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IMMUNOBIOLOGY
e-Blood
Macrophage-specific gene functions in Spi1-directed innate immunity
*Anna Zakrzewska,1 *Chao Cui,1 Oliver W. Stockhammer,1 Erica L. Benard,1 Herman P. Spaink,1 and Annemarie H. Meijer1
1Institute
of Biology, Leiden University, Leiden, The Netherlands
The Spi1/Pu.1 transcription factor plays a
crucial role in myeloid cell development
in vertebrates. Despite extensive studies
of Spi1, the controlled gene group remains largely unknown. To identify
genes dependent on Spi1, we used a
microarray strategy using a knockdown
approach in zebrafish embryos combined with fluorescence-activated cell
sorting of myeloid cells from transgenic
embryos. This approach of using knockdowns with specific green fluorescent
protein-marked cell types was highly
successful in identifying macrophage-
specific genes in Spi1-directed innate
immunity. We found a gene group downregulated on spi1 knockdown, which is
also enriched in fluorescence-activated
cell-sorted embryonic myeloid cells of a
spi1:GFP transgenic line. This gene
group, representing putative myeloidspecific Spi1 target genes, contained all
5 previously identified Spi1-dependent
zebrafish genes as well as a large set of
novel immune-related genes. Colocalization studies with neutrophil and macrophage markers revealed that genes
cxcr3.2, mpeg1, ptpn6, and mfap4 were
expressed specifically in early embryonic macrophages. In a functional approach, we demonstrated that gene
cxcr3.2, coding for chemokine receptor
3.2, is involved in macrophage migration to the site of bacterial infection.
Therefore, based on our combined transcriptome analyses, we discovered
novel early macrophage-specific marker
genes, including a signal transducer
pivotal for macrophage migration in
the innate immune response. (Blood.
2010;116(3):e1-e11)
Introduction
The SPI1 gene, also known as PU.1, encodes a member of the
E26-transformation-specific family of transcription factors and is
expressed specifically in cells of the hematopoietic lineage.1
SPI1/PU.1 transcription factor activity is crucial for development
of cells of the myeloid and lymphoid lineages.2-7 PU.1-null mice
lack macrophages, neutrophils, and B and T cells, and they die
within the first 48 hours.4,5 Distinct hematopoietic cell fates depend
on graded expression levels of SPI1/PU.1, with a high concentration driving macrophage differentiation, and a lower concentration
promoting B lymphocyte development.2 Several target genes of
SPI1/PU.1 have already been identified in mammals, including
PTPN6,8 PTPRC,9 CSF1R,10 IL-1␤,11 MPO,12 and PU.113 itself.
Based on the presence of cofactors and interaction with other
transcription factors, PU.1-regulated gene groups are expected to
be cell type and condition dependent. Microarray technology has
been used to demonstrate reprogramming of PU.1-dependent
expression during wound-induced inflammation in mouse neonates
and to determine PU.1-regulated gene sets in cultured cells
committed to macrophage differentiation and in preleukemic
hematopoietic stem cells.14-16 However, which precise gene groups
are under control of PU.1 at different stages of myeloid and
lymphoid lineage commitment remains unknown.
The transcriptional regulation of hematopoiesis is largely
conserved in human, mice, and zebrafish. The spi1 (pu.1) gene of
zebrafish is first expressed at 12 hours postfertilization (hpf) in cells
of the anterior lateral plate mesoderm (LPM).17 The second site of
spi1 expression is observed at 14 hpf in the posterior LPM.17,18 In
that region, expression of spi1 overlaps with cells expressing
transcription factor gata1. The cells expressing spi1 in the anterior
LPM develop into myeloid cells, whereas the gata1-expressing
cells in the posterior LPM differentiate into erythroid cells. The
expression of spi1 decreases gradually with only a limited number
of spi1-positive cells in the erythromyeloid progenitors (EMPs)19
in the posterior blood island and sometimes over the yolk at
30 hpf.17 SPI1/PU.1 and GATA1 have been shown to antagonize
each other in human myeloerythroid progenitor cells, and the
reciprocal negative regulation of these transcription factors has
been recapitulated in the zebrafish embryo model.20 Furthermore,
expression of exogenous spi1 mRNA in zebrafish cloche mutant
embryos, which lack hematopoietic and vascular tissues, was
sufficient to rescue myelopoiesis but not erythropoiesis.20 The
morpholino-gene knockdown of spi1 resulted in a complete loss of
primitive macrophage development and a reduced development of
neutrophils.21 Similar to mammalian models, these observations
indicate the crucial role for spi1 in myeloid cell development in
zebrafish.
Blood circulation in zebrafish embryos begins between 24 and
26 hpf. The blood cells present in the 1- to 2-day-old zebrafish
embryos consist of erythrocytes and 2 distinct populations of
myeloid cells that both express the pan-leukocytic marker lcp1
(l-plastin).22,23 One of these myeloid cell populations is marked by
expression of mpx (coding for myeloperoxidase), which is a marker
of differentiated neutrophils at later stages.21,24,25 The other population represents the early zebrafish macrophages that are able to
sense, migrate toward, and phagocytose bacteria injected into body
cavities.22 The csf1r (fms) gene is the only known specific marker
Submitted January 5, 2010; accepted April 13, 2010. Prepublished online as
Blood First Edition paper, April 27, 2010; DOI 10.1182/blood-2010-01-262873.
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 USC section 1734.
*A.Z. and C.C. contributed equally to this study.
The online version of this article contains a data supplement.
BLOOD, 22 JULY 2010 䡠 VOLUME 116, NUMBER 3
© 2010 by The American Society of Hematology
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BLOOD, 22 JULY 2010 䡠 VOLUME 116, NUMBER 3
ZAKRZEWSKA et al
of the macrophage lineage, but this gene is also expressed in neural
crest cells.26 There is a growing need for identification of macrophage-specific marker genes, which will facilitate the understanding of the development, behavior, and role of macrophages in the
immune system.
The zebrafish embryo, because of its transparency, is an excellent
model to study live progression of hematopoiesis and innate immune
responses. Importantly, innate immunity can be studied in separation
from adaptive immunity in zebrafish embryos, as development and
maturation of lymphoid cells require several weeks.27 The generation of
a number of transgenic zebrafish with fluorescent proteins under the
control of promoters of genes involved in distinct aspects of hematopoiesis greatly facilitated studies on cell-fate mapping and understanding
the development of various hematopoietic lineages.28,29 Spi1:GFP
transgenic zebrafish embryos between 12 and 28 hpf show a myeloidspecific green fluorescent protein (GFP) expression pattern that largely
overlaps with expression of endogenous spi1 mRNA.30 Flow cytometric
analysis of GFP-expressing cells from adult zebrafish kidney of the
spi1:GFP line revealed that spi1 is also expressed in cells exhibiting
myeloid or lymphoid morphology, thus indicating that this gene may be
involved in definitive hematopoiesis in zebrafish adults.30,31
Recognizing the importance of the Spi1 transcription factor in
regulation of macrophage and neutrophil development, we set out
to identify the genes that directly or indirectly depend on Spi1 and
are expressed in embryonic myeloid cells. Microarray-based
expression analysis of spi1 knockdown embryos and fluorescenceactivated cell sorter (FACS)–sorted myeloid cells from embryos
of spi1:GFP transgenic zebrafish made it possible to identify
249 putative Spi1 target genes. These include several known genes
with roles in hematopoiesis and immune system development as
well as novel genes with immune-related functions. We demonstrate 4 of these genes (cxcr3.2, mfap4, mpeg1, and ptpn6) to be
macrophage specific in early zebrafish embryos. Furthermore, we
show that the function of gene cxcr3.2, coding for a chemokine
receptor, is necessary for macrophage migration to local bacterial
infection with Salmonella typhimurium.
Methods
calcium-free Ringer solution, followed by digestion with 0.25% trypsin.
The obtained cell suspension was centrifuged, rinsed with phosphatebuffered saline (PBS), and resuspended in Leibovitz medium L15
without phenol red, 1% fetal calf serum, 0.8mM CaCl2, penicillin
50 U/mL, and streptomycin 0.05 mg/mL. The single-cell suspension
was subjected to FACS at room temperature using a FACSAria (BD
Biosciences) with the BD FACSDiva software, Version 5.0.3 and
a Coherent Sapphire solid-state laser 488 nm with 13 mW power.
The GFP⫹ and GFP⫺ cell fractions were collected as in L15 medium
but with 10% fetal calf serum. The standard yield from approximately
300 embryos was approximately 2 ⫻ 105 GFP⫹ cells.
Microarray experiment design and analysis
Embryos injected either with 1mM spi1 morpholino20 or 1mM Standard
Control morpholino were harvested at 28 hpf. As a control, embryos
injected with an equal volume of 1% phenol red in Danieau buffer (1 times)
were used. RNA samples from pools of 20 to 30 spi1 and standard control
morphants were labeled with Cy5 and hybridized against a Cy3-labeled
common reference (a mixture of all samples injected with 1 times phenol
red in Danieau buffer). This experiment was performed in triplicate.
FACS-sorted spi1 GFP embryos were analyzed as follows: RNA of the
GFP⫹ fraction was labeled with Cy5 and hybridized against Cy3-labeled
RNA of the corresponding GFP⫺ cell fraction from the same pool of
embryos. This experiment was performed in duplicate. RNA isolation,
synthesis of amino allyl-labeled aRNA, dye coupling, and hybridization
conditions were as described.33 Microarray analysis was performed using
custom-designed 44k Agilent chips (Gene Expression Omnibus platform
accession no. GPL7735) described elsewhere.33 Microarray data were
processed and analyzed as described.33 Significance cutoffs for differentially expressed probe sequences were set at 1.2-fold change at P less than
.01. The data were submitted to the Gene Expression Omnibus database
(National Center for Biotechnology Information; www.ncbi.nlm.nih.gov/
geo) under accession no. GSE19206.
Whole-mount in situ hybridization
Whole-mount in situ hybridization using alkaline phosphatase detection
with BM Purple substrate (Roche Diagnostics) was carried out as described
using phenylthiourea-treated embryos.33 Colocalization experiments by
whole-mount fluorescent in situ hybridization were performed as described.23 Primer sequences used for polymerase chain reaction (PCR)
amplification of templates for probe synthesis are in supplemental Table 1.
Zebrafish strains
Quantitative RT-PCR of embryonic RNA
Zebrafish (Danio rerio) were handled in compliance with the local animal
welfare regulations and maintained according to standard protocols (www.
Zebrafish Information Network.org). The study was approved by the
Institutional Review Board of Leiden University. The spi1:GFP transgenic
line used in this study (Pu1-lynEGFP, a gift from Franscesca Peri, EMBL,
Heidelberg) contains 4 kb of spi1/pu.1 promoter sequence driving myeloid
expression of membrane-targeted enhanced green fluorescent protein
(EGFP), as validated by immunostaining with antibodies against L-plastin
and Pu.1/Spi1 (supplemental Figure 1, available on the Blood Web site; see
the Supplemental Materials link at the top of the online article).
Real-time PCR was performed as described33 using the primer sequences in
supplemental Table 1.
Morpholino knockdown experiments
Morpholino oligonucleotides (Gene Tools) were diluted in 1⫻ Danieau
buffer (58mM NaCl, 0.7mM KCl, 0.4mM MgSO4, 0.6mM Ca(NO3)2,
5.0mM N-2-hydroxyethylpiperazine-N⬘-2-ethanesulfonic acid; pH 7.6)
containing 1% phenol red (Sigma-Aldrich), and approximately 1 nL was
injected into the 1- or 2-cell–stage embryo using a Femtojet injector
(Eppendorf).
Bacterial infection experiments
S typhimurium strain SF1592 expressing red fluorescent protein was grown
as described.33 Morpholino-injected embryos were anesthetized at 28 hpf
with 200 ␮g/mL tricaine (Sigma-Aldrich) and microinjected in the tail
muscle with 5 nL of bacterial suspension in PBS. Injected embryos were
transferred into fresh egg water, incubated at 28°C for 3 hours, and fixed
with 4% paraformaldehyde in PBS. In situ hybridization was performed
with digoxigenin-labeled mfap4 and fluorescein-labeled mpx probes. Leukocytes accumulated within a 50-␮m radius from the injection site were
counted using a Leica MZ16FA stereo microscope.
Results
Embryo dissociation and FACS
Microarray-based identification of early myeloid-specific genes
regulated by Spi1
Dissociation of embryos was performed according to Covassin et al.32 In
short, embryos were dechorionated using pronase treatment, rinsed in
In this study, we used transcriptome analysis to identify the subset
of genes that are directly or indirectly dependent on the Spi1
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BLOOD, 22 JULY 2010 䡠 VOLUME 116, NUMBER 3
GENE FUNCTIONS IN Spi1-DIRECTED INNATE IMMUNITY
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transcription factor and are expressed in myeloid cells of the
zebrafish embryo. Two distinct microarray experiments were
carried out. First, mRNA isolated from 28-hpf embryos injected
with spi1 morpholino was analyzed. The efficiency of spi1
knockdown was confirmed by whole-mount in situ hybridization
with a probe against lcp1 (coding for L-plastin), a gene known to be
regulated by Spi120 (Figure 1A). In the second approach, we
profiled the transcriptome of GFP⫹ early myeloid cells against the
GFP⫺ remaining cell fraction. Both fractions were obtained from
FACS sorting of a single-cell suspension that resulted from
dissociation of embryos of a transgenic zebrafish line, spi1:GFP
(Figure 1A).
We reasoned that the overlap between genes down-regulated in
the spi1 knockdown and enriched in the GFP⫹ myeloid cell fraction
(Figure 1B) contains genes that are both downstream of Spi1 and
expressed in early myeloid cells. After filtering against control
morpholino microarray data, 249 distinct genes were found in the
overlapping group (Figure 1B; supplemental Table 2). Previously,
5 genes were shown to be downstream of Spi1 in zebrafish
embryos, namely, lcp1, mpx, lyz, coro1a, and ncf1.20 All 5 genes
were found to be significantly down-regulated in the spi1 knockdown embryos, while they were also significantly enriched in the
GFP⫹ myeloid cell fraction (supplemental Figure 2), thus providing an internal control and confirming our analysis approach. In
addition, our gene set contained several homologs of previously
validated target genes of SPI1/PU.1 in mammalian systems,
including ptpn6, ptprc, il1␤, and mpx, as well as the homologs of
2 genes, ctss and rgs18, recently identified as novel SPI1/PU.1
target genes in an integrated microarray and chromatin immunoprecipitation (ChIP)-chip analysis of murine macrophages and hematopoietic cell lines.8,9,11,12,16 Furthermore, spi1 itself was identified
in our 2-step microarray approach, consistent with its myeloidspecific expression pattern and known autoregulatory function.1
Novel genes downstream of Spi1 play a role in hematopoiesis
and immune system development
In the microarray data, 249 genes were identified that are expressed
in embryonic myeloid cells and downstream of the Spi1 transcription factor. Next, using Zebrafish Information Network and National Center for Biotechnology Information databases, we determined which of the 249 genes have at least one Gene Ontology
(GO) term annotation (Figure 1C). Interestingly, we found that
93 of the 249 genes lack any GO term annotation either in zebrafish
or when using human homologs of zebrafish genes. To characterize
the annotated gene group (categories 1 and 2, Figure 1C), a GO
enrichment analysis was performed using DAVID tools for functional classification and clustering (Table 1). To this extent, we
converted the zebrafish gene identifiers to their human homologs,
taking advantage of the fact that the GO annotations of human
genes are more complete.34 DAVID analysis of GO-Biological
process of the myeloid genes downstream of Spi1 revealed
significant enrichment in functional groups involved in “hemopoiesis,” “immune system development,” and “myeloid cell differentiation.” In addition, several GO terms related to “apoptosis” and
“anion transport” were also enriched within the investigated gene
group. In the GO-Cellular Component analysis, we found significant enrichment in several categories associated with “vacuole,”
“lysosome,” and “extracellular matrix,” all consistent with myeloid
cell functions. In summary, the DAVID functional analysis supports our microarray approach and confirms that genes dependent
on Spi1 and expressed in myeloid cells share functions required for
the innate immune system.
Figure 1. Microarray experiment setup and analysis. (A) The experimental setup
of the microarray study. The 28-hpf time point for microarray analysis in both
experiments was chosen after the onset of blood circulation in the embryo and to
ensure the abundance of myeloid cells of the innate immune system as shown by
lcp1 in situ staining of embryos treated with the standard control (SC) morpholino.
The lack of lcp1 staining in spi1 morphants confirms the absence of myeloid cells.
GFP⫹ myeloid cells were isolated from embryos expressing membrane-targeted
EGFP under control of the spi1 promoter. The 2-step approach eliminated false
positives because of leaky expression of the spi1:GFP transgene in the brain and
allowed determination of the specific effect of spi1 knockdown on myeloid cells.
(B) Primary analysis of microarray data. For initial comparative analysis we chose an
arbitrary cut-off of absolute fold change larger than or equal to 1.2 and a P value
ⱕ .01. We found 3551 sequences to be down-regulated and 3684 sequences to
be up-regulated in the spi1 knockdown embryos. In the GFP⫹ myeloid cell fraction,
6327 sequences were enriched and 6335 sequences were reduced compared with
the GFP fraction. The overlap between the sequences found down-regulated in spi1
morphants and enriched in the GFP⫹ myeloid cell fraction was estimated and filtered
against the sequence set found down-regulated in control morpholino-injected
embryos. (C) GO term annotation analysis of the 249 genes found in the overlap.
Category 1 indicates zebrafish genes with at least one GO term annotation; category
2, genes without any annotation in zebrafish but having a homolog with known GO
term annotation in humans; and category 3, genes without any GO term annotation
either in zebrafish or in humans.
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ZAKRZEWSKA et al
Table 1. GO enrichment analysis of the myeloid cell–specific gene set downstream of Spi1
Term
Count
%
P
GO_biological process
7
4.49
⬍ .001
GO:0030097—hemopoiesis
8
5.13
⬍ .001
GO:0006820—anion transport
8
5.13
.001
22
14.10
.001
8
5.13
.001
GO:0006817—phosphate transport
GO:0048513—organ development
GO:0048534—hemopoietic or lymphoid organ development
GO:0065008—regulation of biologic quality
17
10.90
.002
GO:0002520—immune system development
8
5.13
.002
GO:0015698—inorganic anion transport
7
4.49
.002
GO:0006915—apoptosis
15
9.62
.004
GO:0012501—programmed cell death
15
9.62
.005
GO:0030099—myeloid cell differentiation
5
3.21
.006
GO:0008219—cell death
15
9.62
.007
GO:0016265—death
15
9.62
.007
GO:0032502—developmental process
39
25.00
.009
GO_cellular component
77
49.36
⬍ .001
GO:0044424—intracellular part
100
64.10
⬍ .001
GO:0005622—intracellular
104
66.67
⬍ .001
GO:0005764—lysosome
9
5.77
⬍ .001
GO:0000323—lytic vacuole
9
5.77
⬍ .001
GO:0005829—cytosol
14
8.97
⬍ .001
GO:0005773—vacuole
9
5.77
⬍ .001
GO:0044445—cytosolic part
7
4.49
⬍ .001
GO:0044420—extracellular matrix part
6
3.85
.002
44
28.21
.003
GO:0005515—protein binding
73
46.79
⬍ .001
GO:0005198—structural molecule activity
15
9.62
.007
GO:0005737—cytoplasm
GO:0044444—cytoplasmic part
GO_molecular function
The sets of significant ENTREZ Gene IDs were converted to human ENSEMBL gene IDs using g:orth function from G:profiler (http://biit.cs.ut.ee/gprofiler/) and subjected to
GO enrichment analysis using the DAVID database (http://david.abcc.ncifcrf.gov/).33 The resulting categories were considered significant with P ⱕ .001 and an E value of 1.32
or higher. The gene groups with fewer than 5 members were discarded.
Quantitative RT-PCR and in situ hybridization confirm the
expression of immune-related genes downstream of Spi1
Quantitative RT-PCR was used to confirm a subset of 10 novel
genes downstream of Spi1. The genes, cxcr3.2, mfap4, mpeg1,
LOC571584, and plek were among the highest expressed in the
GFP⫹ myeloid fraction, showing approximately 20- to 40-fold
increased expression compared with the GFP⫺ remaining cells of
the embryo. In addition, their expression levels were strongly
reduced in spi1 knockdown embryos compared with wild-type
embryos (Figure 2). Gene cxcr3.2 codes for chemokine-C-X-C
motif-receptor 3.2. Its potential human homolog, CXCR3, is a
G protein-coupled receptor involved in chemotactic migration of
leukocytes.35 Gene mfap4 (which we renamed from zgc:77076) is
homologous to human MFAP4 coding for microfibril-associated
glycoprotein 4, which has been implicated in inflammation
through its binding capacity for surfactant proteins.36 Gene
mpeg1 (formerly named zgc:66409) codes for macrophageexpressed gene 1 in human and mice. Gene LOC571584 is
described as similar to macrophage receptor MARCO (macrophage receptor with collagenous structure), which belongs to the
murine class A scavenger receptor family and is a part of the
innate antimicrobial immune system with its ability to bind
Gram-negative and Gram-positive bacteria.37 Gene plek encodes
pleckstrin, which is involved in intracellular signaling and actin
cytoskeleton reorganization.38
Similarly, our quantitative RT-PCR corroborated the differential
expression of genes ptpn6, lgals9l1, zgc:63982, zgc:64051, and
zgc:65942 (Figure 2B) as found in the microarray. Gene ptpn6
encodes protein tyrosine phosphatase, nonreceptor type 6, also
known as shp1. Human PTPN6 is implicated in negative regulation
of signaling from immune cell receptors.8 Gene lgals9l1 encodes a
galactoside-binding, soluble lectin. A human homolog of gene
zgc:63982, ALOX5AP, is required for synthesis of leukotrienes,
implicated in various types of inflammatory responses.39 CD53⫺
leukocyte surface antigen, the homolog of zebrafish Zgc:64051, is
a cell surface glycoprotein whose familial deficiency has been
linked to an immunodeficiency associated with infectious diseases.40 The homolog of gene zgc:65942, GRAP2, encodes a
member of the GRB2/Sem5/Drk family involved in leukocytespecific signaling.41
Further validation of 4 of the novel Spi1-dependent genes was
performed by whole-mount in situ hybridization. In wild-type
embryos at 28 hpf, genes mfap4, mpeg1, and ptpn6 were expressed
in cells localizing anteriorly over the yolk and in the posterior
blood island (Figure 3A,C,E,G,I,K). This expression pattern is very
similar to that of spi1 or the pan-leukocytic marker lcp1 and typical
for myeloid cells. Expression of cxcr3.2 could only be detected by
fluorescent in situ hybridization but also showed a similar pattern
(Figure 3M,O). The knockdown of spi1 resulted in a total loss of
cells expressing genes mfap4, mpeg1, ptpn6, and cxcr3.2 (Figure
3B,D,F,H,J,L,N,P), demonstrating that Spi1 is required for myeloidspecific expression of these genes. All 4 genes as well as 3 of the
myeloid-specific genes confirmed by quantitative RT-PCR (lgals9l1,
LOC571584, and plek) contain Spi1 consensus binding sites in
their upstream promoter regions, suggesting that they might be
direct targets of Spi1.
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BLOOD, 22 JULY 2010 䡠 VOLUME 116, NUMBER 3
GENE FUNCTIONS IN Spi1-DIRECTED INNATE IMMUNITY
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Figure 2. Quantitative RT-PCR validation of novel genes
downstream of Spi1 identified in microarray data.
(A) Relative expression levels in microarray. Values are
mean ⫾ SEM of all oligos for each gene present on the array.
(B) Relative expression levels in quantitative RT-PCR. For
normalization, peptidylprolyl isomerase A-like (ppial), which
showed no changes over the mRNA samples used, was
taken as reference. Results were analyzed using the ⌬⌬Ct
method. Gene expression values for spi1 MO (morpholino)injected group were calculated relative to the phenol redinjected control group, whereas values for GFP⫹ cells were
calculated relative to values of the GFP⫺ cell fraction. Values
of quantitative RT-PCR data are the mean ⫾ SEM of 2 or
3 biologic replicates.
Cell-type specificity of genes cxcr3.2, mpeg1, mfap4, and ptpn6
in zebrafish embryos
Double fluorescent in situ hybridization was used to determine the
identity of the myeloid cells expressing 4 novel Spi1-regulated
genes found in this study, namely, cxcr3.2, mfap4, mpeg1, and
ptpn6. Colocalization studies were performed using csf1r as a
marker for macrophages and mpx as a marker for a distinct myeloid
population at 28 hpf and for differentiated neutrophils at 2 dpf. The
expression of these 2 genes was previously shown to have no
overlap at 28 hpf and at later stages.21,23
In 28 hpf wild-type embryos, the expression of mfap4
colocalized completely with that of csf1r (Figure 4A-C),
although no overlap was observed with expression of mpx
(Figure 5A-C). Similarly, expression of genes cxcr3.2, mpeg1,
and ptpn6 did not overlap with expression of mpx (Figure 5D-L),
and their expression overlapped almost completely (⬎ 90%)
with expression of macrophage marker csf1r (Figure 4D-L). The
minor difference with the csf1r pattern may be the result of the
differences of expression levels between the genes. The important observation is that expression of mpx and the 4 genes
investigated is mutually exclusive in myeloid cells in 28-hpf
embryos. In 2-dpf embryos, the same results of lack of
colocalization with mpx and almost complete overlap with csf1r
expression were obtained for genes mfap4 and mpeg1. Gene
ptpn6 showed a slight overlap with gene mpx, while we were not
able to detect the fluorescent in situ signal for gene cxcr3.2 at
2 dpf (supplemental Figure 3).
Considering that Spi1 controls development of both the myeloid
and the lymphoid lineage, we investigated whether our genes of
interest are also expressed in the thymus of developing larvae. As
cxcr3.2 expression was below the detection limit of in situ
hybridization at 5 dpf, the timing of lymphoid expression could not
be determined for this gene. Gene ptpn6 showed clear expression in
the thymus at 5 dpf (supplemental Figure 4), which was confirmed
by colocalization with the rag1 marker (supplemental Figure 5).
Genes mfap4 and mpeg1 were expressed at lower levels at 5 dpf
than at earlier stages but still displayed a myeloid-specific expres-
sion pattern and were not expressed in the thymus (supplemental
Figure 4).
Based on these data, we conclude that genes mfap4 and mpeg1
are the most specific novel markers for macrophage-specific
expression in zebrafish embryos.
Transcriptional regulation of hematopoiesis on spi1
knockdown and in early myeloid cells
Transcriptional regulation of hematopoiesis is a well-conserved
process between zebrafish and mammals, and during embryogenesis a wave of primitive hematopoiesis precedes definitive
hematopoiesis in vertebrates.42 We constructed a pathway map
of primitive and definitive hematopoiesis in early zebrafish
embryos, on which we superimposed the microarray data
obtained from 28 hpf spi1 knockdown embryos and GFP⫹
myeloid cells from embryos of spi1:GFP transgenic zebrafish
(Figure 6; supplemental Figure 6).
The hematopoiesis map reveals increased expression of most
early hematopoietic marker genes (drl, scl, lmo2, gata2, and hhex)
in the GFP⫹ myeloid cells. We found 3 genes (c-myb, runx3, and
ikzf1/ikaros) involved in differentiation and proliferation of hematopoietic progenitor cells to be enriched in the GFP⫹ myeloid cells.
The observed increase of expression of transcription factor genes
c/ebpa and c/ebp1 in GFP⫹ cells is concomitant with their role in
myeloid development,21 similar to genes spi1 and ptprc (CD45).
The increased expression levels of gene gata1 and other genes
characteristic of erythrocytes (␣-globin, slc11a2, urod, fch, alas2,
klf4, and znfl2a) are consistent with the expression of spi1 in both
the committed myeloid cells and the EMPs.20
In the spi1 knockdown embryos, we found 5 early hematopoietic marker genes (etsrp, scl, lmo2, hhex, and fli1a) to be weakly
up-regulated, possibly as a transcriptional compensatory mechanism against spi1 deficiency. In addition, the myeloid-expressed
c/ebp1 and c/ebpa genes21 were both up-regulated in spi1 knockdown. Analysis of the transcriptional regulation of the hematopoiesis pathway in spi1 knockdown embryos also revealed activation
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ZAKRZEWSKA et al
BLOOD, 22 JULY 2010 䡠 VOLUME 116, NUMBER 3
Cxcr3.2 function in the response of macrophages to local
bacterial infection
The zebrafish embryonic myeloid cells, macrophages, and neutrophils mount a quick and effective defense response against
wounding or invading pathogens. Although macrophage migration
toward sites of bacterial infection and their phagocytotic activity
have been documented in zebrafish embryos using various infection models, the receptors involved in macrophage migration to
sites of infection are still unknown.
Five members of the chemokine receptor family, namely,
cxcr3.1, cxcr3.2, cxcr4a, cxcr4b, and cxcr7b, were present on our
microarray platform. Only one, cxcr3.2, was found to be specific
for early myeloid cells and downstream of Spi1. The remaining
4 genes did not show significant change in expression in either the
spi1 knockdown or the GFP⫹ myeloid cell fraction. Three other
members of the family were tested by quantitative RT-PCR on
mRNA samples used for the microarray. Genes cxcr1, cxcr2, and
cxcr5 showed enriched expression in GFP⫹ myeloid cells compared with the GFP cell fraction; however, their expression levels
were independent of Spi1 (supplemental Figure 7).
Our discovery of a member of the chemokine receptor family as
an early macrophage-specific downstream target of Spi1 prompted
Figure 3. Expression of mfap4, mpeg1, ptpn6, and cxcr3.2 is dependent on
Spi1 transcription factor. (A-L) Whole-mount in situ hybridization. Wild-type
expression of genes mfap4 (A,C), mpeg1 (E,G), and ptpn6 (I,K) as shown in
whole embryos and enlarged tail regions shows a pattern typical for myeloid cells.
Expression of genes mfap4 (B,D), mpeg1 (F,H), and ptpn6 (J,L) is absent in spi1
knockdown embryos. Images were taken with a Leica M165C stereomicroscope
equipped with DFC420 camera. Composite images of different focal planes were
generated using Adobe Photoshop. (M-P) Single fluorescent in situ hybridization
using TSA Cy3 Plus detection. Wild-type expression of gene cxcr3.2, as shown in
the tail region (M) and in the magnified region of the blood island (O), shows a
pattern typical for myeloid cells. Expression of gene cxcr3.2 (N,P) is absent in spi1
knockdown embryos. Images were taken with Leica TCS SPE confocal microscope, using the 532-nm laser line for scanning of signal in the red channel. The
HC PL Fluotar 10.0⫻/0.30 dry objective was used. The images were processed in
ImageJ Version 1.43 software (National Institute of Mental Health). Bars represent 100 ␮m (M-N) and 60 ␮m (O-P).
of expression of several genes specific to the erythrocyte population (gata1, hbae1, hbbe1, hbbe3, slc11a2, fch, and znfl2a),
consistent with earlier observations that the loss of spi1 redirects
myeloid progenitor cells into erythroid precursors.20
Finally, genes specific for the myeloid lineage at this embryonic
stage, including pan-leukocytic markers (lcp1, coro1a), early
macrophage markers (csf1r and as demonstrated in this study,
mpeg1, cxcr3.2, mfap4, and ptpn6), and neutrophil markers (mpx,
ncf1, and lyz), consistently showed enrichment in expression in
GFP⫹ myeloid cells, although they were all but csf1r repressed in
spi1 knockdown.
Figure 4. Expression of genes mfap4, cxcr3.2, mpeg1, and ptpn6 colocalizes
with expression of macrophage-specific marker csf1r. Double fluorescent in situ
with the csf1r gene. Probes for mRNA are used as indicated in the bottom left corner
of each image. Expression of genes, mfap4 (A-C), cxcr3.2 (D-F), mpeg1 (G-I), and
ptpn6 (J-L) overlaps with expression of macrophage-specific marker csf1r. Images
show summed Z-stacks of consecutive confocal images through the posterior blood
island of 28-hpf embryos (lateral view). A Zeiss axioplan microscope with Bio-Rad
MRC1024ES scanhead was used for confocal imaging. Signals in the green and red
channels were scanned sequentially using, respectively, the 488-nm laser line with
522 DF32 filter for detection of emitted light, and the 568-nm laser line with 605 DF32
filter for detection of emitted light: 10⫻/0.30 NA and 20⫻/0.50 NA Plan-Neofluar
objectives were used. Images were processed in ImageJ Version 1.43 software and
show (from left to right) the signal in the green channel, the signal in the red channel,
and the merged signals. (A-F) Bars represent 20 ␮m. (G-L) Bars represent 60 ␮m.
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BLOOD, 22 JULY 2010 䡠 VOLUME 116, NUMBER 3
GENE FUNCTIONS IN Spi1-DIRECTED INNATE IMMUNITY
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infection and indicate a function for cxcr3.2 in the innate immune
response of zebrafish embryos.
Discussion
Figure 5. Expression of genes mfap4, cxcr3.2, mpeg1, and ptpn6 does not
colocalize with expression of neutrophil-specific marker mpx. Double fluorescent in situ with the mpx gene. Probes for mRNA are used as indicated in the bottom
left corner of each image. Expression of genes, mfap4 (A-C), cxcr3.2 (D-F), mpeg1
(G-I), and ptpn6 (J-L) does not colocalize with expression of the mpx gene. Images
show summed Z-stacks of consecutive confocal images through the posterior blood
island of 28-hpf embryos (lateral view). In addition, no colocalization was observed in
the head and yolk regions or in embryos at 48 hpf when mpx expression marks
differentiated neutrophils (data not shown). Imaging conditions are as in Figure 4.
Bars represent 20 ␮m.
us to investigate the role of cxcr3.2 in macrophage response to a
bacterial cue.
Splice donor and acceptor morpholinos, targeting the single
intron of the cxcr3.2 gene, were used to obtain cxcr3.2 knockdown
embryos (Figure 7A). RT-PCR on embryos injected either with
cxcr3.2 donor or acceptor or combination of both morpholinos
showed a significant depletion of normal cxcr3.2 transcript compared with the control embryos (Figure 7B).
To examine the role of cxcr3.2 in macrophage migration toward
a local bacterial infection, S typhimurium was injected in the tail
muscle of 28-hpf cxcr3.2 knockdown or control embryos (Figure
7C; supplemental Figure 8). In wild-type embryos at 3 hours post
injection (hpi) a clear migration of macrophages was observed as
detected by fluorescent in situ with mfap4, identified as a robust
macrophage marker gene in this study. No significant differences
were observed in the general number of mfap4⫹ cells in the tail
region between cxcr3.2 knockdown and control embryos (data not
shown). The number of macrophages that accumulated at the
infection site at 3 hpi was significantly lower in the cxcr3.2
knockdown embryos compared with the control (Figure 7Di-iii).
Interestingly, the migration of the distinct population of myeloid
cells that expresses the neutrophil specific marker mpx was not
affected by cxcr3.2 knockdown (Figure 7Div). These results
demonstrate that cxcr3.2 is necessary for normal migration of
macrophages, but not mpx-expressing cells, to the sites of bacterial
The Spi1/Pu.1 transcription factor plays a crucial role in myeloid
and lymphoid cell development and is conserved between human
and zebrafish. The temporal separation of myeloid and lymphoid
development during zebrafish embryogenesis offers a unique
opportunity to identify the specific gene group expressed in early
myeloid cells and dependent on Spi1.43 Our microarray-based
analysis of this gene group gives insight into the transcriptional
program of developing myeloid cells and resulted in the identification of specific markers of the macrophage lineage, which have
thus far been lacking in zebrafish embryos. Furthermore, it resulted
in the discovery of an early macrophage-specific chemokine
receptor gene, which is the first shown to be involved in migration
of macrophages during the innate immune response of zebrafish
embryos to bacterial infection.
To identify the putative gene group dependent on Spi1 and
expressed in early embryonic myeloid cells, we used a 2-step
microarray approach, where we determined the overlapping group
between genes enriched in GFP⫹ myeloid cells isolated from
28-hpf embryos of spi1:GFP transgenic zebrafish and genes
down-regulated in embryos that lack myeloid cells resulting from
knockdown of Spi1. The 2-step approach was applied to lower the
rate of false positive discovery. The overlap group consisted of
249 genes. Six of these were known previously to be dependent for
their expression on active Spi1 in the zebrafish embryo,20 thus
validating our approach. These genes included spi1 itself, which is
known to have an autoregulatory function.13 In addition to downregulation of known myeloid cell markers and immune-related
genes, the transcriptional regulation of hematopoiesis in spi1
knockdown embryos resulted in the induction of genes involved in
early steps of hematopoiesis and genes regulating erythroid development (eg, gata1), suggesting that a transcriptional compensatory
mechanism is initiated in the absence of spi1. These data are
consistent with the finding that gata1 drives erythrocyte differentiation by antagonizing spi1 activity in both zebrafish and mammalian
models.6,7,20 GO analysis of the Spi1-dependent gene group
identified by our 2-step microarray approach showed enrichment of
genes functionally linked to the innate immune response. In
addition, approximately one-third of the genes have not yet been
functionally characterized either in zebrafish or in humans; therefore, they represent novel genes potentially involved in myeloid
cell development or function, which are of great interest for further
research. Presently, we do not know which genes identified in our
study are direct targets of Spi1; however, several contain the
consensus Spi1 binding sites44 in the upstream promoter region or
represent validated target genes in mammalian systems. This
includes genes, such as ctss and rgs18, recently shown to be part of
a PU.1 regulatory network in cultured murine macrophages,16
suggesting that these genes are also important during early myeloid
development in the embryo model. Further studies using ChIPmicroarray or ChIP-Seq may assist in discovering which of the
genes identified here are indeed the direct targets of Spi1. In
addition, we found that csf1r, which is also part of the PU.1
regulatory network in murine macrophages, was enriched in early
myeloid cells isolated from zebrafish embryos. This gene was not
selected in our strict 2-step microarray approach, as Spi1dependent down-regulation was not detected, probably because of
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ZAKRZEWSKA et al
BLOOD, 22 JULY 2010 䡠 VOLUME 116, NUMBER 3
Figure 6. Hematopoiesis pathway regulation on spi1
knockdown and in myeloid cells of a developing embryo
at 28 hpf. Expression profiles of the spi1 knockdown embryos and GFP⫹ myeloid fractions at 28 hpf are simultaneously mapped on a schematic representation of hematopoiesis in zebrafish embryos based on the current knowledge
of this process19,42,43 and including the novel macrophagespecific markers identified in this study. The primitive wave of
hematopoiesis in zebrafish initiates at 2 distinct sites. At the
anterior lateral plate mesoderm (ALPM), hemangioblasts
(H) differentiate into myeloid cells (M) or endothelial cells
(EC). At the second site, the posterior lateral plate mesoderm (PLPM), which later converts into the intermediate cell
mass (ICM), hemangioblasts give rise to erythroid cells
(E) and also ECs. In a transient wave of hematopoiesis
occurring in posterior blood island (PBI), the EMPs, which
are the first multipotent hematopoietic progenitor cells,
differentiate into either myeloid or erythroid cells. The definitive wave of hematopoiesis starts in the aorta-gonadmesonephros (AGM), where hemogenic endothelium (HE)
gives rise to hematopoietic stem cells (HSC) and ECs. Gene
boxes are color-coded with the microarray data from spi1
knockdown on the left and GFP⫹ myeloid data on the right
side. Expression of gene csf1r was determined by quantitative RT-PCR as the microarray contained only one probe for
that gene, and it did not return a significant result. Upregulation is indicated in yellow, down-regulation in blue, and
no significant change in white. Genes marked by asterisks
are the genes that were both down-regulated in spi1 knockdown embryos and enriched in the spi1-GFP myeloid cell
fraction, and are the focus of this study. It should be noted
that it is currently unknown to what extent the microarray
expression data from 28-hpf embryos can be extrapolated to
later waves of hematopoiesis. Macrophage specificity of
cxcr3.2 in embryos is based on colocalization data at 28 hpf,
and macrophage specificity of mpeg1, mfap4, and ptpn6 is
based on colocalization studies at 28 and 48 hpf.
Spi1-independent expression of csf1r in neural crest cells of developing
embryos.26 Further insight into the precise gene groups under control of
Spi1 or other hematopoietic transcription factors at different stages of
myeloid and lymphoid development might be obtained by extending our
approach of knockdown analysis in combination with fluorescent
marker lines of other leukocyte cell types.
In situ hybridization results for genes cxcr3.2, mfap4, mpeg1,
and ptpn6 confirmed that all 4 genes are expressed in embryonic
myeloid cells in an Spi1-dependent manner as their expression was
lost in spi1 morphants. In addition, quantitative RT-PCR results
validated the microarray data for 7 new myeloid-specific and
Spi1-dependent genes with suggested functions in the immune
response. By double fluorescent in situ colocalization experiments,
we determined that genes cxcr3.2, mfap4, mpeg1, and ptpn6 all
were macrophage specific in early zebrafish embryos before the
onset of adaptive immunity, as their expression did not overlap
with the expression of neutrophil-specific marker mpx, although
they showed clear colocalization with macrophage marker csf1r.
Although no differentiated neutrophils are present at 28 hpf in the
developing embryo, we clearly show that there are 2 distinct
myeloid cell populations present at this stage, where one expresses
markers, such as csf1r, cxcr3.2, mfap4, mpeg1, and ptpn6, whereas
the second cell population is characterized by expression of mpx.
Gene ptpn6 showed some overlap with mpx activity at 2 dpf,
suggesting that this gene is also expressed in a subset of neutrophils
from this stage. The same may be true for cxcr3.2, of which we
could not detect in situ expression at 2 dpf. However, our prelimi-
nary results of fluorescent reporter gene expression driven by the
cxcr3.2 promoter suggest that this gene is also expressed in a subset
of mpx-expressing cells at later stages of embryo development (M.
van der Vaart, unpublished results, January 2010). At 5 dpf, gene
ptpn6 was also expressed in the thymus consistent with its murine
homolog, which is found in cells of other hematopoietic lineages
and is known as a direct target of Spi1 functioning as a negative
regulator of the immune response.8,45 Expression of genes mfap4
and mpeg1 was not observed in the larval thymus at 5 dpf but still
remained myeloid specific. Compared with the only known macrophage-specific marker csf1r, these markers show more robust
expression, and they are not expressed in neural crest cells.
Although expression of mfap4 in mammalian macrophages is not
known, the perforin-like protein encoded by murine mpeg1 is
expressed in mature macrophages and prion-infected brain cells.46
Gene mfap4 encodes a protein with a fibrinogen receptor-like
domain. A role in inflammation for human MFAP4 was suggested
based on its interaction with D-type lectin surfactant proteins,36 but
it might also have a direct function in pathogen recognition similar
to the fibrinogen-related proteins of invertebrates.47 Whereas GFP
transgenic lines marking the early myeloid, neutrophil, and lymphoid lineages are already available to facilitate real-time analyses
in the zebrafish model, macrophage-specific marker lines are in
great demand. The embryonic macrophage markers downstream of
Spi1 discovered here can greatly facilitate future research into the
role of macrophages in the innate immune response by GFP
transgenic lines that are currently in development.
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BLOOD, 22 JULY 2010 䡠 VOLUME 116, NUMBER 3
GENE FUNCTIONS IN Spi1-DIRECTED INNATE IMMUNITY
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Figure 7. Knockdown analysis of cxcr3.2 using splice donor and
acceptor morpholinos revealed a significantly reduced accumulation of macrophages at sites of bacterial infection. (A) Schematic of
the cxcr3.2 gene and MO targeting sites. Red boxes represent the exons
in the cxcr3.2 gene. The intron (⬃ 4 kb) is not drawn to scale. Red and
blue bars represent the splice donor (D) and acceptor (A) MOs, which
bind to the pre-mRNA on the intron/exon boundaries, inducing an
insertion of the intron into the final transcripts. (B) RT-PCR detection of
cxcr3.2 transcripts on MO knockdown. Altered splicing induced by D or
A MOs resulted in an effective knockdown of the original cxcr3.2 mRNA at
24 hpf, whereas the combination of D ⫹ A MOs extended the knockdown
effect until 48 hpf. The altered splice product with insertion of the
approximately 4 kb intron is not amplified by RT-PCR. (C) Schematic of
the experimental setup and representative images showing the attraction
of macrophages and neutrophils to a local infection site. S typhimurium
bacteria were injected into the embryo tail muscle at 28 hpf followed by a
3-hour incubation. Leukocytes were visualized at 3 hpi after double
fluorescent in situ hybridization: (i) mfap4⫹ macrophages; (ii) mpx⫹
myeloid cells; (iii) merged; and (iv) bright-field image; bar represents
200 ␮m. (D) Migration of innate immune cells after MO knockdown.
Accumulation of macrophages marked by mfap4 (i-iii) and accumulation
of the nonoverlapping mpx⫹ myeloid cell population (iv) were quantified
by determining the percentages of cells accumulated at the infection site
with respect to the total number of cells in the tail. Data were collected
from 3 independent experimental setups, in which the cxcr3.2 D MO (i),
A MO (ii), or D ⫹ A MOs (iii-iv) were tested versus the Standard Control
(SC) MO. Each data point represents an individual embryo, and each
graph represents the combined results of 2 replicate experiments with
the indicated MO combination. Lines indicate the mean, and P values
indicate the level of statistical significance by Student t test. The
knockdown of cxcr3.2 did not have a significant effect on migration of
either macrophages or mpx⫹ myeloid cells in response to sterile injury
(data not shown).
To initiate the investigation of the novel Spi1-dependent genes
in macrophage function, we chose to study gene cxcr3.2 in the
context of the well-established zebrafish embryo model of bacterial
infection with S typhimurium.34,48 We found that cxcr3.2 is the only
zebrafish CXC chemokine receptor gene that is both Spi1dependent and expressed specifically in early myeloid cells. The
local infection of zebrafish embryos with S typhimurium in the
cxcr3.2 knockdown resulted in the reduced migration of macrophages, although the total number of macrophages was not altered
in the absence of cxcr3.2. These results led us to conclude that
cxcr3.2 plays a role in macrophage migration toward bacterial cues.
As cxcr3.2 knockdown did not completely abolish macrophage
migration, it is probable that other currently unknown receptors are
also involved in the response to S typhimurium infection. cxcr3.2 is
most similar to human CXCR3 and CXCR5, which are expressed
predominantly on T and B lymphocytes. Consistent with the role of
cxcr3.2 in cell migration during bacterial infection in zebrafish, it
has been shown that CXCR3-positive cells are recruited to sites of
Mycobacterium tuberculosis infection in primates.49 To our knowledge, the possible function of CXCR3/CXCR5 receptors in the
embryonic immune system of vertebrates has not been addressed,
and our study is the first that directly implicates a CXCR3/5
homolog in the innate immune response to bacterial infection.
However, it has been shown that CXCR3 expression can be induced
on CD34⫹ hematopoietic progenitors from human cord blood by
stimulation with granulocyte-macrophage colony-stimulating factor and that chemotactic activity of these progenitor cells could
subsequently be triggered by CXC chemokines.50 These in vitro
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BLOOD, 22 JULY 2010 䡠 VOLUME 116, NUMBER 3
ZAKRZEWSKA et al
studies suggest that CXCR3 may also have an innate immune
function in mammalians, as we have shown here in vivo for its
homolog in the zebrafish embryo model. In conclusion, this study
contributes to our understanding of the Spi1-dependent development of myeloid cells and has identified novel early macrophagespecific marker genes for zebrafish, including a chemokine receptor
gene with a pivotal role in the migration of macrophages during the
innate immune response.
Acknowledgments
The authors thank Francesca Peri for the gift of the Pu1-lynEGFP
transgenic line, Ulrike Nehrdich, Davy de Witt, and Karen Bosma
for fish care, Zakia Kanwal and Michiel van der Vaart for their help
with in situ experiments, Gerda Lamers for assistance with
microscopy, Menno van der Hoorn for help with FACS sorting, and
David Traver for many helpful suggestions.
This work was supported by the European Commission 6th
framework project ZF-TOOLS (LSHG-2006-037220) and the
Smart Mix Program of The Netherlands Ministry of Economic
Affairs and the Ministry of Education, Culture and Science.
Authorship
Contribution: A.Z. and C.C. designed and conducted experiments
and wrote the manuscript; O.W.S. and E.L.B. contributed to the
experiments; and H.P.S. and A.H.M. designed and supervised the
study and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Annemarie H. Meijer, Institute of Biology,
Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands; e-mail [email protected].
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From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
2010 116: e1-e11
doi:10.1182/blood-2010-01-262873 originally published
online April 27, 2010
Macrophage-specific gene functions in Spi1-directed innate immunity
Anna Zakrzewska, Chao Cui, Oliver W. Stockhammer, Erica L. Benard, Herman P. Spaink and
Annemarie H. Meijer
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