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HEMATOPOIESIS
From the National Human Genome Research Institute,
National Institutes of Health, Bethesda, MD, and the Howard Hughes
Medical Institute, Children's Hospital, Harvard Medical School,
Boston, MA.
The CCAAT/enhancer-binding protein (C/EBP) family consists of
transcription factors essential for hematopoiesis. The defining feature
of the C/EBPs is a highly conserved carboxy-terminal bZIP domain that
is necessary and sufficient for dimerization and DNA binding, whereas
their amino-terminal domains are unique. This study reports a novel
c/ebp gene (c/ebp1) from zebrafish that encodes
a protein homologous to mammalian C/EBPs within the bZIP domain, but
with an amino terminus lacking homology to any C/EBP or to any known
sequence. In zebrafish embryos, c/ebp1 expression was
initially observed in cells within the yolk sac circulation valley at
approximately the 16-to 18-somite stage, and at 24 hours postfertilization (hpf), also in circulating cells. Most
c/ebp1+ cells also expressed a known early
macrophage marker, leukocyte-specific plastin (l-plastin).
Expression of both markers was lost in cloche, a mutant
affecting hematopoiesis at the level of the hemangioblast. Expression
of both markers was retained in m683 and
spadetail, mutants affecting erythropoiesis, but not
myelopoiesis. Further, c/ebp1 expression was lost in a
mutant with defective myelopoiesis, but intact erythropoiesis. These
data suggest that c/ebp1 is expressed exclusively in
myeloid cells. In electrophoretic mobility shift assays, c/ebp1 was
able to bind a C/EBP consensus DNA site. Further, a chimeric protein
containing the amino-terminal domain of c/ebp1 fused to the DNA-binding
domain of GAL4 induced a GAL4 reporter 4000-fold in NIH3T3 cells. These
results suggest that c/ebp1 is a novel member of the C/EBP family that
may function as a potent transcriptional activator in myeloid cells.
(Blood. 2001;97:2611-2617) Differentiation of hematopoietic cells is regulated
by coordinate activation and repression of transcription
factors.1 Multiple members of the CCAAT/enhancer-binding
protein (C/EBP) family are essential for myeloid
development.2 Six mammalian members have been identified
to date and have been named C/EBP In vitro analyses and murine targeted deletion models have shown
that C/EBP Among the C/EBP family members, C/EBP The zebrafish, Danio rerio, offers a powerful model system
in which to study hematopoiesis due to its external fertilization, transparent embryos, and rapid embryonic development.16
Erythrocytes, myeloid cells, T lymphocytes, and thrombocytes have been
identified in the zebrafish.17-21 Recently, macrophage
development was shown to originate in the ventrolateral mesoderm
anterior to the cardiac field.17 Macrophages were shown to
develop and stream along the yolk sac to enter the circulation.
Twenty-six hematopoietic mutants have been identified to
date.18,19 A number of bloodless mutants have been
described and are characterized by few or no blood cells at the onset
of circulation. The mutation in the bloodless mutant,
cloche, has been shown to affect both hematopoiesis and
vasculogenesis and is believed to occur at the level of the
"hemangioblast"22-24; characterization of other
bloodless mutants has shown that the mutations affect later steps along
the erythroid pathway.25 However, data on mutants that
specifically affect the myeloid lineage have not yet been published.
To facilitate analysis of myelopoiesis in D rerio, a
zebrafish kidney complementary DNA (cDNA) library was screened for
c/ebp cDNAs using a probe encoding the conserved bZIP region
of human C/EBP Library screening and sequence analysis of cDNAs
Radiation hybrid mapping
Zebrafish maintenance and breeding Zebrafish were maintained and bred essentially as described28 under an approved animal use protocol of the National Institutes of Health. After breeding, embryos were maintained in egg water (0.006% Instant Ocean in distilled water) with 2 parts per million methylene blue to prevent fungal growth.Whole-mount in situ hybridization Embryos used for in situ hybridization were obtained from breedings of the wild-type EK strain (Ekkwill)29 and 3 hematopoietic mutants, cloche (clo39),22 spadetail (sptb104),30 and m683.25 Embryos were staged as described.28 When growing embryos for harvest at more than 24 hours postfertilization (hpf), embryos were grown in 0.003% 1-phenyl-2-thiourea (Sigma, St Louis, MO) to prevent melanization. Embryos were dechorionated in 2 mg/mL pronase followed by extensive washing in 30% Danieau solution (58 mM NaCl, 0.67 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca(NO3)2, and 4.5 mM HEPES, pH 7.5) and killed in 0.2% 3-amino benzoic acidethylester (Tricaine). Whole-mount in situ hybridization was performed essentially as described with the following modifications. Embryos less than 24 hpf were not treated with proteinase K. Embryos between 24 and 36 hpf were treated for 5 minutes in 10 µg/mL proteinase K and embryos 36 hpf to 2 days postfertilization (dpf) were treated for 10 to 20 minutes. Hybridization and washing were performed at 55°C for all probes. RNA antisense probes were synthesized according to the manufacturer's instructions with either UTP-digoxigenin (c/ebp1) or UTP-fluorescein (l-plastin) (Boehringer Mannheim, Indianapolis, IN). Probes labeled with digoxigenin were visualized with BM-purple (Boehringer Mannheim) and probes with fluorescein with fast red (Boehringer Mannheim). The c/ebp1 cDNA used for in situ hybridization started at nt +3 and included over 600 bp of 3' untranslated sequence. The plasmid was digested with EcoRI and T7 RNA polymerase was used to synthesize the antisense probe. The plasmid containing l-plastin (kindly provided by B. Thisse)17 was linearized with NotI and transcribed with T7 RNA polymerase. Embryos were examined with an Olympus dissecting microscope or a Nikon Microphot-FXA compound microscope and photographed with a Spot (Diagnostic Instruments, Sterling Heights, MI) or Quantix (Photometrics, Tucson, AZ) CCD camera. In some cases, images focused in 2 different planes were merged using Adobe Photoshop to allow both yolk sac and tail regions to be visualized in the same image.Electrophoretic mobility shift assays For DNA-binding assays, a double-stranded probe containing an optimal C/EBP binding site was prepared by annealing a self-complementary oligonucleotide with a GATC overhang (shown in bold), 5'-GATCTGCAGATTGCGCAATCTGCA-3'12 and labeling using Klenow polymerase and 32P-dCTP. pBKCMV-c/ebp1 with a T3 promoter upstream of c/ebp1, and pCMV-C/EBP containing a T7
promoter upstream of human C/EBP,31 were
used to in vitro transcribe and translate proteins (TNT reticulocyte lysate system, Promega, Madison, WI) according to manufacturer's directions. Products from 40 ng of each plasmid were combined with 1 µg poly[dI-dC], 1 ng labeled probe (1 × 105 cpm),
and varying amounts of unlabeled probe as indicated. A probe with a
mutated C/EBP binding site,12
5'-GATCTGCAGAGACTAGTCTCTGCA-3' (the GATC
overhang is shown in bold and changes from the wild-type probe are
underlined) was used as a negative control. DNA-binding assay samples
were separated on a 4% polyacrylamide/0.25 × TBE (tris borate EDTA)
gel. 35S-methionine-labeled proteins were analyzed on a 4%
to 12% bis-tris gradient gel (Invitrogen, Carlsbad, CA), fixed,
treated with Amplify (Amersham-Pharmacia), and dried followed by autoradiography.
Transfection and activation assays The cDNAs encoding putative activation domains were placed in a pcDNA3-based vector containing the region encoding the DNA-binding domain (DBD) of GAL4 (amino acids [aa] 1-147) (plasmid kindly provided by N. Perkins, University of Dundee). A PCR fragment encoding the region amino terminal of the bZIP domain of c/ebp1 was ligated downstream of the GAL4 DBD using EcoRI and BamHI. The primers used to amplify the region of c/ebp1 were 5'-CCGGAATTCATGTCGGTGTCTGACAACATC-3' (nt 1-21, EcoRI site in bold letters) and 5'-CGCGGATCCGCGCACAGGCGGAGCGCAGACG-3' (nt 262-241, BamHI site in bold letters). PCR fragments encoding the first 194 aa and the first 102 aa of human C/EBP were amplified from
a human C/EBP plasmid and placed downstream of the GAL4 DBD with EcoRI and BamHI. Both PCR products were
obtained using the forward primer:
5'-CCGGAATTCATGTCCCACGGGACCTACTCA-3' (nt 1-21, EcoRI site in bold letters). The product encoding aa 1-194 was amplified with the reverse primer:
5'-CGCGGATCCCTTGTGTAAGGGGCCAGCCGG-3' (nt 586-565, BamHI site in bold letters) and the product encoding aa
1-102 was amplified with the reverse primer:
5'-CGCGGATCCCAGCGCCTTCCTGTCTGGGCC-3' (nt 306-286, BamHI site in bold letters). The amplified regions and the
restriction site junctions were sequenced in both directions for all
constructs. NIH3T3 cells were grown in Dulbecco modified Eagle medium
containing 10% fetal calf serum (Life Technologies, Rockville, MD) at
37°C under 5% CO2. Approximately 5 × 105
cells were cotransfected with 125 to 250 ng of a cytomegalovirus promoter-driven  -galactosidase expression vector and the indicated quantities of plasmids in 6-well dishes using Superfect (Qiagen, Valencia, CA) according to the manufacturer's instructions.
Transfected cells were harvested 24 to 48 hours after transfection for
luciferase and -galactosidase assays. Cells were lysed by the
addition of 500 µL reporter lysis buffer (Promega) into each well.
Cells were scraped from each well and lysates were incubated at room
temperature with gentle shaking for 10 minutes followed by
centrifugation at 14 000 rpm for 5 minutes to pellet debris. Twenty
microliters of lysate was used to measure luminescence from luciferase
or from a chemiluminescent -galactosidase assay in a Tropix TR717 microplate reader (Applied Biosystems, Foster City, CA) following the
manufacturer's protocol (Promega). Luminescence units were normalized
for transfection efficiency using -galactosidase activity.
Isolation and mapping of c/ebp1 in zebrafish A zebrafish kidney cDNA library was screened with a 264-bp PCR product encoding the conserved bZIP domain of human C/EBP to
identify C/EBP family members from zebrafish. One c/ebp
cDNA, represented by 25% of the clones, was not orthologous to
any mammalian C/EBP genes and was named
c/ebp1.
The start of the open reading frame for c/ebp1 was
determined based on the rules of Kozak.32
c/ebp1 encodes a 170-aa protein including a bZIP domain
containing 4 leucines in a heptad periodicity. The bZIP domain of
c/ebp1 was between 26% and 50% identical and 66% and 79% conserved
when compared to the bZIP domains of the human C/EBP family members.
However, the amino-terminal region of c/ebp1 showed no significant
homology with any of the human C/EBPs (Figure
1A). Further, BLAST analysis did not
reveal homology between the amino-terminal region of c/ebp1 and any
open reading frame in GenBank. To clarify the evolutionary relationship
of c/ebp1 to other C/EBP family members, a phylogenetic tree was generated using an alignment of the conserved bZIP region of c/ebp1 with those of all the human C/EBP family members and the recently cloned zebrafish c/ebpa, c/ebpb,
c/ebpd, and c/ebpg genes (S.E.L. et al,
manuscript in preparation) (Figure 1B). In this analysis, c/ebp1 was
excluded from the branches containing the human C/EBP family members
and thus does not appear to have a human ortholog.
The c/ebp1 gene was mapped to the zebrafish genome to aid
future mutant screening analyses. PCR primers within the coding sequence of c/ebp1 were used to type the Goodfellow
radiation hybrid panel.26 The PCR primers used did not
amplify other zebrafish c/ebps or sequences in hamster
genomic DNA (data not shown). The c/ebp1 gene was mapped to
linkage group 24 (LG24) between 71 and 72.1 cM from the top of LG24
with a LOD score of 10.51 (Figure 2).
Expression of c/ebp1 in normal embryos Expression of c/ebp1 throughout embryonic development was analyzed by RNA in situ hybridizations using digoxigenin-labeled antisense RNA. At approximately the 16- to 18-somite (17-18 hpf) stage of development, expression was first detected in a few cells overlying the yolk sac in the embryo (data not shown). By the 20- to 21-somite stage (19.5 hpf), approximately 25 cells located between the epithelial layer and the yolk sac mass were stained (Figure 3A). At approximately 24 hpf, 25 to 50 stained cells could be seen overlying the yolk sac (Figure 3B,D) and stained cells could be seen within the caudal portion of the axial vein. The exact locations of the c/ebp1+ cells over yolk sac and in the axial vein are not fixed, because they are cells in circulation. The stained cells appeared small and round, differing from the yolk syncytial layer underneath, consistent with circulating hematopoietic cells (Figure 3D). The majority of cells appeared to have a scant amount of cytoplasm with a large, circular nucleus (Figure 3E). At 2 dpf, stained cells were still visible within the circulation and the surrounding mesenchyme around the caudal part of the axial vein (Figure 3C). Further, cells were seen in the mesenchyme of the head. There is minimal staining at 3.5 days, with only a few circulating cells visible (data not shown).
The expression pattern appeared similar to that seen with a known
myeloid marker, l-plastin, which is expressed in circulating cells along the yolk sac initially at the 16- to 18-somite stage (17-18 hpf) (data not shown) and subsequently in the axial vein and the
head.17 It is difficult to compare expression patterns between 2 embryos hybridized separately with l-plastin and
c/ebp1 probes, due to the fact that cells expressing either
of these 2 genes are in circulation, and therefore, likely to change
their locations from embryo to embryo. However, when embryos at 24 hpf were double-labeled with the c/ebp1 and l-plastin
probes, the majority of cells visualized expressed both
c/ebp1 and l-plastin (Figure
4). Individual cells appeared to express
different ratios of l-plastin and c/ebp1, as
shown by arrows in Figure 4. These data suggest that c/ebp1
is expressed specifically in myeloid cells from the 16- to 18-somite
stage through 2 dpf stages of development.
Expression of c/ebp1 in hematopoietic mutant embryos To determine if c/ebp1 gene expression is altered in hematopoietic mutants, RNA in situ hybridization was performed with mutant embryos. The bloodless mutant, cloche, has been shown to be affected at a very early stage in both hematopoietic and vascular development.22 The mutation occurs upstream of scl, a gene encoding a transcription factor required for hematopoietic stem cell differentiation.24 Expression of c/ebp1 was absent in cloche embryos as was l-plastin expression (Figure 5).
In a bloodless mutant, m683, scl expression was intact, but gata-1 expression was absent, consistent with a defect beyond the stem cell level.25 In this mutant, expression of both c/ebp1 and l-plastin was normal, suggesting a defect in the erythroid pathway with normal myeloid development. Therefore, c/ebp1 expression parallels l-plastin expression in hematopoietic mutants, consistent with a common lineage for the cells marked by c/ebp1 and l-plastin. Another mutant, spadetail (sptb104), which affects somitic mesoderm specification,30,33 has defects in erythropoiesis with normal vascular development34 and normal expression of pu.1, another early myeloid marker.35 The c/ebp1 gene was expressed later than pu.1 and was maintained in the spadetail mutant (sptb104) (data not shown), again consistent with c/ebp1 as a marker of the myeloid lineage. Finally, a mutant was recently isolated from N-nitroso-N-ethylurea (ENU) mutagenesis screening that has lost expression of l-plastin (S.E.L. et al, results to be described elsewhere). In this mutant, c/ebp1 expression was also lost while expression of the stem cell marker, cbfb,25 and the erythroid marker, gata1, was maintained. Characterization of this mutant suggests a specific defect in the myeloid pathway with intact hematopoietic progenitor and erythroid pathways. Therefore, the loss of c/ebp1 expression in this mutant points strongly toward expression of c/ebp1 specifically in cells of myeloid lineage. Analysis of c/ebp1 as a transcriptional activator Sequence comparison between c/ebp1 and mammalian C/EBP family members revealed significant homology within the bZIP domain, but no homology at the amino terminus. Consistent with the high homology within the bZIP domain, full-length c/ebp1 was able to bind a palindromic C/EBP binding site by an electrophoretic mobility shift assay (Figure 6). This binding was specific because it was competed away with addition of an excess amount of unlabeled self DNA, but not with a mutated site probe. In the same experiment, human C/EBP also bound the C/EBP site specifically (data
not shown).
To assess whether the amino terminus of c/ebp1 could function as a
transcriptional activator, a chimera (GAL4-c/ebp1 [1-87]) containing
the DBD of GAL4 fused to the region of c/ebp1 amino terminal to the
bZIP domain (aa 1-87) was used to activate a GAL4 DNA recognition
site-driven luciferase reporter gene (GAL4-luc) (Figure
7A,B). The GAL4-c/ebp1 (1-87) expression
construct induced the GAL4-luc reporter up to 4000-fold in NIH3T3 cells
relative to induction of the GAL4-luc reporter by the GAL4 DBD without an activation domain (Figure 7B). A dose response was seen when increasing amounts of GAL4-c/ebp1 (1-87) were used to induce the GAL4-luc reporter (Figure 7B). To compare the transcriptional activation activity of c/ebp1 to human C/EBP
In this report, we have identified a novel myeloid-restricted member of the C/EBP family in zebrafish containing a highly conserved bZIP domain and a unique amino-terminal transactivation domain. The existence of 2 copies of many mammalian genes in zebrafish has led to the belief that an additional genome-wide duplication occurred during evolution of teleost fish compared to other vertebrates.37 For example, each HOX-bearing chromosome in humans has 2 paralogous chromosomes in zebrafish.38 However, c/ebp1 is not likely to be the result of this tetraploidization event or a tandem duplication based on its sequence divergence from the other c/ebp family members. No c/ebp1 ortholog has been found in the nearly completed human genome, and therefore, the mammalian ortholog of c/ebp1 may have been lost in humans while being maintained in the zebrafish. The myeloid specificity of c/ebp1 expression in zebrafish embryos is supported by the following observations: (1) c/ebp1 is only expressed in a fraction of circulating cells over the yolk sac and the axial vein; (2) most of the c/ebp1+ cells also express the myeloid marker, l-plastin, another myeloid marker; (3) its expression is abolished in cloche, a mutant affecting hematopoiesis at the level of stem cells; and (4) its expression is retained in mutants affecting specifically the erythroid lineage, whereas it is lost in a mutant affecting specifically the myeloid lineage. It is possible that this gene is also expressed in lymphoid and megakaryocytic lineages, although the location and the timing of the expression make such possibilities unlikely.20,39,40 Most of the cells expressing c/ebp1 also express the macrophage marker, l-plastin. However, there are cells that express varying ratios of c/ebp1 and l-plastin. The cells with these different expression patterns may represent myeloid cells at slightly different stages of development or may represent separate subsets of myeloid cells. Further studies with additional hematopoietic markers, as they become available, will help to elucidate the identity of the cells observed in this analysis. In our transcriptional activation assay, the amino-terminal 87 aa of
c/ebp1 act as a strong transcriptional activation domain, far more
potent than that of human C/EBP c/ebp1 binds a consensus C/EBP site with high affinity and its
amino terminus acts as a potent transcriptional activation domain in a
mammalian system. Therefore, c/ebp1 likely acts as a transcriptional
activator in vivo. Like the mammalian C/EBPs, c/ebp1 may heterodimerize
with other c/ebps, such as c/ebp The myeloid-specific expression pattern of c/ebp1 is
similar to the expression of mammalian C/EBP In this study, we have identified a novel member of the c/ebp family with myeloid-specific expression. This factor has a previously undescribed amino-terminal domain with potent transcriptional activation properties. Our findings suggest that c/ebp1 may play a role in myelopoiesis in the zebrafish. In the near future, the use of c/ebp1 as a marker in directed mutagenesis screens should lead to the identification and characterization of myeloid mutants and thus to the identification of genes crucial to myeloid development.
We thank Maria Anderson and the physical mapping core for radiation hybrid mapping; Brant Weinstein, P. J. Bennett, and Amy Chin for hematopoietic mutant embryos; Bernard Thisse, Neil Perkins, and Julie Lekstrom-Himes for constructs; and Lin Lei, Trevor Blake, and the laboratories of Brant Weinstein and Ajay Chitnis for helpful discussions and technical assistance.
Submitted September 18, 2000; accepted January 7, 2001.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Pu Paul Liu, NHGRI, NIH, 49 Convent Dr, Rm 3A18, Bethesda, MD 20892; e-mail: pliu{at}nhgri.nih.gov.
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© 2001 by The American Society of Hematology.
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A. C. Ward, D. O. McPhee, M. M. Condron, S. Varma, S. H. Cody, S. M. N. Onnebo, B. H. Paw, L. I. Zon, and G. J. Lieschke The zebrafish spi1 promoter drives myeloid-specific expression in stable transgenic fish Blood, November 1, 2003; 102(9): 3238 - 3240. [Abstract] [Full Text] [PDF]
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G. J. Lieschke, A. C. Oates, M. O. Crowhurst, A. C. Ward, and J. E. Layton Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult zebrafish Blood, November 15, 2001; 98(10): 3087 - 3096. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
, 
, 
, 
.
/
5, the region is not significantly acidic in comparison to described
activation domains such as VP16 with a charge of