Antisense Targeting of CXXC Finger Protein 1 Inhibits Genomic Cytosine Methylation and Primitive Hematopoiesis in Zebrafish*

CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is a component of the Set1 histone methyltransferase complex. Mice lacking CFP1 suffer a peri-implantation lethal phenotype, and CFP1-deficient embryonic stem cells are viable but unable to differentiate and exhibit a 60-80% decrease in genomic cytosine methylation. A zebrafish homolog of CFP1 has been identified, is ∼70% similar to murine CFP1, and is widely expressed during development. Zebrafish embryos treated with a zCFP1 antisense morpholino oligonucleotide had little or no circulating red blood cells and exhibited abnormal yolk sac morphology at 48 h post-fertilization. Many of the antisense-treated zebrafish also exhibited cardiac edema, and 14% were dead at 24 h post-fertilization. Morphant zebrafish also exhibited elevated levels of apoptosis, particularly in the intermediate cell mass, the site of primitive erythropoiesis, as well as aberrations in vascular development. Genomic DNA isolated from morphant embryos exhibited a 60% reduction of global genomic cytosine methylation. A similar phenotype was observed with an independent zCFP1 antisense morpholino oligonucleotide, but not following injection of an unrelated control oligonucleotide. The morphant phenotype was rescued when mRNA encoding murine CFP1 was co-injected with the antisense oligonucleotide. Genomic data base analysis reveals the presence of a second version of zebrafish CFP1 (zCFP1b). However, the morphant phenotype observed following specific depletion of zCFP1 indicates that these related genes have nonredundant functions controlling normal zebrafish hematopoiesis and epigenetic regulation. These findings establish the importance of CFP1 during postgastrulation development.

Deletion of the CXXC1 gene in mice results in a peri-implantation embryonic lethal phenotype (14), and embryonic stem (ES) cells lacking CFP1 fail to differentiate upon removal of leukemia inhibitory factor (15). CXXC1 Ϫ/Ϫ ES cells also exhibit a 3-fold increase in apoptosis and a 60 -80% loss of global genomic cytosine methylation, including reduced cytosine methylation within repetitive elements, single copy genes, and imprinted genes (15). In addition, CFP1 is a component of the mammalian Set1 histone H3-Lys 4 methyltransferase complex, and co-localizes to nuclear speckles with both Set1 and human trithorax (2,16). Absence of CFP1 in ES cells leads to perturbed patterns of histone modifications consistent with reduced levels of heterochromatin. Hence, CFP1 is an important regulator of both cytosine methylation and histone methylation.
Consistent with the early death of CXXC1 Ϫ/Ϫ embryos, epigenetic regulation is critical for cellular differentiation and embryogenesis (17)(18)(19). A dramatic reduction in CpG dinucleotide methylation occurs during early development in preimplantation mouse embryos (20,21). At the time of implantation, de novo methylation occurs at most CpG motifs except for CpG islands, which remain unmethylated and transcriptionally active (22,23). In addition, ES cells undergo a global epigenetic reprogramming upon induction of in vitro differentiation, and inhibition of histone deacetylase enzymes prevents cellular dif-ferentiation (17). Deletion of the Dnmt1 gene in mice results in an embryonic lethal phenotype (18), and Dnmt1 Ϫ/Ϫ ES cells exhibit impaired erythroid colony differentiation and no myeloid colony differentiation (24). Overexpression of DNMT1 leads to DNA hypermethylation and embryonic lethality (25). The DNMT3a and DNMT3b de novo methyltransferases are also critical for mammalian development (19). Dnmt3b Ϫ/Ϫ mice die in utero at day E14.5-E18.5, and Dnmt3a Ϫ/Ϫ mice develop to term but are runted and die within 4 weeks of age. Lastly, disruption of genes that encode histone methyltransferases, such as G9a (26), ESET (27), or human trithorax (28), results in embryonic lethal phenotypes in mice.
Given the inability of CXXC1 Ϫ/Ϫ ES cells to differentiate, we hypothesize that CFP1 is also required for stem and progenitor cell function later in development. In particular, we reason that CFP1 is important for hematopoietic stem and progenitor cell function. Hematopoiesis requires appropriate epigenetic regulation to achieve a balance between stem cell self-renewal and lineage commitment. Stem cell differentiation involves global remodeling of chromatin structure and a progressive accumulation of heterochromatin and restriction of gene expression and developmental potential (29). Treatment of hematopoietic stem cells with inhibitors of DNMT enzymes (deoxyazacytidine) and histone deacetylase proteins (trichostatin A) leads to altered cell fate and an expansion of stem cells in vitro (30). Finally, the transcription factor IKAROS is a critical regulator of lineage commitment during hematopoiesis. The absence of IKAROS leads to a 40-fold decrease in hematopoietic stem cell function (31). IKAROS associates with the 2-MDa chromatin remodeling complex NURD, which contains histone deacetylase proteins and an ATP-dependent chromatin remodeling enzyme (32).
The importance of epigenetics to hematopoietic cell development was recently illustrated by the finding that DNA demethylation and alterations in chromatin structure occur prior to induction of gene expression at the lysozyme locus during myeloid cell differentiation (33). There is also a large body of literature describing a correlation between the level of gene-specific DNA methylation and the appearance or extinction of lineagerestricted gene expression throughout hematopoiesis (34 -37). In addition to the role of DNA methylation in normal hematopoiesis, aberrant DNA methylation is associated with the development of leukemia. A common mechanism is hypermethylation of tumor suppressor genes such as DAP-kinase, HIC1, p15, and p16, leading to the loss of gene expression (38 -42). Hypermethylation of DNA has also been linked to chromosome instability and increased chromosomal translocations that are frequently associated with hematopoietic malignancies (43). Agents that inhibit DNA methylation, such as azacytidine, are being used as chemotherapeutic agents in the treatment of hematopoietic malignancies such as myelodysplastic syndrome, chronic myelogenous leukemia, and acute myelogenous leukemia (44 -46). Altered histone modifications are also associated with leukemia. For example, the gene encoding the human trithorax histone H3-K4 methyltransferase is involved in chromosomal translocations in the majority of pediatric leukemia (47,48).
Because of the early embryonic death of CXXC1 Ϫ/Ϫ mice, zebrafish were used as an alternative model system with which to assess the role of CFP1 during post-gastrulation development, particularly hematopoiesis. The zebrafish homolog of CFP1 (zCFP1) was identified, and its function during embryogenesis was examined using antisense morpholino oligonucleotides. Targeting of zCFP1 with morpholino oligonucleotides results in decreased global genomic cytosine methylation and disrupted primitive hematopoiesis. These studies demonstrate that zCFP1 is required for proper epigenetic regulation of primitive hematopoiesis.

EXPERIMENTAL PROCEDURES
Zebrafish Maintenance-Zebrafish (Danio rerio) were maintained under standard conditions (49) in accordance with the Indiana University policy on animal care and use. Development of embryos proceeded at 28.5°C, and developmental stages were determined by the time of fertilization and morphologic features. Embryos were treated with 0.2 mM phenylthiourea (Sigma-Aldrich) to prevent melanization, and embryo ages are given as hours post-fertilization (hpf) or days post-fertilization.
Isolation of Zebrafish CFP1 cDNA-First strand cDNA was prepared from 5 g of total RNA isolated from 24-hpf zebrafish embryos using Tri-reagent (Molecular Research Center, Cincinnati, OH) and Superscript II RNase H Ϫ reverse transcriptase (Invitrogen) as per the manufacturer's instructions. Amplification of the zebrafish CXXC1 (zCXXC1) cDNA was performed by RT-PCR using the following oligonucleotides: zebrafish forward, 5Ј-TTGTTAGCTGTCTGCCCACGG-3Ј; and zebrafish reverse, 5Ј-TACTGATTACTGAGATGGC-3Ј. The PCR product was cloned into pCR4-TOPO (Invitrogen) per the manufacturer's instructions and then into pBluescript SK.
Microinjection-Morpholino oligonucleotides were microinjected into embryos at the 1-2 cell stage. The injection volume into each embryo was 1-2 nl (4.3-8.6 ng/embryo). Injected embryos were imaged using a Leica MZ12.5 dissecting microscope equipped with a SPOT RT camera (Diagnostic Instruments, Sterling Heights, MI). Synthetic capped murine CXXC1 mRNA was transcribed from murine CXXC1 cDNA in the pBluescript SK vector using the T7 mMessage mMachine (Ambion, Austin, TX). Messenger RNA was co-injected with morpholino oligonucleotides as described above into 1-2 cell stage embryos at 50 pg/nl (50 -100 pg/embryo).
Acridine Orange and o-Dianisidine Staining and TUNEL Assays-Apoptosis in whole zebrafish embryos was visualized by staining with acridine orange or TUNEL assay. Live embryos were removed from their chorions, stained with 5 g/ml acridine orange (Molecular Probes, Eugene, OR) for 2 min, and washed three times in embryo medium (49). Epifluorescence images were collected using a Nikon Eclipse TS100 microscope (Nikon, Inc., Melville, NY) equipped with DIC optics and a SPOT RT camera (Diagnostic Instruments). For TUNEL analysis of apoptosis, whole zebrafish embryos were dechorionated and fixed in 4% paraformaldehyde. TUNEL assay was then performed using a fluorescein in situ cell death detection kit (Roche Applied Science) according to the manufacturer's instructions. The embryos were imaged using a Nikon Eclipse TS100 microscope equipped with SPOT RT camera (Diagnostic Instruments).
Staining of hemoglobin was performed as described (50). Briefly, unfixed embryos were dechorionated and stained with a solution of o-dianisidine (0.6 mg/ml) (Sigma-Aldrich), 0.01 M sodium acetate (pH 4.5), 0.65% hydrogen peroxide, and 40% (v/v) ethanol in the dark for 15 min. The embryos were imaged using a Nikon Eclipse TS100 microscope equipped with a SPOT RT camera (Diagnostic Instruments).
Video Capture of Blood Flow-Zebrafish were placed in embryo medium in glass-bottomed microwell dishes (MatTek Corp., Ashland, MA) for imaging. Blood flow videos were acquired at 30 frames/s using a DAGE MTI CCD 72 camera (Michigan City, IN) mounted on a Nikon Diaphot microscope equipped with DIC optics. To enhance visibility of the blood flow, the images were processed in Adobe Photoshop using contrast-stretch and unsharp-mask effects. An AVI format movie was generated from the image sequence using Quicktime Pro (Apple) and then converted to MPG1 format using a TMP-GEnc MPEG encoder (Pegasys Inc., Tokyo, Japan).
In Situ Hybridization and RT-PCR Analysis of Gene Expression-Plasmids containing cDNA clones of zebrafish gata1, scl (stem cell leukemia gene), and myeloperoxidase were provided by Dr. Todd Evans (Albert Einstein College of Medicine), and a gata2 cDNA clone was provided by Dr. Michael Trautman (Indiana School of Medicine). Plasmid DNA was linearized by restriction enzyme digestion to generate template for riboprobe synthesis. Digoxigenin-labeled riboprobes were synthesized by in vitro transcription using digoxigenin RNA labeling mix (Roche Applied Science) and T7, Sp6 (New England Biolabs, Beverly, MA), and T3 polymerases (Roche Applied Science). Whole mount in situ hybridizations were performed on embryos fixed in 4% paraformaldehyde as previously described (51). The embryos were mounted in glycerol and examined using a Leica MZ12 dissecting microscope equipped with Axiovision imaging software (52).
Embryonic Vascular Development-Transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under the control of the fli-1 promoter, TG(fli1:EGFP)y1 (53) were purchased from the zebrafish international resource center (Eugene, OR) and used to visualize developing vascular struc-tures. Injected embryos were imaged using a Leica DMIRE2 microscope equipped with a Leica DFC480 imaging system and software (Leica Microsystems, Inc.).
Analysis of Cytosine Methylation-Global genomic cytosine methylation was analyzed using a methyl acceptance assay as described (15,54). Briefly, 500 ng of genomic DNA, isolated as described previously (55), was incubated with 2 Ci of [ 3 H]methyl-S-adenosyl L methionine (PerkinElmer Life Sciences; 15 Ci/mmol) and 3 units of SssI methylase (New England Biolabs, Ipswich, MA) in 120 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM EDTA, and 1 mM dithiothreitol for 1 h at 30°C. In vitro methylated DNA was isolated by filtration through Whatman DE-81 ion exchange filter, and incorporated radioactivity was measured by scintillation counting.
Statistical Analysis-Statistical significance was assessed by one-tailed t tests, with a p value of Ͻ0.05 interpreted as statistical significance.

Identification and Characterization of a Zebrafish
Homolog of the CXXC1 Gene Encoding CFP-Homologs of mammalian CFP1 have been identified in Drosophila, Caenorhabditis elegans, and both Saccharomyces cerevisiae and Schizosaccharomyces pombe (1,56). Based upon DNA alignment between sequences available from the Welcome Trust Sanger Institute Ensembl zebrafish data base (contig 12623.1 on chromosome 11) and the murine CXXC1 genomic DNA (57), the zebrafish CXXC1 gene was identified (zCXXC1). The zCXXC1 cDNA sequence was predicted from expressed sequence tag clones within the NCBI Genbank TM data base (accession numbers TC142796, TC142797, and TC130677) and confirmed by determining the nucleotide sequence of a cDNA clone recovered by RT-PCR of RNA isolated from 24-hpf zebrafish embryos. Zebrafish CFP1 protein (Genbank TM accession number NP_956627) shares ϳ60% identity and ϳ70% similarity with murine CFP1 (Fig. 1). Mammalian CFP1 contains several protein domains, including two plant homology domains, a CXXC DNA-binding domain, acidic and basic domains, and a coiled-coil domain. The plant homology domain has been identified in over 40 chromatin-associated proteins (58,59) and often mediates protein-protein interactions (60 -62). The CXXC domain of CFP1 is required for binding to DNA sequences that contain unmethylated CpG dinucleotides (1); the acidic domain functions as a transcription activation domain (63); and the acidic, basic, and coiled-coil domains are required for subnuclear localization to euchromatic nuclear speckles (2). All of the domains of the mammalian CFP1 protein are conserved in the zCFP1 protein.
The expression pattern of zCFP1 in developing zebrafish embryos was analyzed by both RT-PCR and in situ hybridization. RT-PCR analysis detects expression of the zCXXC1 gene at all developmental stages examined between 2 hpf and 7 days post-fertilization ( Fig. 2A). Using the zCCCX1 cDNA described above, a digoxigenin-labeled zCXXC1 probe corresponding to the first 460 nucleotides of the transcript was generated and hybridized with zebrafish embryos at 12, 18, and 24 hpf (Fig.  2B). The sense control probe is shown as a negative control.
These studies reveal that zCFP1 is widely expressed throughout embryonic zebrafish development.
Zebrafish Injected with zCXXC1 Morpholino Oligonucleotide Exhibit Developmental Defects-To examine the functional role of CFP1 in zebrafish, two nonoverlapping antisense morpholino oligonucleotides were designed against the zCXXC1 mRNA and injected into 1-2 cell embryos. A control morpholino oligonucleotide that does not target any known transcript was similarly injected and used as a negative control. Zebrafish embryos were examined at 24, 48, and 96 hpf. Injection of the zCXXC1 morpholino oligonucleotide resulted in abnormal yolk sac extension morphology com-  DECEMBER 1, 2006 • VOLUME 281 • NUMBER 48 pared with embryos injected with the control morpholino oligonucleotide (Fig. 3). Embryos treated with the zCXXC1 morpholino oligonucleotide were also smaller at 48 and 96 hpf (Fig. 3, C-F) and exhibited cardiac edema (Fig. 3, E and  F). The abnormal yolk sac extension was observed in 98% of embryos injected with the zCXXC1 morpholino oligonucleotide but in only 3% of embryos injected with the control morpholino oligonucleotide (Table 1). Cardiac edema was observed in 66% of embryos injected with the zCXXC1 morpholino oligonucleotide but in only 11% of embryos injected with the control ( Table 1). Injection of the zCXXC1 morpholino oligonucleotide resulted in increased mortality of the embryos, with 23% of injected embryos being dead at 24 hpf (Table 1) and greater than 50% of embryos dying by 96 hpf (data not shown). A similar phenotype was observed following injection of a second independent zCXXC1 morpholino oligonucleotide, with 71% of embryos exhibiting a yolk sac extension defect and 59% showing cardiac edema, thus confirming the specificity of the effect (Table 1). Furthermore, the severity of the phenotype was dose-dependent (Table 2).

CFP1 Is Required for Zebrafish Hematopoiesis
CFP1 Regulates Cytosine Methylation in Zebrafish Embryos-CFP1 binds to unmethylated CpG motifs (1) and regulates the methylation state of the murine genome (15). It was therefore hypothesized that zCFP1 may also regulate cytosine methylation in zebrafish. To determine this, zebrafish embryos were injected with the zCXXC1 morpholino oligonucleotide or the control morpholino oligonucleotide at the 1-2 cell stage and collected at 24 hpf, and genomic DNA was isolated from pooled samples. Global cytosine methylation levels were analyzed by a    methyl acceptance assay (15,54), which measures the ability of a genomic DNA sample to accept methyl groups. The ability to act as a methyl acceptor is inversely proportional to the degree of genomic cytosine methylation initially present in each DNA sample. Genomic DNA obtained from embryos injected with the zCXXC1 morpholino oligonucleotide accepted 2.5-fold more methyl groups than DNA from untreated embryos or embryos injected with the control morpholino oligonucleotide (Fig. 4), indicating an ϳ60% decline in global genomic cytosine methylation in embryos treated with the zCXXC1 morpholino oligonucleotide. This is likely an underestimate of the magnitude of this defect, because the assay was performed on a pool of injected embryos, only a subset of which would be expected to be severely morphant (Tables 1 and 2). However, this result is comparable with the 60 -80% decline of global genomic cytosine methylation observed in CXXC1 Ϫ/Ϫ murine ES cells (15).
These results indicate that zCFP1 regulates cytosine methylation in zebrafish embryos. Zebrafish Injected with zCXXC1 Morpholino Oligonucleotide Exhibit Severe Anemia and Are Rescued by Murine CXXC1 mRNA-Zebrafish injected with the zCXXC1 morpholino oligonucleotide exhibit abnormal morphology of the yolk sac extension (Fig. 3), which is located posterior to the intermediate cell mass (ICM) (50,64). The ICM functions similarly to yolk sac blood islands of mammals and is the location of primitive hematopoiesis in zebrafish embryos (65). This led us to investigate the state of hematopoiesis in embryos injected with the zCXXC1 morpholino oligonucleotide. These embryos exhibited a severe reduction of erythropoiesis at 24 hpf and 48 hpf, as revealed by reduced o-dianisidine staining of hemoglobin (Fig.  5). In addition, video image capture reveals a dramatic decline of circulating cells in morphant zebrafish (see supplemental data).
To further assess the specificity of this aspect of the morphant phenotype, murine CXXC1 mRNA was co-injected with the zCXXC1 morpholino oligonucleotide. The murine mRNA was used instead of zebrafish mRNA because the murine mRNA is not targeted by the zCXXC1 morpholino oligonucleotide. 59% of embryos co-injected with both the zCXXC1 morpholino oligonucleotide and the murine CXXC1 mRNA exhibited a normal phenotype indistinguishable from embryos injected with the control morpholino oligonucleotide, compared with only 10% of embryos injected with the zCXXC1 morpholino oligonucleotide alone (Fig.  5B). In addition, the morphant phenotype of the co-injected embryos was less severe, with 27% of embryos exhibiting some circulating red blood cells (moderate) and only 11% exhibiting an absence of red blood cells (severe) at 48 hpf, compared with 12 and 65%, respectively, for embryos injected solely with the zCXXC1 morpholino oligonucleotide. Hence, murine CXXC1 mRNA rescues the hematopoietic abnormalities of embryos injected with the zCXXC1 morpholino oligonucleotide, and this phenotype is therefore a specific consequence of targeting the zCXXC1 transcript.

Analysis of Hematopoietic Markers in Zebrafish Embryos Injected with zCXXC1 Morpholino Oligonucleotides-
Zebrafish embryos injected with the zCXXC1 morpholino oligonucleotide exhibit severe anemia at 24 and 48 hpf (Fig.  5). To further characterize this hematopoietic defect, in situ hybridization was performed to analyze the expression of  The magnification is 20ϫ. B, zebrafish were analyzed at 48 hpf for red blood cell production using o-dianisidine stain, and the percentage of fish that were dead or displayed severe anemia, moderate anemia, or normal blood cell production was determined. The bar graph represents the averages of three independent experiments. The asterisks denote values that are significantly different (p Ͻ 0.05) from control samples, and the number signs denote values that are significantly different (p Ͻ 0.05) from rescue samples. DECEMBER 1, 2006 • VOLUME 281 • NUMBER 48 early hematopoietic markers in embryos injected with zCXXC1 morpholino oligonucleotide. At 24 hpf, the stem cell leukemia gene (scl), which is important for primitive and definitive hematopoiesis (66), is expressed in the ICM (50). In situ hybridization analysis revealed that scl is expressed normally at 24 hpf in zebrafish embryos injected with zCXXC1 morpholino oligonucleotides as compared with embryos treated with control morpholino oligonucleotide (Fig. 6, A and B).

CFP1 Is Required for Zebrafish Hematopoiesis
GATA-1, a member of the GATA-binding protein family, is required for both primitive and definitive hematopoiesis (67) and is expressed normally in the anterior ICM at 24 hpf in morphant zebrafish (50) (Fig. 6, C and D). Another member of the GATA-binding protein family, GATA-2, is expressed in hematopoietic progenitors. Transcripts of gata-2 are appropriately detected in the wedge region of the anterior ICM and the posterior ICM at 24 hpf (68) in embryos injected with either control or zCXXC1 morpholino oligonucleotides (Fig. 6, E and F).
To investigate whether a marker of the myeloid lineage is expressed normally in embryos injected with zCXXC1 morpholino oligonucleotide, in situ hybridization analysis of zebrafish myeloperoxidase was performed. Expression of myeloperoxidase is appropriately detected in the anterior and posterior ICM at 24 hpf (69) in embryos treated with the zCXXC1 morpholino oligonucleotide (Fig. 6, G and H). Thus, both erythroid-and myeloid-specific markers are expressed normally in these embryos despite a dramatic reduction of circulating hematopoietic cells in morphant zebrafish.
Targeting of zCFP1 Causes a Defect in Vascular Development-Because of evidence that a common hemangioblast progenitor gives rise to both hematopoietic and endothelial cells (70), we wanted to determine whether the targeting of zCFP1 additionally caused abnormal development of the vasculature in zebrafish embryos. The TG(fli1:EGFP) y1 transgenic zebrafish, which expresses EGFP under control of the fli1 promoter (53), was used to investigate the role of zCFP1 on vascular development. The zCXXC1 or control morpholino oligonucleotides were injected into TG(fli1:EGFP) y1 embryos at the 1-2 cell stage, and embryos were examined at 48 hpf. The control morpholino oligonucleotide did not affect the development of the vasculature (Fig. 7, A, C, and E), because well defined and organized vessels indistinguishable from untreated embryos (data not shown) can be seen throughout the body of the embryo. Injection of the zCXXC1 morpholino oligonucleotide, however, resulted in fewer and less organized intersomitic vessels throughout the body of the embryo (Fig. 7, B, D,  and F). Hence, targeting of zCFP1 results in disrupted vascular development.

Injection of zCXXC1 Morpholino Oligonucleotide Causes Increased Apoptosis in Zebrafish
Embryos-The absence of CFP1 in murine ES cells results in a 3-fold increase in the rate of apoptosis (15). Thus, embryos injected with the zCXXC1 morpholino oligonucleotide were examined for the level of apoptosis by staining with acridine orange. Some apoptosis occurs normally in the developing zebrafish embryos as can be seen at 24 hpf and 48 hpf in embryos treated with control morpholino oligonucleotide (Fig. 8, A, C, E, and G). However, there is an increase in the number of fluorescent cells in embryos treated with the zCXXC1 morpholino oligonucleotide (Fig. 8, B, D, F,  and H), indicating that the targeting of zCFP1 causes an increase in apoptosis. Importantly, a site of strong fluorescence is observed at the end of the anterior ICM and the beginning of the posterior ICM in embryos injected with the zCXXC1 morpholino oligonucleotide (Fig. 8, C, D, G, and H). This corresponds to the site of primitive erythropoiesis. Similar results were obtained upon detection of apoptosis using the TUNEL assay (Fig. 8, I and J). Increased TUNEL signal is detected throughout the body of morphant embryos, including within the ICM.

DISCUSSION
Mammalian CFP1 is a critical epigenetic regulator of both cytosine methylation and histone methylation and is required for early mouse embryogenesis and in vitro murine ES cell differentiation (14 -16). However, despite being widely expressed throughout development (1,57), nothing was known regarding the function of CFP1 during post-gastrulation development. Zebrafish was chosen as a model system to address this question because of the rapid embryonic development of this organism and the ability to efficiently ablate target gene expression by injection into early embryos of antisense morpholino oligonucleotides.
A cDNA encoding zCFP1 was isolated and found to be closely related to mammalian CFP1. Inhibition of zCFP1 expression by antisense morpholino oligonucleotides resulted in a dramatic phenotype. Morphant embryos were small and exhibited increased mortality, a 60% decrease in global genomic cytosine methylation, elevated apoptosis, defects in vasculogenesis, and a dramatic decline in circulating erythroid cells. These findings are strikingly similar to the phenotype observed in murine ES cells that lack CFP1. These cells exhibit a 3-fold increase in the rate of apoptosis, a 60 -80% decline in global genomic cytosine methylation, and an inability to achieve in vitro differentiation, including hematopoietic development (15). Thus, CFP1 function is critical for epigenetic regulation of development in a wide range of vertebrates.
Following analysis of zCFP1 function, re-examination of zebrafish data bases revealed a second zebrafish homolog, denoted zCFP1b (Genbank TM accession number NP_956893). This protein exhibits a similar degree of homology with mammalian CFP1 as does zCFP1 (BLAST score of 509 versus 507), including conservation of all the previously described protein domains in mammalian CFP1. The cDNA sequences produced by the two zCXXC1 genes are ϳ75% identical, and the protein sequences exhibit 70% identity and 84% similarity (data not shown). Importantly, the region of the cDNA used for in situ hybridization analysis of zCXXC1 expression exhibits only 35% similarity with the zCXXC1b sequence. Multiple expressed sequence tag clones representing the zCXXC1b gene have been identified (data not shown), indicating that both zCXXC1 genes are expressed. Importantly, because of sequence divergence, the morpholino oligonucleotides used in this study to deplete zCFP1 expression do not target transcripts produced from the zCXXC1b gene. Hence, the appearance of a morphant phenotype following zCFP1 depletion indicates that these two zebrafish proteins have nonredundant functions. Zebrafish is a pseudo-tetraploid organism (71), and duplicate genes often exhibit distinct expression patterns and distinct functional roles (72,73). Examples of this phenomenon include the hedgehog, tinman-related, and muscle segment homeobox (MSX) proteins (74 -76). Additional studies will be required to further explore the functional relationship between the zCFP1 and zCFP1b proteins.
The failure of primitive hematopoiesis is the most obvious phenotype following injection of zCXXC1 antisense oligonucleotides. However, this is likely not the cause of elevated mortality of these embryos, because bloodless mutant zebrafish survive up to 5 days in the absence of circulating blood cells (68). Nevertheless, the observed decline in circulating blood cells reveals the importance of zCFP1 for this postgastrulation developmental process. Hematopoiesis may be  DECEMBER 1, 2006 • VOLUME 281 • NUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 37041 particularly sensitive to the loss of the CFP1 epigenetic regulator, because this developmental program must continue throughout the lifespan of the organism and would thus be susceptible to the effect of CFP1 depletion, even if this occurs following completion of gastrulation.

CFP1 Is Required for Zebrafish Hematopoiesis
Despite the requirement of murine ES cells for CFP1 to initiate in vitro differentiation, zebrafish treated with zCXXC1 morpholino oligonucleotides induce early markers of hematopoiesis at normal times and locations during development. However, these embryos exhibit greatly elevated levels of apoptosis in the ICM, suggesting that the decline in circulating blood cells is due to a failure of terminal erythroid differentiation. A similar phenotype was observed in murine ES cells deficient in DNMT1 (77). These cells grow normally in an undifferentiated state but undergo rapid apoptosis upon induction of in vitro differentiation. In addition, murine ES cells lacking CFP1 exhibit a 3-fold increase in the rate of apoptosis in the undifferentiated state (15). Taken together, these data suggest that terminal primitive erythropoiesis in zebrafish requires CFP1 and that CFP1-depleted cells undergo apoptosis in response to epigenetic perturbations. Detection of appropriate early hematopoiesis markers in morphant zebrafish was unexpected, given the inability of murine ES cells lacking CFP1 to differentiate. Perhaps this reflects a partial redundancy with zCFP1b function during the early stages of stem cell lineage commitment and progenitor cell development.
Although not fully described, it appears that epigenetic modifications play an important role in zebrafish development. The zebrafish genome is heavily methylated at CpG dinucleotides, and a zebrafish homolog of DNMT1 has been described (78). Similar to mammals, global cytosine methylation is modulated during early zebrafish development. Sperm DNA is heavily methylated but becomes actively demethylated immediately following fertilization (79). Global cytosine methylation is restored between 2 and 6 hpf. In addition, cytosine methylation is correlated with gene silencing in zebrafish. For example, a CpG island near the no tail gene becomes methylated between 14 and 48 hpf, coincident with silencing of this locus, whereas several other CpG islands remain unmethylated (80). Furthermore, transgene expression correlates with the state of cytosine methylation. A transgene introduced in the methylated state is actively demethylated and then acquires de novo cytosine methylation by 12 hpf, similar to the dynamics of global zebrafish cytosine methylation (81). Treatment with azacytidine, an inhibitor of cytosine methylation, leads to transgene demethylation and induction of expression (81).
Treatment of zebrafish embryos with azacytidine during the 2-3-hpf period leads to developmental defects, including loss of the tail and abnormal patterning of somites (82). However, post-gastrulation exposure to azacytidine failed to perturb embryonic development, although hematopoiesis was not specifically described in this report (82). This is in contrast to the correlation of reduced genomic cytosine methylation and reduced hematopoiesis following injection of zCXXC1 morpholino oligonucleotides, suggesting that the morphant phenotype observed following CFP1 depletion may not be entirely explained by the observed reduction in global cytosine methylation patterns. Similarly, the phenotype of CFP1-deficient murine ES cells is more severe than can be explained by the observed partial deficiency in genomic cytosine methylation (15). For example, CXXC1-null murine embryos die earlier in gestation (peri-implantation) compared with embryos that lack DNMT1 (8.5-9.5 dpc) but exhibit a less severe loss of cytosine methylation (15,77). In addition, the existence of CFP1 homologs in organisms that lack cytosine methylation (such as yeast) suggests an additional function for CFP1 that is independent of cytosine methylation.
The yeast homolog of CFP1 (Spp1) is a component of the Set1 (COMPASS) histone H3-Lys 4 methyltransferase complex (56,83,84). Recent work by our laboratory has determined that mammalian CFP1 associates with the mammalian analog of the Set1 histone H3-Lys 4 methyltransferase complex (16). Very little is known regarding the role of covalent histone modification in the zebrafish, although acetylation of zebrafish histone H4 has been reported to be associated with gene activity (85), and inhibition of the zebrafish homolog of histone deacetylase 1 leads to cardiac defects (although hematopoiesis and the peripheral vasculature were normal) (86). Similar to the situation in mouse embryos, the morphant phenotype observed in zebrafish treated with zCXXC1 antisense oligonucleotides may result from interference with both the cytosine methylation and histone methylation machinery. Although no studies have been published describing histone methylation in zebrafish, data base searching reveals zebrafish homologs for each component of the human Set1/CFP1 histone H3-Lys 4 methyltransferase complex. The zebrafish homolog of human Set1 (zSet1) (XP_684451.1) exhibits 60% identity over 1007 amino acids (BLAST score of 427); zebrafish Ash2 (XP_686474.1) exhibits 82% identity with human Ash2 over 410 amino acids (BLAST score of 718); zebrafish Wdr5 (XP_709140.1) exhibits 90% identity with human Wdr5 over 318 amino acids (BLAST score of 648); zebrafish Rbbp5 (NP_956539.1) exhibits 90% identity with human Rbbp5 over 510 amino acids (BLAST score of 907); and zebrafish Swd2 (NP_956893.1) exhibits 87% identity with human Swd2 over 305 amino acids (BLAST score of 557) (data not shown). Thus, it is likely that an analogous Set1⅐CFP1 histone methyltransferase complex is present in zebrafish and that the observed morphant phenotype following depletion of CFP1 is a consequence of perturbations of both cytosine methylation and the histone code. Additional studies will be required to evaluate perturbations of histone modifications and chromatin structure in zebrafish embryos treated with zCXXC1 antisense oligonucleotides.
The importance of CFP1 has previously been documented for ES cell differentiation and pregastrulation murine development, but this is the first demonstration of a role for CFP1 during post-gastrulation development and zebrafish embryogenesis. These studies further implicate CFP1 as a crucial factor for cellular differentiation and hematopoiesis. Although the underlying mechanisms of CFP1 action remain under investigation, these findings emphasize the importance of CFP1 for vertebrate development.