. Bioinformatic indicating that if redundancy is the explanation, it involves analysis identified a total of 31 complete ETS genes in the ETS factors outside this subfamily in zebrafish. zebrafish genome. Gene expression profiling showed that 12 Etsrp, an ETS domain protein discovered in zebrafish, has of them are expressed in blood and/or endothelial lineages been shown to be necessary and sufficient for the initiation of during embryogenesis. Functional analysis of erg and fli1 vasculogenesis.18 Here, we have clearly demonstrated, by demonstrated that they are required for angiogenesis, possibly using antisense MO knockdown and a genetic homozygous via direct regulation of VE-cadherin, a key regulator of mutant, that Etsrp is absolutely required for anterior heman- endothelial intercellular junctions. Combined use of antisense gioblast formation in zebrafish. This requirement could be knockdown and a genetic mutant clearly demonstrated that cell-autonomous, because etsrp is coexpressed with scl and etsrp plays an early role during hemangioblast development flk1 in the same cell population, the anterior lateral plate to drive both myeloid and endothelial differentiation. mesoderm, during embryogenesis. As etsrp is dispensable for erythroid differentiation, it suggests that the anterior heman- The ETS Family and Genome Duplication gioblast, only giving rise to myeloid and endothelial lineages, Our phylogenetic tree analysis indicates that the zebrafish and the posterior hemangioblast population, giving rise to genome contains 3 pairs of duplicated ETS genes, ie, elk4 and erythroid and endothelial lineages, are differentially pro- elk4l1, elf2 and elf2l1, spic and spicl1, and 1, erf, that has 3 grammed. It is highly possible that both cell-intrinsic differ- diverged copies (supplemental Table I and Figure 1). The ences and local environments account for the differential zebrafish-specific ETS gene duplicates may result from regulation of these 2 hemangioblast populations. Our studies zebrafish-specific gene duplication or mammalian-specific have identified etsrp as a critical regulator required for gene loss, or both. Several lines of evidence suggest that generation of the hemangioblasts that differentiate into my- whole-genome duplication and subsequent massive loss of eloid and endothelial lineages. Consistent with our finding, duplicated genes occurred in the teleost.26–29 Syntenic anal- mice carrying a mutation in Er71, the homolog of etsrp in

Downloaded from http://circres.ahajournals.org/ by guest on November 25, 2015 Liu and Patient ETS Genes in Zebrafish 1153 mammals, have very recently been shown to lack both whether activated forms of Erg or Fli1 could induce expres- hematopoietic and endothelial lineages.34 sion of the ligands (supplemental Figure VI, D through I). Sumanas et al recently reported the dependence of myeloid However, we found that vegf expression was unaffected and, gene expression on etsrp in the anterior hemangioblast even though fgf8 expression was upregulated, it was in the tail population in zebrafish embryos.35 They showed, by etsrpMO bud and not the vasculature. In contrast, both activated ETS knockdown that myeloid markers, such as lcp1, mpx, and factors, upregulated flk1 expression (supplemental Figure VI, pu.1, were reduced and that overexpression of etsrp could A through C), raising the possibility that they might sensitize upregulate lcp1 and pu.1 expression. However, anterior endothelial cells to VEGF signaling. expression of endothelial markers, such as flk1, was not In conclusion, by using systematic bioinformatic analysis analyzed. Together with our data, we can conclude that etsrp and gene expression profiling, we have comprehensively lies at the top of the genetic hierarchy programming both characterized the whole ETS gene family in zebrafish and blood and endothelium in the anterior hemangioblast popu- identified 12 of the 31 ETS genes as expressing in hemato- lation. A recent study by Xiong et al36 showing that etsrp can poietic or endothelial tissues. Functional assays have identi- partially rescue both hematopoietic and endothelial defects in fied 3 genes, etsrp, erg, and fli1, that are specifically required cloche embryos is consistent with our finding here. for hemangioblast development and angiogenesis. Our studies provide new insights into how tissue-specific differentia- Erg, Fli1, and Angiogenesis tion and maintenance is achieved by the ETS domain tran- Although erg is dispensable for the hemangioblast program, scription factors during hematopoiesis, vasculogenesis, and its specific expression in endothelial cells at later stages of angiogenesis. development suggests that it might be involved in angiogenesis. In addition, fli1 knockout mice showed a central nervous Acknowledgments system hemorrhage phenotype, suggesting that it is not We thank Aldo Ciau-Uitz for helpful comments. redundant in angiogenesis.23,24 In contrast, the very recently reported ErgMld mutation did not cause a vasculature pheno- Sources of Funding type in mice.37 A possible explanation is that this Mld2 This work was funded by the Medical Research Council (United Kingdom). mutation, which caused a substitution in the ETS DNA- binding domain but did not affect DNA binding affinity, Disclosures might not therefore be a null allele. Many ETS genes have None. been implicated in angiogenesis.11 Pham et al have demonstrated a combinatorial function for ETS transcription factors, References fli1, fli1b, ets1, and etsrp, in the developing vasculature in the 1. Davidson AJ, Zon LI. The ‘definitive’ (and ‘primitive’) guide to zebrafish zebrafish.22 The additive interaction of erg and fli1 discov- hematopoiesis. Oncogene. 2004;23:7233–7246. 2. Keller G. The Hemangioblast. In: Marshak DR, Gardner RL, Gottlieb D, ered in this study supports such combinatorial functions eds. Stem Cell Biology. New York: Cold Spring Harbor Laboratory Press; among ETS factors in vessel development. 2001:329–348. Downregulation of VE-cadherin, a key molecule involved 3. Sabin FR. Studies on the origins of blood-vessels and of red blood- in cell–cell junctions and cell survival, by erg and fli1 corpuscles as seen in the living blastoderm of chicks during the second day of development. Contrib Embryol. 1920;36:213–262. knockdown could explain why a hemorrhage occurred in both 4. Shalaby F, Rossant J, Yamaguchi TP, Gertenstein M, Wu XF, Breitman morphants. Targeted null mutation of VE-cadherin impairs ML, Schuh AC. Failure of blood-island formation and vasculogenesis in the organization of vascular-like structures in embryoid Flk-1 deficient mice. Nature. 1995;376:62–66. 39 5. 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Downloaded from http://circres.ahajournals.org/ by guest on November 25, 2015 Supplemental Table 1. Zebrafish ETS domain genes

Gene ID Gene Name Chromosome Description Subfamily Human Homologue

BC092935 ets1 18 ETS1 ETS ETS1 NM_001023580 ets2 10 ETS2 ETS ETS2 NM_001037375 etsrp 16 ETS1 related protein (Etsrp) ETS ER71 NM_001008616 erg 10 Transcripional regulator ERG ERG ERG NM_131348 fli1 18 Friend leukemia integration 1 transcription factor ERG FLI1 NM_001008780 fli1b 16 Friend leukemia integration 1b transcription factor ERG FLI1 XM_001341479 fev 9 ETS oncogene family ERG FEV NM_131587 gabpa 9 GA-binding protein alpha chain (GABP-alpha subunit) ELG GABPa NM_131832 tel1/etv6 6 translocation ets leukemia/ets variant gene 6 TEL TEL1/ETV6 XM_001340202 tel2 n.a. translocation ets leukemia 2 TEL TEL2 NM_131425 pea3 12 polyomavirus enhancer-binding activator 3 PEA PEA3 NM_131205 erm 6 ets related protein PEA ERM NM_001030182 er81/etv1 15 ets variant gene 1 PEA ER81/ETV1 NM_214720 etv5 9 ets variant gene 5 PEA ETV5 NM_131159 elf1 14 E74-like factor 1 ELF ELF1 NM_199219 elf2 1 E74-like factor 2 ELF ELF2 NM_001004116 elf2l1 14 E74-like factor 2-like 1 ELF ELF2 XM_684268 elf3 22 E74-like factor 3 ESE ELF3 NM_001039822 ehf 25 ets homologous factor ESE EHF XM_001343041 pdef 6 Prostate Epithelium-specific Ets Transcription Factor PDEF PDEF spleen focus forming virus (SFFV) proviral integration NM_198062 spi1/pu.1 7 oncogene spi1 SPI SPI-1/PU.1 spleen focus forming virus (SFFV) proviral integration NM_001004621 spic 4 oncogene spi-c SPI SPI-C spleen focus forming virus (SFFV) proviral integration XM_001341111 spicl 1 n.a. oncogene spi-c-like SPI SPI-C CAK04878 erf 19 ETS2 repressor factor ERF ERF XM_681312 erfl1 16 ETS2 repressor factor-like 1 ERF ERF NM_001044927 erfl2 19 ETS2 repressor factor-like 2 ERF ERF XM_689171 erfl3 16 ETS2 repressor factor-like 3 ERF ERF XM_691202 elk1 8 Ets-like protein 1-like 1 TCF ELK1 NM_001030108 elk3 4 Ets-like protein 3 TCF ELK3 NM_130930 elk4 11 Ets-like protein 4 TCF ELK4 XM_001332642 elk4l1 11 Ets-like protein 4-like 1 TCF ELK4

1 Supplemental Figure Legends

Figure S1. ets1, ets2, fev, gabpα, elf1, elf2, pu.1, elk4 are expressed in haematopoietic and/or endothelial cells during zebrafish embryogenesis. Scl, the known hemangioblast marker in zebrafish was used for comparison (A-D). ets1 (E-

H), ets2 (I-L), fev (M,N), gabpa (O,P), elf1 (Q,R), elf2 (S,T), pu.1 (U,V) and elk4

(W). Of note, elk4 is not expressed at 6s stage. Anterior to the left in all panels.

Figure S2, Expression of ets1, ets2, fev, gabpα, elf1, elf2, pu.1, elk4 was reduced or lost in cloche mutant embryos at 26 hpf. (A) ets1, (B) ets2, (C) fev, (D) gabpa, (E) elf1, (F) elf2, (G) pu.1 and (H) elk4. Note that endothelial or blood specific expression was reduced or absent in the cloche embryos (arrows). Anterior to the left and dorsal to the top.

Figure S3. etsrp but not fli1 and erg is essential for anterior hemangioblast formation in zebrafish embryos. ALM expression of scl was not affected in fli1- (B,

10/10) and erg- morphants (C, 10/10), but was significantly reduced in etsrp- morphants (D, 12/12). pu.1 expression in ALM was not affected in fli1- (F, 10/10) and erg- morphants (G, 10/10), but was completely lost in etsrp morphants (H, 10/10). flk1 expression in ALM was only slightly affected in fli1- (J, 6/10) and erg- morphants (K, 4/9), but was absent in etsrp morphants (L, 10/10). PLM expression of scl was not affected in erg- (N, 11/12) and fli1- (O, 10/10) morphants, but was reduced in etsrp- morphants (P, 10/10), whereas flk1 expression in PLM was slight affected in erg- (R, 4/10) and fli1- morphants (S, 6/11), but completely lost in etsrp morphants (T, 10/10). gata1 expression in PLM was normal in erg- (V, 9/9), fli1- (W,

2 10/11) and etsrp- (X, 10/10) morphants. Dorsal view and anterior to the top; controls

and MO-injected embryos were at 10s (A-X).

Figure S4. Knockdown of erg and fli1 causes downregulation of ve-cadherin.

(A,B, C) Live embryo at 3 dpf showed a hemorrhage in the head of erg morphants (B,

arrows, 24/58) and of the fli1 morphants (C, arrow, 8/24), with otherwise perfectly

normal overall morphology. Downregulation of ve-cadherin was seen in erg- (E,

arrows, 10/13), fli1- (F, arrows, 14/14) and the double morphants (G, arrows, 23/24)

at 50 hpf. Lateral views with anterior towards left (A-G).

Figure S5. Hemangioblast gene expression is unaffected in fev or quadruple (erg,

fli1, fli1b and fev) knockdowns. scl expression was normal in the fev morphant (B,

10/10) and the quadruple knockdown (C, 9/10) at 10s. Dorsal views, anterior to the top.

Figure S6. (A-I) Activated erg (B, 10/10) and fli1 (C, 10/12) upregulate flk1

expression, and also fgf8 (E, 10/12; F, 13/14) but not vegf (H, 10/10; I, 9/10).

Embryos are at 24 hours postfertilization (hpf), anterior to the left. CA-Erg and CA-

Fli1 were constructed by subcloning the full length inserts into pBUT2VP16 vector made in the lab 1. Fli1 and Erg coding sequences were fused at their C-terminus with the viral constitutively active transactivation domain, VP16. The constructs were verified by sequencing. Capped mRNAs (50-100 ng/embryo) for injection were synthesized using the mMessage mMachine kit according to the manufacturer’s protocol (Ambion, Austin, USA).

Supplemental References

1. Gering M, Rodaway AR, Gottgens B, Patient RK, Green AR. The SCL gene specifies haemangioblast development from early mesoderm. Embo J. 1998;17:4029-4045.

4 Liu_FigS1

1s 6s 10s 26h A B C D

scl E F G H

ets1

I J K L

ets2 6s 26h 6s 26h M N O P

fev gabpa Q R S T

elf1 elf2 U V W

pu.1 elk4 Liu_FigS2

A wt C wt F wt

clo clo clo

ets1 elf2 wt fev B G wt D wt

clo clo clo

gabpa pu.1 ets2 E wt H wt

clo clo

elk4 elf1 Liu_FigS3

Control fli1MO ergMO etsrpMO A B C D

scl E F G H

ALM pu.1 I J K L

flk1 M N O P

scl Q R S T

PLM flk1 U V W X

gata1 Liu_FigS4

A WT B ergMO C fli1MO

D WT E ergMO

ve-cadherin

F fli1MO G ergMO+fli1MO Liu_FigS5

A WT B fevMO C 4MOs

scl

Liu_Fig S6

control CA-Erg CA-Fli1 A B C

flk1

D E F

fgf8

G H I

vegf Genome-Wide Analysis of the Zebrafish ETS Family Identifies Three Genes Required for Hemangioblast Differentiation or Angiogenesis Feng Liu and Roger Patient

Circ Res. 2008;103:1147-1154; originally published online October 2, 2008; doi: 10.1161/CIRCRESAHA.108.179713 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2008 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571

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