eena Promotes Myeloid Proliferation through Stimulating ERK1/2 Phosphorylation in Zebrafish*

The EEN (extra eleven nineteen) gene is one of the fusion partners of mixed-lineage leukemia, located on chromosome 19p13. Here we cloned two een genes (designated as eena and eenb) in zebrafish, which are assigned to linkage groups 8 and 2, respectively. Whole-mount in situ hybridization assay showed that eena and eenb have overlapping but distinct expression patterns during embryogenesis. Ubiquitous or targeted overexpression of eena, but not eenb, into wild-type or transgenic embryos (green fluorescent protein-labeled myeloid progenitors) induced a significant proliferation and ectopic distribution of myeloid progenitors in the yolk sac. Using a morpholino antisense gene knockdown approach, we showed that the number of myeloid progenitors and their downstream mature myelomonocytic cells was significantly decreased in the eena- deficient embryos. Mechanistically, overexpression of eena selectively stimulated ERK phosphorylation and increased the level of transcription factor c-Fos in vitro and in vivo, whereas eena lacking the Src homology 3 domain completely abolished these effects. Furthermore, a MAPK/ERK kinase (MEK) inhibitor, PD98059, blocked the eena-induced cell proliferation and activation of ERK signaling. The results suggest that eena plays an important role in the development of the myeloid cell through activation of the ERK pathway and may provide a valuable reference for future studies of the role of EEN in leukemogenesis.

The EEN (extra eleven nineteen) gene is one of the fusion partners of mixed-lineage leukemia, located on chromosome 19p13. Here we cloned two een genes (designated as eena and eenb) in zebrafish, which are assigned to linkage groups 8 and 2, respectively. Whole-mount in situ hybridization assay showed that eena and eenb have overlapping but distinct expression patterns during embryogenesis. Ubiquitous or targeted overexpression of eena, but not eenb, into wild-type or transgenic embryos (green fluorescent protein-labeled myeloid progenitors) induced a significant proliferation and ectopic distribution of myeloid progenitors in the yolk sac. Using a morpholino antisense gene knockdown approach, we showed that the number The EEN (extra eleven nineteen) was originally cloned from a case of infant acute myeloid leukemia M5 subtype with a chromosomal translocation at the breakpoint of t(11;19) (q23; p13), in which EEN is fused with myeloid-lymphoid leukemia (MLL) (1). EEN is the founding member of the endophilin Src homology 3 (SH3) 3 domain-containing protein family, which includes two subfamilies, endophilins A and B. In mammals, the A subfamily consists of three members, endophilin A1 (EA1), A2 (EEN/EA2), and A3 (EA3), whereas the recently identified B subfamily includes B1 (also called SH3GLB1) and B2 (also called Bif1/SH3GLB2) (2)(3)(4). They all have an identical domain structure, consisting of the following domains: a relatively conserved N-terminal BAR (BIN/amphiphysin/Rvsp) domain; a conserved C-terminal region containing the SH3 domain, which shared 70% homology with the adaptor protein GRB2; and a variable region in between, which, despite its lack of extensive sequence homology, containing clusters of proline and other turninducing amino acids, suggesting that it might act as a flexible joint linking the BAR domain and SH3 domain (5,6).
Most of the studies of the endophilin family have been focused on their functions in endocytosis in neuronal cells. The members of this family have been shown to form complexes with several proteins, such as amphiphysin, synaptojanin, and dynamin, all of which are implicated in presynaptic vesicle trafficking (7)(8)(9). They are also binding partners of the G proteincoupled ␤ 1 -adrenergic receptor (10). In addition, endophilin-Ca 2ϩ channel complex is required for synaptic vesicle endocytosis (11). Recent studies demonstrated that EEN could bind BPGAP1 and is involved in the activation of epidermal growth factor receptor endocytosis and ERK1/2 signaling (12). EEN is also engaged in Ras signaling and cellular transformation through binding EBP (13). EEN is the only member in the family expressed in hematopoietic tissues, including bone marrow and fetal liver (1). However, as a myeloid leukemia-associated gene, the role of EEN in myeloid development and leuke-mogenesis has not been fully explored. In mammals, it is relatively difficult to access early developmental processes such as primitive myelopoiesis because they occur within the mother's body.
Zebrafish has become an ideal organism for the study of normal and abnormal hematopoiesis. Hematopoiesis has been shown to be very similar to that of higher vertebrates, and homologues of a large number of genes involved in mammalian hematopoiesis have been identified in the zebrafish (14,15). In zebrafish, as in mammals, primitive and definitive hematopoieses arise successively in the intermediate cell mass (ICM) and the ventral wall of the dorsal aorta in the developing embryos (16).
In this study, we have cloned two zebrafish orthologues of the mammalian EEN gene, termed eena and eenb, and investigated the evolutionary relationship of these genes and expression patterns during early embryonic development. Our results suggest that zebrafish eena and eenb result from a gene duplication event at the een locus and that this may lead to their functional divergence. We also showed that eena and eenb were expressed maternally and ubiquitously, implying a role in early embryonic development. However, at the later stages, eenb was only observed in hatching gland, whereas eena expression was still ubiquitous and richly detected in the ICM at 22 h post-fertilization (hpf). Furthermore, we focused on the function of eena and eenb in myeloid development in vivo because EEN has been found fused to MLL in acute myeloid leukemia. Here, we showed that knockdown of Eena caused a significant suppression in the myeloid cells development, whereas overexpression of eena, but not eenb, led to an increased number of myeloid progenitors through stimulating ERK phosphorylation and the subsequent up-regulation of zebrafish c-fos mRNA. In addition, the N-terminal part of eena, which lacked the SH3 domain, acted as a potential dominant negative mutant and dramatically abolished these effects. Our data indicate that eena regulates myeloid development through activation of the ERK signal pathway, which provides a novel insight into the role of EEN in leukemogenesis caused by MLL-EEN.

EXPERIMENTAL PROCEDURES
Zebrafish Strains-Zebrafish strains were maintained at 28.5°C as described by Westerfield (17). Transgenic line TG(zpu.1:EGFP) was kindly provided by Drs. John Kanki and A. Thomas Look at the Dana-Farber Cancer Institute (18). Embryos were staged as described by Kimmel et al. (19). Developmental stages refer to hours (hpf) or days (dpf) post-fertilization.
Cloning and Plasmids Construction-Zebrafish eena and eenb genes were identified based on homology to human EEN. The specific primers were designed according to genomic sequence in the UCSC (University of California, Santa Cruz) data base (see primer sequences in supplemental Table 1). The amplified PCR product was purified and subcloned into pCS2 ϩ for in vitro synthesis of capped mRNA for microinjection. Then eena and eena⌬SH3 fragments were digested with BamHI and XhoI sites from pCS2 ϩ vector and cloned into an I-sceI-containing plasmid vector between a 9.0-kb zebrafish pu.1 (zpu.1) promoter sequence (18) and an SV40 polyadenylation site, which resulted in the constructs of pu.1-eena and pu.1-eena⌬SH3 for microinjection, respectively. The plasmid of pEGFP-EEN was constructed by Liu et al. (20).
Multiple Sequence Alignment, Phylogenetic Trees, and Gene Structure Analysis-Protein sequences for endophilins of zebrafish and other species were obtained from GenBank TM to determine the phylogenetic relationships. 4 Multiple alignments of amino acid sequences for een genes were obtained by using the ClustalX 1.83 alignment program and subsequently analyzed with the Neighbor Joining method to construct a phylogenetic tree by using Mega3.1 software (21,22). A bootstrap test was performed with 1,000 repetitions.
Genomic sequences of human EEN and zebrafish eena and eenb were obtained by searching the corresponding genome sequence data bases, which have complete information on loci and putative exon arrangement of een genes, as well as on transcript and protein sequences. The exon and intron arrangements of zebrafish eena and eenb genes were defined based on the information deposited in the DNA data base, and checked by amino acid sequence alignment of the corresponding cDNAs.
Identification of Syntenic Relationship-The Sanger Zv6 zebrafish genome assembly was searched for sequences with similarity to human EEN protein using tBLASTn. We applied the reciprocal best "hit" method (23) for syntenic analysis and compared genes neighboring the zebrafish een to the genes neighboring the human EEN. The map positions of the corresponding human genes were obtained on line.
Whole-mount in Situ Hybridization-To localize eena and eenb, as well as c-fos mRNAs in zebrafish embryo, whole-mount in situ hybridization was carried out. pCS2 ϩ containing eena or eenb 3Ј-UTR (352 and 150 bp, respectively) was utilized to generate antisense RNA probes for eena and eenb using digoxigenin-11-uridine 5Ј-triphosphate (Roche Applied Science). Embryos were fixed in 4% paraformaldehyde at the stages indicated, and whole-mount in situ hybridization was performed as described (24). These probes were detected with alkaline phosphatase-conjugated antibodies (1:5,000) (Roche Applied Science) using the substrates 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (purple color) (Vector Laboratories, Burlingame, CA). At least 10 embryos were used at each stage, and all showed similar expression patterns. After staining, embryos were mounted in 3% methylcellulose and captured under the Nikon SMZ1500 microscope equipped with a Nikon DXM1200F digital camera and ACT-1 software.
Microinjection of Zebrafish Embryos-The expression vectors pCS2 ϩ -eena and pCS2 ϩ -eenb were linearized with NotI and transcribed in vitro with SP6 RNA polymerase in the presence of m7G (59) scripts and were microinjected into one-cell stage embryos. Embryos were microinjected with 100 pg of EGFP mRNA or diethyl pyrocarbonate water as controls, using an air pressure injector and glass capillaries. To specifically express Eena in myeloid progenitors, the pu.1-eena and pu.1-eena⌬SH3 plasmids were prepared with endotoxin-free midiprep kit (Promega) and injected into TG(zpu.1:EGFP) embryos with 100 pg of DNA, 0.5ϫ I-sceI buffer and 0.5 units/l I-sceI meganuclease (New England Biolabs). Injection experiments were performed three times. EGFP expression was directly observed under a fluorescent microscope (Zeiss Lumar V12 stereomicroscope equipped with an AxioCam MRC5digital camera and Axio-Vision Rel.4.5 software). The morpholino antisense oligonucleotide (MO) targeted against eena was synthesized by Gene-Tools LLC (Philomath, OR). The sequence was as follows: 5Ј-CTTCTTTAAGC-CCGCCACGGACATC-3Ј. The 5-base mismatch MO sequence was 5Ј-CTTgTTTAAcCCCcCCAaGGAgATC-3Ј. MOs were diluted to obtain 1 mM stock solution in 1ϫ Danieau buffer and were microinjected at a volume of 2 nl into one-cell stage embryos.
Cell Culture and Transfection-NIH3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum (Invitrogen), 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin at 37°C in a 5% CO 2 humidified incubator. For transfection, NIH3T3 cells were seeded on sterile glass coverslips (BDH) in 6-well plates (BD Biosciences) and grown for 24 h before transfection. Transfections were performed using 2-6 g of plasmid cDNA and SuperFect transfection reagent (Qiagen, Germany) according to the manufacturer's instructions. Cells were harvested for RT-PCR at 24 h after transfection.
RT-PCR and Immunofluorescence-After injection or transfection, embryos or cells were harvested at 22 hpf or 24 h, respectively, and lysed in TRIzol (Invitrogen), followed by standard total RNA extraction. The expression of nuclear transcription factors was detected by serial dilution semiquantitative RT-PCR assay using indicated primers (supplemental Table 1).
Embryos were fixed after 22 hpf with 4% paraformaldehyde at 4°C overnight and perforated with 0.3% Triton X-100 for 10 min, then blocked with 10% fetal bovine serum for 1 h, followed by incubation with anti-EGFP antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-phosphohistone H3 antibody or anti-c-Fos antibody (Cell Signaling Technology, Beverly, MA) at 4°C overnight. The embryos were incubated with fluorochrome-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies for 1 h (Molecular Probes, Invitrogen).
Statistical Analysis-Embryos were mounted in 95% glycerol, and the number of cells in the anterior yolk sac was counted under the Zeiss microscope (80ϫ). Data were reported as mean values Ϯ S.E. and statistically compared using oneway analysis of variance followed with least significant difference or Student's t test. Statistical significance was accepted when p Ͻ 0.05.

RESULTS
Characterization and Phylogenetic and Syntenic Analyses of the Zebrafish een Orthologues-After searching the zebrafish genome data base, we identified two een genes, which we refer to as eena and eenb, respectively. The zebrafish eena cDNA (NM_201497) consisted of 1,662-bp and encoded a predicted protein of 351 amino acids (ϳ38 kDa). And eenb cDNA (NM_200301) was 1,460-bp and encoded a predicted protein of 365 amino acids (ϳ39 kDa) (Fig. 1A). At the nucleic acid level, within the coding region, the cDNAs of zebrafish eena and eenb were 72% identical, whereas within the 3Ј-UTR, the similarity dropped to 8%.
We then aligned the complete cDNA coding sequence of eena and eenb with the genomic sequence to reveal the exonintron structure. eenb spans 30.89 kb in the genome and contains 10 exons, each aligning with the 10 human exons. In contrast, eena spans 37.49 kb in the genome and has retained only 9 exons, with exons 1-8 aligning with human exons 1-8 and exon 9 aligning with human exon 10, suggesting that eena is missing a sequence homologous to human exon 9, which encodes a variable region acting as a joint linking the BAR and SH3 domain. Comparisons of exon lengths of the two zebrafish een genes with the human EEN indicated a high degree of crossspecies conservation (supplemental Table 2). We next examined the relationship of eena and eenb amino acid sequences in zebrafish to mammalian endophilin family members in more detail. Results suggested that the highest BLASTP bit scores were with EEN (supplemental Table 3). In the BAR and SH3 domains, zebrafish Eena and Eenb were showed a very good conservation in comparison with mammalian EENs (supplemental Fig. 1). Using the neighbor-joining method, we constructed a phylogenetic tree with several known tetrapod sequences, including human, mouse, chick, and Xenopus (Fig. 1B). We were also able to identify two candidate copies of predicted een in the fugu, medaka, and stickleback genomes.
To more precisely confirm the evolutionary conservation of EEN, we investigated the distribution of genes located adjacent to the EEN locus on chromosomes in zebrafish and human genomes. As shown in Fig. 1C, four genes, HLRC1, CREB3L3, STAP2, and SEMA6B, were located close to each other on human chromosome 19 near EEN, and their zebrafish orthologues can be found in the vicinity of eena on linkage group 8, indicating that zebrafish eena was an orthologue of human EEN. Human genes MBD3A, SF3A2, CHICO, CCDC94, and STAP2 close to EEN also have their zebrafish orthologues on linkage group 2 near eenb, supporting the idea that eenb was also an orthologue of human EEN.
Taken together, it shows that the two zebrafish copies arose from a duplication event in the teleost lineage after the divergence from the tetrapod lineage but before the teleost radiation. This observation was consistent with the notion of a whole genome duplication in the teleost lineage (26). Therefore, the above results strongly support that zebrafish eena and eenb are co-orthologues of the mammalian EEN.
eena and eenb Expression during Embryogenesis-The temporal and spatial expression patterns of eena and eenb were investigated in zebrafish embryos from 0.75 to 120 hpf by in situ hybridization using digoxigenin-labeled antisense RNA probe. As shown in Fig. 2, strong signals were detectable in the blastomeres of the two-cell stage and the sphere period (4 hpf), indicating that both eena and eenb transcripts were maternally derived and suggesting a role in early embryonic development. Although the eena and eenb genes exhibited similar patterns of expression during the early stage, differential expression of the een genes was observed after the 18-somite stage (18 hpf). At that stage, high levels of eena transcripts were still strongly detected throughout the entire embryo, predominantly expressed in neural tissues (brain, spinal cord, and eye) and skeletal muscle, identical to that of EEN in mice described previously (27) (Fig. 2A). At 22 hpf, hematopoietic expression of eena was observed in the posterior ICM ( Fig. 2A, red arrow in inset), a region analogous to the blood island of the yolk sac in mammals (28). By 28 hpf, very strong expression was also seen in the brain and weaker staining in the tail and the trunk. After 2 dpf, expression decreased and became mostly restricted to the notochord and optic capsule ( Fig. 2A, black arrow) and then became undetectable by 5 dpf.
By comparison, we found that eenb was restricted to the hatching gland during 18 and 22 hpf (Fig. 2B, black arrow), which has not been reported previously, then gradually disappeared after 30 hpf (data not shown). By 3-5 dpf, eenb expression was no longer observed. The eenb mRNA was not detected in ICM.
To control for specificity, embryos of each stage were also probed in parallel with a sense probe, which gave no signal at any stage (data not shown). Our data provide the first insight into the embryonic expression profile of een genes in zebrafish.
een mRNA Overexpression in Wild-type or Transgenic TG(zpu.1:EGFP) Zebrafish Embryos-It has been shown that eena was expressed in the ICM region, suggesting its possible role in zebrafish hematopoiesis. It was therefore interesting to explore the possible function of een on hematopoietic cells. We first microinjected synthetic zebrafish een mRNA into wild-type embryos at the one-cell stage and examined the hematopoietic marker expression, including scl (hematopoietic progenitors), gata1 (primitive erythroid lineage), and pu.1 (primitive myeloid precursors) (29,30).
As shown in supplemental Fig. 2, A and B, no obvious morphological changes were detected, and the expression of scl and gata1 was similar in 100 pg of either EGFP-, eena-, or eenb-injected embryos. However, compared with the EGFP-injected or eenb-injected embryos, injection with eena mRNA induced ectopic pu.1 expression (Fig. 3, A and B, red arrows and insets) and caused an increase in the number of pu.1-expressing myeloid cells at 14 and 22 hpf embryos (Fig. 3, A and B). To quantify this effect, we measured the number of pu.1-positive cells in the yolk sac. At 22 hpf, the mean number of control EGFP-injected or eenb-injected was 57.1 Ϯ 2.92 (n ϭ 83, pooled from three independent experiments) and 53.6 Ϯ 2.36 (n ϭ 91), respectively, whereas 40% eena-injected embryos reached to 80.1 Ϯ 3.54 (n ϭ 103). This value was significantly higher than the other two (p Ͻ 0.001) (Fig. 3C).
The above results showed that eena overexpression affected early myeloid cells but not erythroid cells. To investigate whether Eena function is limited to the myeloid cells, we also examined the expressions of two non-myeloid markers krox-20 (ectoderm hindbrain-specific marker) (31) and myoD (mesodermal muscle-expressing marker) (32), after overexpression of eena or eenb mRNA. Like scl and gata1 expression, expression of krox-20 and myoD was not affected in een overexpression embryos (supplemental Fig. 2, C and D).
To further verify that the increased cells were indeed pu.1positive cells but not from trans-differentiation of other cell types, we employed the well characterized transgenic line TG(zpu.1:EGFP), in which EGFP was expressed in the myeloid progenitors under the zpu.1 promoter (18), and injected capped mRNAs encoding Eena or Eenb into embryos. As expected, about 43% eena-overexpression embryos (n ϭ 108) significantly increased the number of pu.1ϩ/EGFPϩ cells to about 1.5-fold higher than that of the control (n ϭ 101) (Fig. 3, D and E, the mean number of pu.1ϩ/EGFPϩ cells was 68.7 Ϯ 4.35 and 40.5 Ϯ 2.53, respectively), and the cells were also exhibited abnormal distribution in the yolk sac (Fig. 3D,  white arrowheads). In addition, to test if mammalian EEN and zebrafish Een proteins are functionally conserved, we injected human EEN mRNA into TG(zpu.1:EGFP) embryos. Overexpression of human EEN resulted in the significant expansion of pu.1ϩ/ EGFPϩ cells, similar to the effect of zebrafish eena overexpression (supplemental Fig. 3, white arrowheads). These results suggest that Eena and EEN proteins are functional orthologues.
Morpholino Knockdown of eena Results in Significant Suppression of Myeloid Cells-To determine whether eena is required for zebrafish myeloid development, we first determined whether the eena MO was able to inhibit the synthesis of endogenous Eena protein by Western blot analysis. Microinjection of eena MO at the dose of 8 and 16 ng per embryo resulted in a dose-dependent reduction of Eena expression (Fig. 4A,  lanes 3 and 4), whereas injection of a 5-bp mismatch control MO demonstrated an appropriate levels of Eena protein as wild-type control (Fig. 4A, lanes 1 and 2).
Next, we assessed the myeloid development in Eena-knockdown embryos using several myeloid markers pu.1, l-plastin, and mpo (29). Compared with the control (Fig. 4B, upper panels), abrogation of Eena protein led to a severe loss of pu.1 in the anterior lateral plate mesoderm, where the primitive myeloid cells developmentally originates (14) (Fig. 4B, bottom panels). Nearly 90% of Eena-knockdown embryos at 22 hpf showed no or a few pu.1-positive cells in this area (n ϭ 100). Consequently, the number of cells expressing l-plastin (primitive macrophage marker) was also dramatically reduced in 85% of Eena morphants at 22 hpf (n ϭ 92, Fig. 4C). The mean number of l-plastin-expressing cells was about 83.2 Ϯ 3.35 versus 136.6 Ϯ 2.96 in Eena-knockdown and control embryos, respectively (Fig. 4, C  and F). Consistently, mpo-expressing granulocytes were nearly completely absent in 80% of Eena-deficient embryos within anterior yolk sac and posterior ICM (Fig. 4D). The number of mpo-expressing cells in the anterior yolk sac was 46.2 Ϯ 3.57 versus 16.6 Ϯ 4.13 in control and eena morphants, respectively (Fig. 4F, n ϭ 60). The result is consistent with previous observation that the l-plastin and mpo-expressing cells were absent or dramatically decreased in the pu.1-deficient embryos (33). In contrast, expression of scl was unaffected by Eena deficiency (Fig. 4E).
eena Enhances pu.1-expressing Myeloid Cells Expansion via Its SH3 Domain-The I-sceI was originally utilized in Medaka to induce stronger promoter activity in F0 founder and to decrease nonspecific expression and increase the stable integration of transgene into the genome (34). For example, it has been used to study the BMP signaling under the control of zlmo2 promoter in zebrafish hematovascular development, which decreased the mosaicism substantially caused by transient transgenic expression and acquired a specific expression pattern in ϳ30% of the injected embryos (35). Taking advantage of the I-sceI approach, we constructed eena expression vector driven by zpu.1 myeloid promoter flanked by I-sceI meganuclease recognition sites, named as pu.1-eena, to further determine whether eena indeed act as activator of myeloid cells expansion. As a control, the SH3 domain-deleted eena was also made as described in Fig. 5A, and referred to as pu.1-eena⌬SH3, to determine the function of SH3 domain in myeloid development.
eena Enhances Myeloid Proliferation through Stimulating ERK1/2 Phosphorylation-A number of studies have shown that activation of MAPK is involved in a diverse set of responses affecting cell proliferation and differentiation, adaptation to environmental stress, and apoptosis (37,38). To investigate whether the regulation of myeloid cell proliferation by Eena during embryonic development is through the cross-talk with MAPK pathway, Western analysis was first performed. As shown in Fig. 5E, at 22 hpf, the level of phospho-ERK was significantly up-regulated by about 5-6-fold in pu.1-eenainjected embryos compared with controls, whereas overexpression of eena⌬SH3 showed normal levels of ERK phosphorylation. In contrast, eena had no effects on the activation of phospho-JNK and phospho-p38 (Fig. 5E). These data suggested that overexpression of Eena might induce proliferation and migration of pu.1-positive cells via selective activation of the ERK signal pathway.
Effects of eena on Activation of Nuclear Transcription Factors-It has been reported that, in the nucleus, c-Fos, c-Myc, and c-Jun are probably major downstream targets in the MAPK pathway (39). To determine whether these transcription factors were influenced, we used a combination of in vitro and in vivo experiments. In vitro, NIH3T3 cells were transfected with the vector control or expression plasmid encoding human wild-type EEN. Cells overexpressing the EEN showed 2-3-fold higher level of murine c-Fos mRNA than that of control vectors (Fig. 6A). On the other hand, the levels of c-Jun mRNA was not changed (Fig. 6A). To test whether zebrafish eena can produce a similar effect on c-Fos expression in cultured mammalian cells, we employed Western blot assay to compare c-Fos expression level in NIH3T3 cells transfected with pEGFP, pEGFP-eena, or pEGFP-eenb plasmid, respectively. As shown in the Fig. 6B, zebrafish eena highly induced c-Fos expression. In vivo, serial dilution semiquantitative single embryo RT-PCR was also performed to analyze the mRNA expression of zebrafish c-fos (zc-fos), c-jun (zc-jun) and c-myc (zc-myc). Consistently, the results showed that the levels of zc-fos mRNA were ϳ2-fold higher in eena-injected embryos than that of control or eena⌬SH3-injected embryos. Conversely, the expressions of zc-jun and zc-myc mRNAs were not changed (Fig. 6C).
The above result implied c-Fos is an important factor for myeloid cells proliferation and led us to further examine its expression in these cells of 22-hpf embryos after injected with pu.1-eena, using zc-fos as probe of whole-mount in situ hybridization. In the control embryos, only a diffuse staining was observed in the head and the trunk. After injected with pu.1-eena, however, a high level of zc-fos mRNA expression was detected in the individual pu.1positive myeloid cells (Fig. 6D, left panel, black arrowheads). To verify these hybridization results, immunofluorescence assay was used to determine the EGFP and c-Fos protein in pu.1positive cells with specific anti-EGFP (Fig. 6D, green channel) and anti-c-Fos (red channel) antibodies in vivo. In agreement with observations, c-Fos was also up-regulated in the pu.1ϩ/ EGFPϩ cells (Fig. 6D, middle and right   c-Fos and quantitating the number of pu.1ϩ/EGFPϩ cells in the yolk sac. The results showed that eena-induced phosphorylation of ERK and up-regulation of zc-fos transcripts were significantly blocked by PD98059. Western blot analysis using a specific antibody against zebrafish een protein showed that the levels of Eena protein was similar in pu.1-eena-injected embryos treated with and without PD98059 and was about 1.5-2-fold higher than endogenous expression level (Fig. 7A). Quantification assay confirmed that the number of pu.1ϩ/ EGFPϩ cells was strongly increased in ϳ45% of the Eena-overexpressed embryos but was reduced after treatment with PD98059 (Fig. 7B). Therefore, we propose a model where Eena induced myeloid cells proliferation via the ERK-c-Fos signaling (Fig. 7C).

DISCUSSION
More than 30 different genes have been shown to be fused with the MLL gene, and it is still unclear whether these fusion partners act through a loss-of-function or gain-of-function mechanism in leukemogenesis, and there are data supporting both scenarios (41,42). Several observations indicated that these fusion partners play essential roles in determining the oncogenic capacity. For example, chimeric mice generated from embryonic stem cells carrying a single MLL-AF9 "knock-in" allele succumbed to acute myeloid leukemia (AML), whereas mice generated from MLL heterozygote embryonic stem cells remained disease-free (43). Furthermore, our previous work demonstrated that the leukemia-associated fusion protein AML1-ETO could aberrantly transactivate the EEN gene, and higher levels of EEN transcripts were observed in primary M2 leukemia cells carrying the AML1-ETO fusion gene (44). Therefore, fully understanding the function of EEN will undoubtedly contribute to further mechanistic insights into MLL-EEN-mediated or AML1-ETO-associated leukemogenesis.
It has been well documented that there is not a one-to-one correspondence between zebrafish and human chromosomes, and several genes unique in mammals have two or more copies in teleost fish (45,46). In this study, we have identified two highly similar zebrafish een genes and investigated their spatiotemporal expression and the possible functions of these proteins in hematopoiesis. Several lines of evidence suggest that the two een genes are co-orthologues of the mammalian EEN. The conserved synteny suggests that portions of these two zebrafish chromosomes are derived by genome duplication or by segmental duplication of a chromosome sharing a common ancestor with human chromosome 19. Changes in gene order within this conserved synteny support the frequent occurrence of inversions and other intrachromosomal rearrangements in these regions because of the divergence of teleost and tetrapod ancestors (47).
After a gene is duplicated, one of the copies usually accumulates nonsense mutations and becomes a pseudogene, although sometimes both paralogue copies are retained in the genome (48). Zebrafish with partial genome duplication had become a critical model system to study the functional divergence of duplicated genes (49). The finding that both of the duplicated een genes are preserved in zebrafish also raised an important question regarding their functional relationship. In this study, we first explored the possible difference in the spatio-temporal expression patterns of the zebrafish eena and eenb genes. At 18 hpf, expression of eenb was restricted to the hatching gland, whereas expression of eena was still detected ubiquitously and expressed in hematopoietic cells. Therefore, the differential expression patterns of the zebrafish eena and eenb genes imply a hierarchical subfunctionalization that may account for the retention of both the duplicated eena and eenb genes in the zebrafish genome and also indicate that eena may have more general and/or different functions from eenb in hematopoietic cells. Previous data suggest that EEN is the only member in the endophilin family expressed in hematopoietic cells capable of transforming NIH3T3 and enhancing the self-renewal capacity and proliferative potential of clonogenic hematopoietic progenitors in vitro (1,44). Our previous study also showed that the HL-60 cells transfected with pEGFP-EEN grew much faster than the control cells (44). Consistently, in this study, we found that knockdown of zebrafish eena resulted in the significantly decreased number of myeloid cells. Furthermore, targeted expression of eena led to proliferation of pu.1-positive myeloid cells in vivo. Evidence suggested that overexpression of cortactin in NIH3T3 fibroblasts and endothelial cells resulted in an enhanced cell migration and invasive potential (50,51). It has been shown that cortactin is found to interact with dynamin (52), which is also a target for EEN (6,9). Likewise, we found that targeted overexpression of zebrafish Eena led to abnormal migration of pu.1-positive myeloid cells in the yolk sac. Therefore, it raises the possibility that Eena may couple dynamin to cortactin to regulate myeloid cell migration in zebrafish.
As described previously, endophilin family members encode proteins with highly conserved SH3 domains that share the highest homology to the SH3 domain found in Grb-2/SE-5, a member of the family of adaptor proteins conserved from fruit fly to mammals (53-55). These adaptor proteins couple recep-tor tyrosine kinases to the Ras signaling pathway and participate in both normal and pathogenic pathways through the interaction of their SH3 domains with proline-rich peptide sequences of the target proteins (56). Mutations in the SH3 domain of the Grb2/SE-5 protein abolish its binding to downstream effectors and result in a loss of function (54,55). Based on the high sequence homology between the SH3 domains of EEN and Grb-2 proteins, we speculated that eena might play a role in mitogenic signaling via its SH3 domain. Coincident with the hypothesis, our results showed that Eena overexpression specifically increased the phosphorylation of ERK1/2 and promoted the transcription of its downstream target zc-fos. However, Eena lacking the SH3 domain completely abolished these effects. MEKs are the only known upstream activators of ERKs, and PD98059, a specific MEK1/2 inhibitor, partially blocked the phosphorylation of ERK1/2 and subsequent down-regulation of c-Fos and also inhibited the myeloid cell proliferation induced by eena (Fig. 7). Therefore, our results clearly demonstrated that eena overexpression led to the proliferation of myeloid cells mainly via the ERK-c-Fos signal pathway and that the SH3 domain plays an important role. The AP-1 (activator protein-1) complex of transcription factors, which is composed of the Jun and Fos proteins, is related to many oncogenic signal transduction pathways (57). Our previous results indicated that overexpression of EEN transactivates AP-1 in the NIH3T3 and HL60 cell lines, as well as in COS7, K562, and U937 cells by luciferase reporter assay (44). In this study, we showed that EEN selectively induced the up-regulation of c-Fos in NIH3T3 cells again. In addition, we provided further evidence that overexpression of eena did stimulate up-regulation of zc-fos mRNA but not zc-jun in vivo, suggesting that the evolutionarily conserved EEN-ERK-c-Fos signal pathway played a significant role in the proliferation of myeloid cells.
Accumulating evidence suggests that endocytosis of certain receptor tyrosine kinases and G protein-coupled receptors is obligatory for MAPK activation (58 -62). For example, epidermal growth factor receptor-mediated MAPK activation is inhibited by a mutant version of dynamin that blocks endocytosis (60,61). Given the involvement of EEN in endocytosis (8,9,11,12), along with the findings that overexpression of eena activate the MAPK pathway and induce myeloid proliferation, our studies raise an intriguing question as to whether eena may serve as a potential link between the endocytic machinery and the MAPK signal transduction machinery that need to be addressed.