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PHAGOCYTES
Morphologic and functional characterization of granulocytes and macrophages
in embryonic and adult zebrafish
Graham J. Lieschke, Andrew C. Oates, Meredith O. Crowhurst, Alister C. Ward, and Judith E. Layton
The zebrafish is a useful model organism
for developmental and genetic studies.
The morphology and function of zebrafish myeloid cells were characterized.
Adult zebrafish contain 2 distinct granulocytes, a heterophil and a rarer eosinophil,
both of which circulate and are generated
in the kidney, the adult hematopoietic
organ. Heterophils show strong histochemical myeloperoxidasic activity, although weaker peroxidase activity was
observed under some conditions in eosinophils and erythrocytes. Embryonic zebrafish have circulating immature heterophils by 48 hours after fertilization
(hpf). A zebrafish myeloperoxidase homologue (myeloid-specific peroxidase; mpx)
was isolated. Phylogenetic analysis suggested it represented a gene ancestral to
the mammalian myeloperoxidase gene
family. It was expressed in adult granulocytes and in embryos from 18 hpf, first
diffusely in the axial intermediate cell
mass and then discretely in a dispersed
cell population. Comparison of hemoglobinized cell distribution, mpx gene expression, and myeloperoxidase histochemistry in wild-type and mutant embryos
confirmed that the latter reliably identified a population of myeloid cells. Studies
in embryos after tail transection demonstrated that mpx- and peroxidase-expressing cells were mobile and localized to a
site of inflammation, indicating functional
capability of these embryonic granulocytes. Embryonic macrophages removed
carbon particles from the circulation by
phagocytosis. Collectively, these observations have demonstrated the early onset
of zebrafish granulopoiesis, have proved
that granulocytes circulate by 48 hpf, and
have demonstrated the functional activity
of embryonic granulocytes and macrophages. These observations will facilitate
the application of this genetically tractable organism to the study of myelopoiesis. (Blood. 2001;98:3087-3096)
© 2001 by The American Society of Hematology
Introduction
Zebrafish (Danio rerio) have emerged as a useful model organism
for studying a wide variety of physiological systems. Approximately 26 zebrafish mutants have genetic lesions primarily affecting hematopoiesis. Most of these were recognized on the basis of
anemia1,2; hence, it is not surprising that as the mutated genes
underpinning these mutants were cloned, it was noted that they are
genes primarily involved with erythropoiesis. Zebrafish mutants
exist with lesions in genes encoding heme biosynthetic enzymes,3-5
a structural protein,6 and a novel iron transporter.7 Another mutant
has defective vasculogenetic and hematopoietic function,8 suggesting a genetic lesion at the level of the embryonic hemangioblast.
The study of early hematopoietic commitment and erythropoiesis
in these mutants has generated a useful range of reagents.9,10
Unlike erythropoiesis, which generates one mature cell type,
myelopoiesis is a complex process that generates several cell types:
monocytes–macrophages and several types of granulocytes. Teleosts, including cyprinids such as Danio, also have a process of
multilineage myelopoiesis for host defense.11 However, less is
known about zebrafish myelopoiesis than about erythropoiesis.
Macrophages have been recognized in zebrafish as early as the
13-somite stage (15 hours after fertilization [hpf]). They emerge
from the anterior lateral plate mesoderm, migrate over the yolk sac,
phagocytose cell corpses, and clear bacteria from the circulation.12
Two markers of early macrophage commitment were characterized: draculin, which had an expression pattern overlapping that of
early markers of erythroid commitment and also marked the rostral
population of mobile macrophages, and L-plastin, which marked
an early macrophage population as it spread over the yolk sac and a
dispersed axial population of cells presumed to be tissue macrophages.12 A zebrafish homologue of c-fms, the receptor for colonystimulating factor-1, has been isolated. Zebrafish csf1r shows
several differences from its murine counterpart: in zebrafish, csf1r
is expressed in neural crest cells and in macrophages; unlike the
murine osteopetrosis mutant lacking colony-stimulating factor-1,
the zebrafish csf1r mutant panther does not have adult macrophage
deficiency.13
The kidney is the primary adult hematopoietic organ for
zebrafish granulopoiesis14 and a site of granulopoiesis from as early
as 96 hpf.15 Granulocytes circulate in adult and embryonic zebrafish.15,16 Like other teleosts,11 adult zebrafish have at least 2
granulocyte lineages.16 In the zoological literature, these are
usually called heterophil or neutrophil granulocytes (presumed to
be functionally orthologous with the mammalian neutrophil) and
eosinophil granulocytes. Teleost basophil granulocytes are also
From the Ludwig Institute for Cancer Research, The Royal Melbourne Hospital,
Parkville, Victoria, Australia; and the Department of Molecular Biology,
Princeton University, NJ.
Fellowship.
Submitted May 8, 2001; accepted July 11, 2001.
Supported by National Health and Medical Research Council of Australia
project grant 134510 (A.C.W.). G.J.L. is a Wellcome Senior Research Fellow in
Medical Sciences in Australia. A.C.O. was the recipient of a Ludwig Institute
Postdoctoral Fellowship. M.O.C. is the recipient of an Australian Postgraduate
Research Award. A.C.W. is the recipient of a Viertel Senior Research
BLOOD, 15 NOVEMBER 2001 䡠 VOLUME 98, NUMBER 10
Reprints: Graham J. Lieschke, Ludwig Institute for Cancer Research, PO Box
2008, The Royal Melbourne Hospital, Parkville, Victoria, 3050, Australia;
e-mail: [email protected].
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.
© 2001 by The American Society of Hematology
3087
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3088
LIESCHKE et al
described in some species.11,17 Tissue myeloblasts have been
identified in zebrafish embryos on the second day of life in axial
tissues in the vicinity of the yolk sac, apparently entering the
circulation by 34 hpf.15 No molecular marker of zebrafish granulocytes has yet been described, though the recently cloned zebrafish
CCAAT–enhancer-binding protein homologue c-ebp1 is a candidate,18 because mammalian C-EPB⑀ is seen primarily in myeloid
and lymphoid cells and plays an important role in mammalian
granulocyte development. However, c-ebp1 expression largely
overlaps that of L-plastin18; hence, it is unlikely to be granulocyte specific.
Our ultimate goal was to exploit the strengths of zebrafish
genetics to study developmental myelopoiesis, particularly granulopoiesis. Therefore, it was necessary to comprehensively characterize embryonic and adult zebrafish myelopoiesis. In this report, we
describe 2 types of adult zebrafish granulocytes and embryonic
zebrafish granulocytes and macrophages. Histochemical detection
of granulocytes by myeloperoxidase histochemistry was evaluated.
For more specific identification, we cloned and characterized a
myeloid-specific zebrafish peroxidase gene. Rather than presume
that morphologic and enzymatic parallels indicate a parallel
physiological function, as they do in other vertebrates, we devised
simple functional tests of granulocytes and macrophages in zebrafish embryos. Our studies provide a basis for further exploiting
the strengths of this model organism in the study of myelopoiesis.
BLOOD, 15 NOVEMBER 2001 䡠 VOLUME 98, NUMBER 10
ratio of 25 mL stock solution to 87.5 ␮L H2O2. The stain reaction was
allowed to proceed under observation for 1 to 10 minutes and, when focal
staining of cells was evident, was stopped by removing embryos and
washing them repeatedly in tap water. Myeloperoxidase-positive cells were
characterized by a blue–black precipitate immediately after staining, but
within the first 24 hours of storage the color changed to brown, with some
leaching of the precipitate during further storage in 4% paraformaldehyde
in PBS. To stain cells on glass slides, the same reagents were used with a
staining period of 30 seconds; slides were counterstained with Giemsa stain
and were examined under oil without coverslips. For histochemical staining
of hemoglobin, embryos were placed in freshly prepared o-dianisidine stain
solution (40% ethanol with 0.01 M sodium acetate, 0.65% H2O2, and 0.6
mg/mL o-dianisidine [D-9143; Sigma]) for 15 minutes and then were
washed in water.
Electron microscopy
Embryos and tissues fixed in freshly prepared 2.5% glutaraldehyde were
processed as described.22 For peroxidase electron microscopy, the conditions selected for studies of carp leukocytes17 were used, except that
fixation was in 2.5% glutaraldehyde with a reaction mixture containing 0.05
M Tris and 0.01% H2O2 saturated with 3,3⬘-diaminobenzidine hydrochloride, pH 7.6. Postfixation was with 1% OsO4 in 0.1 M phosphate buffer (1
hour, 4°C), and then samples were processed as previously described.22
Measurements of cytoplasmic granule dimensions were made on photographic prints at 17.5 ⫻ 103 and 157.5 ⫻ 103 magnification. Data presented
are mean ⫾ SD (range) for the number of measurements indicated.
Isolation of a zebrafish peroxidase gene
Materials and methods
Zebrafish
Wild-type zebrafish stocks obtained from a local pet shop were held in the
Ludwig Institute Aquarium Facility, and standard husbandry practices were
used. Embryos were grown in 0.003% 1-phenyl-2-thiourea (P-7629; Sigma,
Castle Hill, New South Wales, Australia) to suppress melanization. The
m39 cloche deletion allele19 and the b104 spadetail null allele20 were used.
Collection of zebrafish tissues
Adult zebrafish were anesthetized in water containing 25 to 100 mg/L
benzocaine (E-1505; Sigma), then were dried and killed by cervical
transection. Smears of approximately 1 ␮L blood (collected as it pooled
near the still beating heart) were stained with May-Grünwald-Giemsa
(BDH, Kilsyth, Victoria, Australia). Organs were dissected under a Leitz
dissecting microscope. The posterior kidney was displayed by opening the
peritoneal cavity and removing the abdominal organs, and then it was
removed by scraping it from the ventral surface of the upper abdominal
cavity with fine forceps. To prepare single-cell suspensions, kidneys or
spleens collected from 4 to 8 animals were pooled in 500 ␮L 0.9⫻
phosphate-buffered saline (PBS) and were passed through a 70-␮m sieve
(Falcon 2350; Becton Dickinson, Franklin Lakes, NJ) using a 1-mL syringe
plunger handle. Cell counts in trypan blue confirmed cell viability. Cytospin
preparations were of 104 to 105 cells resuspended in 200 ␮L 50% fetal calf
serum in 0.9⫻ PBS. Tissues were fixed in 10% formaldehyde for histology,
4% paraformaldehyde for in situ hybridization, and 2.5% glutaraldehyde
for electron microscopy.
Database searching identified 2 EST clones with homology to mammalian
leukocyte peroxidases (fj81h09 and fj80f04), which, though similar in their
partially overlapping 5⬘ sequences (GenBank accession numbers AW419670
and AW34911, respectively), had unrelated 3⬘ sequences (AW420468 and
AW420365). Assuming that the 5⬘ sequences of fj81h09 and fj80f04
represented transcripts of the same gene, the 2 primer pairs (5⬘CGGTTCTGTGGATTGTCT-3⬘ with 5⬘-CACGACCACCAGGAGCAA3⬘; and 5⬘-GGATTGTCTGCTCCTCAGA-3⬘ with 5⬘-GCCACCGTCACCAGTCTC-3⬘) were used to amplify 125 and 385 nt fragments, respectively,
from an adult zebrafish kidney cDNA library (gift of L. Zon, Boston, MA).
Sequencing confirmed them to be fragments of a cDNA with homology to
mammalian leukocyte peroxidases. The 385 nt fragment was used for
generating riboprobes and for serial screening the adult kidney cDNA
library under high-stringency conditions (0.1 ⫻ SSC and 0.1% sodium
dodecyl sulfate at 62°C), resulting in 16 positive clones. These clones were
internally sequenced to confirm their identities—clone 16 was sequenced
bidirectionally in full, and selected clones were sequenced regionally to
define several transcript variations. Intron–exon boundaries were compared
between zebrafish and murine genes (GenBank accession number X15378).23
Potential single nucleotide polymorphisms were distinguished from sequencing errors either by their representation in several clones or by bidirectional
sequencing, but they were not confirmed on genomic DNA. GenBank
accession numbers for the zebrafish nucleotide sequences are AF326958
and AF378824-6. Linkage group assignment of fj80f04 was based on
GenBank sequence AW420365 using the primers 5⬘-TGGTTAGAGAATGCCTTATGT-3⬘ and 5⬘-CACAGCATGGACTACCGA-3⬘ and polymerase
chain reaction conditions of 94°C (30 seconds), 55°C (30 seconds), and
72°C (60 seconds) for 40 cycles. The map location was calculated with
SAMapper1.0.
Histochemical staining
Myeloperoxidase staining of whole zebrafish embryos was based on the
method of Kaplow.21 Briefly, embryos were fixed for 60 seconds (1 in 10
dilution of 37% formaldehyde stock in 100% ethanol) and washed for 15 to
30 seconds in water; excess water was carefully removed. Fixed embryos
were placed in freshly prepared incubation mixture, made by mixing a
pre-prepared stock solution (30% ethanol with 3 mg/mL benzidine dihydrochloride [B-3383; Sigma], 1.32 mM ZnSO4, 0.123 M sodium acetate, and
0.0146 M sodium hydroxide) with hydrogen peroxide (20 vol [6%]) in a
Phylogenetic analysis
Sequence identity values were determined using the CLUSTAL algorithm
in MegAlign application of the DNASTAR suite of programs (Madison,
WI; http://www.dnastar.com) with PAM250 residue weight tables and no
manual adjustments. Dendrograms of peroxidase protein domains were
based on a previous analysis24 using catalytic and region 1 domains as
demarcated therein. For the zebrafish peroxidase, the sequences of the
conceptual translation used were residues 110 to 622 (catalytic domain) and
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BLOOD, 15 NOVEMBER 2001 䡠 VOLUME 98, NUMBER 10
ZEBRAFISH GRANULOCYTES AND MACROPHAGES
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Controls with sense riboprobes prepared in parallel with initial mpx
antisense analyses showed no staining and, hence, subsequently were
not repeated.
Leukocyte function assays
For the minor trauma assay, zebrafish embryos (2-7 days after fertilization
[dpf]) were anesthetized in egg water with 2.5 to 10 mg/L benzocaine, and
the tail was transected near its tip. Embryos were then placed in egg water
without anesthetic until analysis at different time points up to 2 days
after trauma.
To demonstrate phagocytic function, embryos were microinjected with
india ink (Hunt Manufacturing, Statesville, NC) diluted approximately 1:10
in PBS so as to flow freely through the micro-injection needle, using a
finely drawn glass capillary tube and Narishige micromanipulators (Tokyo,
Japan). The most successful outcomes resulted from injections directly into
the chambers of the heart or the veins as they converged toward the heart.
Immediately after a successful injection, the circulation was outlined by
black ink; these embryos were selected for analysis.
Results
Morphology of adult zebrafish leukocytes
Figure 1. Light microscope appearance of adult zebrafish granulocytes. (A) A
heterophil (black arrow) and an eosinophil (arrowhead) in a single field of a
May-Grünwald-Giemsa–stained zebrafish blood smear. (B) Detail of mature heterophils with double- and triple-lobed nuclei (May-Grünwald-Giemsa–stained blood
smear). (C) Detail of mature eosinophils, illustrating characteristic pink cytoplasm and
peripheral nucleus (May-Grünwald-Giemsa–stained blood smear). (D) Hematoxylin
and eosin section of posterior kidney showing an interstitial niche of myelopoiesis
lying between renal tubules (t) and blood vessels (v), including several heterophil
granulocytes recognizable by their indented or segmented nuclei (arrow). (E-J)
Myeloperoxidase histochemical staining of cytospin preparations of single-cell
suspensions prepared from adult zebrafish kidneys. Giemsa-stained preparation (E)
showing mature heterophil granulocytes serving as negative control for (F), which
was stained histochemically for myeloperoxidase and counterstained with Giemsa (to
display nuclear morphology), showing myeloperoxidase-positive cells with black
cytoplasmic staining at various stages of heterophil development (eg, arrow).
*Erythrocyte with weakly peroxidase-staining gray cytoplasm. Myeloperoxidasepositive heterophil granulocytes of various stages of development: promyelocyte (G),
myelocyte (H), metamyelocyte (I), and segmented mature form (J). Myeloperoxidase
positivity is strongest at the myelocyte and metamyelocyte stages of development.
Scale bar ⫽ 10 ␮m in all panels except D, where it equals 24 ␮m.
58 to 109 (region 1). The dendrogram was constructed based on an
alignment generated from Clustal X 1.8125 using default settings and
viewed with Treeview, using linoleate diol synthase from Gaeumannomyces graminis as an outgroup. Bootstrap values derive from 1000 bootstrap
trials. GenBank accession numbers of proteins included in the analysis are
as listed previously.24
In situ hybridization analyses
Whole-mount in situ hybridization analyses were performed as previously
described using a hybridization temperature of 70°C.10,26 The 385 nucleotide fragment of the zebrafish mpx catalytic domain was subcloned into
pBluescript (Stratagene, La Jolla, CA), and riboprobes corresponding to it
were transcribed using T7 polymerase with EcoRV-linearized template
(antisense) and T3 polymerase with BamHI-linearized template (sense).
Adult zebrafish have 2 types of circulating granulocytes (Figure
1A). The more common had a pale cytoplasm and multilobulated
segmented nucleus, typical of the heterophil granulocyte of other
cyprinid teleosts (Figure 1A-B).11,17 The less common had an
eosinophilic cytoplasm with a peripheral nonsegmented nucleus
(Figure 1A, C), typical of the cyprinid eosinophil granulocyte.11,17
Heterophil granulocytes comprised more than 95% of circulating
granulocytes in most animals (n ⬎ 12), though in 2 animals with no
apparent disease eosinophils comprised 24% and 49% of
blood leukocytes.
The kidney is the primary hematopoietic organ of adult
zebrafish.14 Sections of adult kidney showed nests of hematopoietic
tissue between renal tubules and blood vessels (Figure 1D). The
adult posterior kidney contained myeloid cells at all stages of
development, with the heterophil granulocyte lineage the more
abundant (Figure 1E).
To determine whether zebrafish heterophils contained granules
analogous to those of mammalian neutrophils, histochemical
staining for myeloperoxidase (an enzyme characteristic of neutrophil primary granules) was performed. Heterophil series cells
showed strong myeloperoxidase activity (Figure 1E-F). Histochemical demonstration of myeloperoxidase-containing granules facilitated recognition of immature heterophil granulocyte cells of
promyelocyte, myelocyte, and metamyelocyte and segmented
stages of development (Figure 1G-J, respectively). However,
histochemical staining for myeloperoxidase activity was not totally
specific to heterophils. Weak peroxidase activity was evident in
erythrocytes (Figure 1F). Eosinophil granulocytes, recognizable by
their characteristic nuclear morphology and location, were negative
for histochemical myeloperoxidase activity under cytospin staining
conditions. Myeloperoxidase-stained cytospin preparations showed
a 5:1 heterophil–eosinophil ratio (n ⫽ 3), confirming that heterophils were the more numerous zebrafish granulocyte. Large
vacuolated macrophages and immature monocytoid cells were also
negative for histochemical myeloperoxidase activity.
Electron microscopy confirmed the presence of 2 distinct types
of zebrafish granulocytes. Heterophils were characterized by
electron-dense, elongated, cigar-shaped, cytoplasmic granules (Figure 2A-D). Immature heterophils contained numerous mitochondria and a prominent endoplasmic reticulum (Figure 2B). In mature
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LIESCHKE et al
BLOOD, 15 NOVEMBER 2001 䡠 VOLUME 98, NUMBER 10
heterophils, the elongated and sometimes segmented nucleus was
typically peripheral. Heterophils contained up to 110 granules in
their cytoplasmic cross-section. These highly distinctive elongated
granules were 0.42 ⫾ 0.13 ␮m in length (range, 0.23-0.8; n ⫽ 139)
and contained 1 or 2 axes of regularly arrayed electron-dense
lamellations with a periodicity of 3.9 ⫾ 0.4 nm (n ⫽ 20) in groups
of 6.0 ⫾ 1.8 (range, 2-10; n ⫽ 20) lamellae (Figure 2D). Eosinophils were characterized by a cytoplasm packed with larger round
or elliptical granules of longest diameter 0.66 ⫾ 0.26 ␮m (range,
0.17-1.42, n ⫽ 99) with a broad, marbled variation in electron
density (Figure 2A, E-G). Immature eosinophils were densely
packed with rough endoplasmic reticulum (Figure 2E). The correlation between electron microscope granule appearance and light
microscope cell type was secure by virtue of the relative abundance
of the 2 cell types (Figure 2A) and by their characteristic nuclear
morphology and location (Figure 2C, F). Both granulocyte types
were also identified in electron micrographs of adult zebrafish
spleen (data not shown).
We also performed peroxidase electron microscopy with diaminobenzidine (DAB) substrate to localize peroxidase activity within
leukocytes.17 It was difficult to discern whether the density of the
already electron-dense granules of heterophil granulocytes increased under the peroxidase–DAB staining conditions used.
However, there was marked increase in the relative electron density
of the granules of eosinophil granulocytes, indicating that they
contained a peroxidasic activity (Figure 2H-I). In addition, under
these staining conditions, an increase occurred in the relative
electron density of the cytoplasm of erythrocytes, confirming the
presence of a weak peroxidasic activity in their cytoplasm.
To search for zebrafish basophil granulocytes and tissue mast
cells, we surveyed toluidine blue-stained tissue sections of whole
adult zebrafish. No cells with positive cytoplasmic granules were
observed in any organ.
Macrophages were also evident in sections of adult kidney and
spleen (Figure 2A, J-K). They were large cells with numerous
cytoplasmic phagosomes. Large phagosomes containing material
of a density and appearance similar to those of erythrocyte
cytoplasm were commonly observed in splenic and kidney macrophages, suggesting that hemophagocytosis is not unusual in normal
adult zebrafish.
Initiation of granulopoiesis in zebrafish embryos
Figure 2. Electron microscope appearance of adult zebrafish granulocytes and
macrophages. (A) Electron micrograph overview of hematopoietic area of adult posterior
zebrafish kidney, illustrating heterophil granulocytes (eg, black arrow) as the most prevalent
cell, a rarer eosinophil granulocyte (white arrow), and a macrophage (white arrowhead)
containing numerous cytoplasmic phagosomes including electron-dense material of similar
appearance to erythrocyte cytoplasm. (B-D) Electron microscope appearance of zebrafish
heterophil granulocytes. (B) Immature heterophil promyelocyte with large nucleus and few
electron-dense, cigar-shaped cytoplasmic granules. (C) Heterophil metamyelocyte with
cytoplasm densely packed with electron-dense granules and peripheral nonsegmented
nucleus. (D) Higher-power view of the cigar-shaped, electron-dense heterophil granulocyte
cytoplasmic granules, showing their axial electron-denser lamellations. (E-G) Electron
microscope appearance of zebrafish eosinophil granulocytes. (E) Immature eosinophil
promyelocyte with large nucleus and few round cytoplasmic granules of variable electron
density. (F) Eosinophil metamyelocyte with cytoplasm densely packed with round and
oval-shaped granules and peripheral nonsegmented nucleus. (G) Higher-power view of
the characteristic granules of eosinophils, larger than heterophil granules (D) and with
marbled variable electron density. (H, I) Electron micrograph incubated for peroxidase (I)
and negative control (H) showing the peroxidase reactivity of eosinophil granules (white
arrow), evidenced by their darker color in panel I than in panel H, and erythrocyte cytoplasm
(black triangle) under conditions of this stain. The already electron-dense heterophil
granules (black arrow) are not discernibly darker under the peroxidase reaction conditions.
(J, K) Macrophage in kidney (J) and spleen (K) of adult zebrafish, with phagosomes
suggestive of erythrophagocytosis. Scale bar ⫽ 5 ␮m in all panels except D and G, where it
equals 0.5 ␮m.
Zebrafish embryos of 24 hpf and older were surveyed by electron
microscopy to determine when granulocytes first appeared during
zebrafish development. Cells containing characteristic heterophil
granules were reliably found in tissues of 48-hpf zebrafish embryos
(Figure 3A-B) and within axial vessels (Figure 3C-D). This
indicates that primitive granulocytes circulate in zebrafish embryos
at 48 hpf and that cells within embryos containing these granules
are indeed granulocytic leukocytes. No cells containing the granules of an eosinophil granulocyte were detected in embryos of up to
5 dpf.
These observations in fixed embryos correlated with observations made in vivo in 1-phenyl-2-thiourea–treated living embryos
of 2 dpf and older and in adults under Nomarski illumination.
Particularly when the heart rate was slowed by anesthesia, large
round cells that rolled slowly along the vessel walls were observed
within the ventral venous plexus, occasionally lodging momentarily, while erythrocytes streamed past in the center of the vessel.
Embryos were surveyed for peroxidase-positive cells by myeloperoxidase histochemical staining of whole zebrafish embryos
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BLOOD, 15 NOVEMBER 2001 䡠 VOLUME 98, NUMBER 10
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confirmed that it initiates myelopoiesis (G.J. Lieschke et al,
manuscript submitted). Consistent with this, histochemically myeloperoxidase-positive cells were demonstrated scattered throughout
2-dpf spt embryos (Figure 4L-M).
Figure 3. Initiation of granulopoiesis in zebrafish embryos. (A-D) Immature
heterophil granulocytes in a 48-hpf zebrafish embryo. Panels A and C are low-power
electron microscope views of immature heterophil granulocytes in axial tissue (A) and
in an axial vessel (C). The heterophils boxed in panels A and C are recognizable by
the characteristic cigar-shaped, electron-dense granules in cytoplasm and are shown
at higher power in panels B and D. Scale bars ⫽ 2 ␮m (A), 1 ␮m (B).
(Figure 4A-E). Peroxidase enzymatic activity was not detected in
24-hpf embryos (Figure 4A), but peroxidase-positive cells were
scattered throughout 33-hpf embryos (Figure 4B), particularly over
the surface of the yolk and in the ventral vein region. By 48 hpf and
beyond, peroxidase-positive cells were most evident in the ventral
venous plexus (Figure 4C-D) but were scattered throughout the
entire embryo. Peroxidase positivity was cellular (Figure 4E).
Consistent with the weak peroxidasic activity observed in adult
erythrocytes, in embryos of 33 hpf and older, weaker peroxidase
activity was evident in the pooled red blood cells in the region of
the heart (Figure 4B-C). Parallel staining with myeloperoxidase
and o-dianisidine for hemoglobin revealed that the strongly peroxidase-positive cells never pooled within vessels as did erythrocytes
and were significantly larger (Figure 4F-G) than erythrocytes,
indicating that the dispersed population of strongly peroxidase-positive cells was different from that of hemoglobinized
erythrocytes.
We evaluated this histochemical assay for granulocyte identification in 2 zebrafish mutants with perturbed hematopoiesis. The
mutant cloche (clo) fails to initiate hematopoiesis in the lateral
plate mesoderm and intermediate cell mass.8,10,26 However, because peroxidase activity was not detectable histochemically
before 33 hpf in wild-type embryos, embryos were studied at 2 and
3 dpf, when clo homozygous mutant embryos were unequivocally
identifiable by their pericardial edema. Although clo embryos
showed a marked reduction in the number of peroxidase-positive
cells, a few positive cells were detected in some clo embryos,
particularly in the regions of the posterior intermediate cell mass
and the ventral venous plexus (Figure 4J-K). Because there is
residual expression of some markers of erythropoiesis in clo at this
stage,10,26 embryos were stained in parallel for hemoglobin using
o-dianisidine. Although numbers of hemoglobinized cells in the clo
embryos were reduced (Figure 4H-I), the reduction in number of
peroxidase positive-cells was greater (Figure 4I, K). The mutant
spadetail (spt) fails to initiate erythropoiesis,10 but our studies
Figure 4. Initiation of granulopoiesis in zebrafish embryos. (A-E) Whole-mount
myeloperoxidase histochemistry of zebrafish embryos at 26 (A), 31 (B), and 48 (C)
hpf, showing discrete histochemically positive myeloperoxidase reactive cells over
the yolk sac and in the ventral venous plexus (B-E, black arrows). Discrete cellular
staining is shown at higher power under Nomarski illumination in the ventral venous
plexus of 48-hpf embryos (D, E). Diffuse, less intense staining over the surface of the
yolk (arrowhead B, C) is believed to be nonspecific staining in pooled erythrocytes. (F,
G) Comparison of histochemical staining for myeloperoxidase (F) and o-dianisidine
staining (G) for hemoglobin in the head and gill region of 6-dpf embryos. Myeloperoxidase-positive cells are scattered and discrete, whereas hemoglobin-positive cells
are pooled in large blood vessels. (H-K) Comparison of histochemical staining for
myeloperoxidase (J, K) and o-dianisidine staining (H, I) for hemoglobin in wild-type
(H, J) and cloche (I, K) 3-dpf (H, I) and 2-dpf (J, K) embryos. Cloche (clo) embryos
retain some globin expression in the ventral vein region (H, I, arrowhead). Myeloperoxidase-positive cells were fewer and more sparsely scattered and were generally
located in the posterior ICM immediately caudal of the tip of the yolk sac extension (K,
arrow). (L, M) Myeloperoxidase histochemical staining of a 48-hpf spadetail (spt)
embryo (M) and age-matched sibling wild-type embryo (L), showing myeloperoxidasepositive cells (arrows) scattered throughout the spt embryo.
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LIESCHKE et al
Figure 5. A zebrafish myeloid-specific peroxidase (mpx) gene. (A) Domain
homology alignment of Danio rerio (dr) peroxidase, compared with human (hs) and
murine (mm) myeloperoxidase (MPO), eosinophil peroxidase (EPX), lactoperoxidase
(LPO), and thyroid peroxidase (TPO). Bold numbers within domains indicate
percentage amino acid identity to the zebrafish peroxidase in region 1 (shaded) and
the catalytic domain (open). Numbers under the box diagrams representing each
protein indicate the amino acid number of the junction shown, counted from the
initiation methionine, except for zebrafish peroxidase, where the italicized numbering
starts from the first amino acid of the conceptual translation of the incomplete cDNA
clone 16. (B) Three variant carboxyl termini of the Danio rerio peroxidase. The upper
sequence (exemplified by clone 16) represents the most prevalent cDNA form
recovered from library screening (GenBank AF378824). Clone 11 contained a
38-nucleotide deletion of the 38 nucleotides 1930-1967 (position marked by ),
resulting in a change in reading frame and the variant conceptual translation shown
(GenBank AF378825). Clone 14 differed from clones 16 and 11, again diverging after
nucleotide 1929 (marked by *), resulting in the variant conceptual translation shown
(GenBank AF378826). Six clones appeared to have a retained intron of 587-91
nucleotides after nucleotide 1735, and other clones identified 3 more apparently
retained introns (length)—after nucleotide 1065 (91 nt), after nucleotide 1325 (90 nt),
and after nucleotide 1496 (92 nt). (C) Phylogenetic analysis of the Danio rerio
peroxidase with its closest mammalian homologues. Analysis was confined to the
catalytic domain of each protein. The dendrogram was constructed using Clustal X
and Treeview, building on the analysis of the entire peroxidase family as given in
Daiyasu and Toh,24 using linoleate diol synthase (LDS) from Gaeumannomyces
graminis (gg) as an outgroup. Bootstrap values (n ⫽ 1000) are indicated at nodes as
percentages. SPO, salivary peroxidase; bt, bovine.
Isolation and characterization of a zebrafish peroxidase gene
To address the nonspecificity of the histochemical myeloperoxidase stain and to develop an independent way of identifying
zebrafish granulocytes, we isolated a zebrafish peroxidase gene
fragment. Three overlapping clones (clones 9, 15, 16) combined to
describe a 2814-nucleotide cDNA fragment encoding the carboxyl
terminal 678 amino acids of a peroxidase protein, embracing the
region 1 and catalytic domains. Sequence comparison over the
catalytic domain showed 51% and 52% amino acid identity with
that of human and murine myeloperoxidases (Figure 5A) and 53%
identity with human and murine eosinophil peroxidases. There was
also a higher degree of identity in the region 1 domain (58%-64%).
Several other clones appeared likely to represent splice variants
(Figure 5B). Two clones (11, 13) contained a 38-nucleotide
deletion after nucleotide 1929 that resulted in a frame shift and a
121-amino acid variant carboxyl tail (Figure 5B). Clone 14 also
diverged after nucleotide 1929 for its entire remaining length of
743 nucleotides, resulting in a short, 8-amino acid carboxyl
terminus (Figure 5B). These variations occurred near the point at
BLOOD, 15 NOVEMBER 2001 䡠 VOLUME 98, NUMBER 10
which mammalian myelosinophil and eosinophil peroxidases end
when the sequences are aligned.
The remaining 10 partially sequenced clones contained multiple
instances of 4 apparently retained introns that aligned exactly with
the boundaries between exons 9 and 10, 10 and 11, 11 and 12, and
12 and 13 of murine myeloperoxidase,23 though the introns were of
different sizes in the 2 species. This indicates a high degree of
conservation in the genomic structure of this region of these
peroxidase genes.
To determine whether zebrafish peroxidase was orthologous to a
particular mammalian peroxidase gene, we undertook phylogenetic
analysis on the basis of their catalytic domains (Figure 5C). The
zebrafish peroxidase lay basal to the 3 closely related mammalian
peroxidases (myeloperoxidase, eosinophil peroxidase, and lactoperoxidase), thereby complicating the naming of the zebrafish peroxidase we
isolated. This phylogeny suggests that the gene duplication and diversification that occurred in mammals to create these various peroxidases
occurred after the evolutionary divergence of fish and tetrapods. We also
built a phylogenetic tree for the region 1 domains—this, too, placed
zebrafish peroxidase outside the group of closely related mammalian
peroxidases, including outside the thyroid peroxidases, on a node with a
bootstrap value of 69.4%, supporting this evolutionary hypothesis. We
have, therefore, called the zebrafish peroxidase gene we isolated
myeloid-specific peroxidase (mpx), taking into account its expression
pattern described below and avoiding suggestion of the simple orthologous relation that might be inferred from the name myeloperoxidase.
The EST clone fj80f04, which corresponded to our mpx clone
16 at the 5⬘ and 3⬘ ends, was mapped on the T51 radiation hybrid
map to zebrafish linkage group 10 between the SSLP markers
z8146 and z9473, flanked by the mapped ESTs fj59e03 and
fa97h07. There are no closely mapped genes or annotated ESTs to
suggest a syntenic relation between this part of the zebrafish
linkage group 10 and the human genome in the vicinity of
myeloperoxidase on human chromosome 17q23.1.27,28
We also identified 11 potential single-nucleotide polymorphisms; all lay within the protein-coding sequence, though this is
not a complete analysis because not all clones were sequenced in
full. This variation probably reflects the fact that the library was
prepared from RNA from multiple animals of a noninbred strain.
Nine of 11 were conservative polymorphisms [nt# (nt/nt)]: 765(C/
T), 777(T/C), 879(C/G), 897 (C/T), 903(A/G), 984(C/T), 1008(C/
A), 1050(C/T), and 1890(A/G). Two nonconservative variations
were 16(G/C), changing Arg54 to Thr, and 1576(G/A), changing
Gln526 to Lys.
Expression of zebrafish mpx
The expression pattern of mpx was evaluated by whole-mount in
situ hybridization. Cells scattered throughout the adult kidney and
spleen showed strong cytoplasmic expression (Figure 6A-B); no
mpx-expressing cells were seen in other tissues such as the gut (not
shown) or gill arches (Figure 6C). Erythrocytes within vessels were
negative for mpx expression (Figure 6A, C), indicating greater
specificity of this method of detection of leukocyte peroxidase
gene expression.
Expression patterns of mpx in zebrafish embryos are shown in
Figure 6D-K. Expression was first seen at 18 to 19 hpf, diffusely
through the axial intermediate cell mass (Figure 6F). Prolonged
staining incubations of younger embryos did not detect earlier mpx
expression. In older embryos, mpx-expressing cells were scattered
throughout the embryo, particularly over the yolk sac, related to the
axial blood vessels above the posterior yolk sac extension and the
posterior ventral vein plexus, and in the head and pharyngeal
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BLOOD, 15 NOVEMBER 2001 䡠 VOLUME 98, NUMBER 10
ZEBRAFISH GRANULOCYTES AND MACROPHAGES
3093
Figure 6. Expression of zebrafish mpx in adult and embryonic
zebrafish by in situ hybridization. (A-C) Whole-mount in situ
hybridization analysis of mpx expression in adult kidney (A),
spleen (B), and gill vasculature (C). Black arrows indicate blue
cytoplasmic staining indicating gene expression in heterophils of
the adult kidney and spleen but not in erythrocytes in the gill
vessels. Control sections with sense riboprobes showed no
staining under identical hybridization and development conditions. Scale bar (A-C) ⫽ 14 ␮m. (D-E) Expression of mpx in a
30-hpf embryo (E) as detected by an antisense riboprobe (E,
arrow), along with a control embryo hybridized to a sense
riboprobe (D). Panels D and E are composite images bringing
several focal planes together to demonstrate at higher magnification the typical appearance of the scattered mpx-positive cells
indicated by arrows in panels G to K. (F-K) Expression of mpx in
zebrafish embryos from 19 to 72 hpf. Note that expression occurs
first diffusely throughout the intermediate cell mass at 19 hpf (F,
black arrowhead), but from 24 hpf expression is discrete in cells
scattered throughout the embryo, particularly over the surface of
the yolk and in relation to the axial vasculature (G-K, black
arrowheads).
regions of the embryo (Figure 6D-E, G-K). Although mpx mRNA
expression was demonstrated at 19 hpf (12-14 hours before the
first detection of enzymatic activity by histochemical staining),
after 30 hpf the distribution of mpx-expressing cells essentially
recapitulated the pattern demonstrated by myeloperoxidase
histochemistry.
Functional studies of phagocytes in zebrafish embryos
To determine whether the peroxidase-positive granulocytes of
zebrafish embryos were functional, we devised an acute inflammation assay in which the tip of the embryo’s tail was sectioned and
the resultant process of inflammation followed. We initially
evaluated the behavior of histochemically detected peroxidasepositive cells in this assay using embryos at 6 dpf. Before and
immediately after the trauma, there was no aggregation of peroxidase activity at the trauma site (Figure 7A-B), but after 8 hours,
peroxidase activity accumulated at the trauma site (Figure 7C). The
assay was highly reproducible (Figure 7D). Accumulated peroxidase activity was punctate and related to cells (Figure 7E), though
there appeared to be some spread of enzymatic activity beyond the
margins of cells. Patterns of o-dianisidine and peroxidase histochemical staining were significantly different at 6 hours and 2 days
after trauma (Figure 7F-G). Hemoglobinized cells were smaller
than peroxidase-positive cells, and, after 2 days, peroxidasepositive cells remained, despite less hemoglobinized cell accumulation. The specificity of this effect for the peroxidase-positive cell
population was further confirmed when embryos were evaluated
for mpx mRNA expression after trauma. In 2-dpf embryos, before
and immediately after trauma, there were no mpx-expressing cells
at the trauma site, but by 8 hours after trauma, mpx-expressing cells
had accumulated (Figure 7H-J). Heterophil granulocytes were
readily located on electron microscope examination of transverse
sections immediately proximal to the trauma site. Immature
heterophils were found within vessels (Figure 7K) and at sites
otherwise unusual for such cells, such as between muscle fibers (eg,
Figure 7L), suggesting migration of these immature heterophil
granulocytes through tissues and toward the inflammatory site.
To evaluate whether macrophages were functional at an early
age of development (2 dpf) and to develop an assay for displaying
functional macrophages in vivo, we micro-injected embryos with a
suspension of carbon particles. Circulating carbon was cleared and
within 1 hour had accumulated in axial cells of the ventral venous
plexus (Figure 8A-B). Histologic examination of embryos confirmed that the carbon had been taken up into the cytoplasm of
phagocytic cells (Figure 8C-D) and not merely localized in
intravascular embolic aggregates.
Discussion
The nomenclature of piscine granulocytes has long been a source of
confusion.11 Our studies indicate that like other cyprinid teleosts,17,29 adult zebrafish have at least 2 types of granulocytes, a
neutrophilic–heterophilic granulocyte and an eosinophilic granulocyte. The term “acidophilic granulocyte” has also been used,30 but
we have avoided it because it has been confused with eosinophilic
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3094
LIESCHKE et al
BLOOD, 15 NOVEMBER 2001 䡠 VOLUME 98, NUMBER 10
Figure 7. Functional studies of granulocytes in zebrafish embryos. (A-E) Acute inflammation assay for granulocyte function in embryonic zebrafish at 6 dpf, based on
tail transection and whole-mount histochemical staining for myeloperoxidase. Whole-mount zebrafish embryos were subjected to myeloperoxidase histochemical
staining before (A), immediately after (B), and 8 hours after (C) transection near the tail tip. Note the scattered population of darkly staining peroxidase-positive cells in
the ventral vein region in nontraumatized embryos and the accumulation of peroxidase activity at the site of acute inflammation (B and C arrowheads). Panel D displays
an array of 16 embryos, indicating the assay is highly reproducible. At higher power, under Nomarksi illumination, the aggregated myeloperoxidase activity is seen to be
distinctly cellular (E). (F, G) Comparison of the pattern of myeloperoxidase histochemical staining (F) and o-dianisidine staining (G) for hemoglobin in embryos 6 hours
(left tail in panels) and 2 days (right tail in panels) after tail transection, showing the longer persistence of peroxidase-positive cells compared with
hemoglobin-containing cells. (H-J) Identical assay to those in panels A to C, performed in 48-hpf embryos but using whole-mount in situ hybridization for mpx-expressing
cells rather than histochemistry. The pattern of mpx-expressing cells (black dots) is shown before (H), immediately after (I), and 8 hours after (J) tail transection. Note the
accumulation of mpx-expressing cells at the site of transection (shown by the black arrow) after (J), but not before (H, I), 8 hours. (K, L) Electron micrographs showing
granulocytes, recognizable by their characteristic cytoplasmic cigar-shaped, electron-dense granules, in the vicinity of the trauma site in embryos subjected to tail
transection 8 hours earlier. Granulocytes are seen within a vessel (K) and at an unusual site between skeletal muscle fibers (L). Scale bar ⫽ 2 ␮m (K, L).
Figure 8. Functional studies of macrophages in zebrafish embryos. (A, B)
Dissecting microscope appearance of embryos before (A) and 2 hours after (B)
intravascular micro-injection with a carbon particle suspension. Arrows in panel B
indicate the accumulation of black carbon within cells in the ventral venous plexus.
(C, D) Light microscope appearance of hematoxylin and eosin-stained sections of
uninjected embryos (C) and embryos injected previously with carbon suspension (D).
Cell with carbon in the cytoplasm, indented by the nuclei, indicated by white
arrowheads. Other structures are gut and anal canal (black arrowhead), notochord
(n), and yolk (y). Embryos were aged 2 dpf. Scale bars ⫽ 10 ␮m (C, D).
granulocytes and the more abundant heterophilic–neutrophilic
granulocytes. Even within the recent literature, these terms for
granulocytes have been applied inconsistently or even incorrectly.16 It is very important that these names, which refer to the
staining reaction of the cells, not result in mistaken inferences
about the function of these cells in host defense.
The electron-dense, lamellated, cigar-shaped granules of the
more prevalent zebrafish heterophil closely resemble those of the
carp heterophil.11,17,29 Because zebrafish heterophils display myeloperoxidase activity, it is probable that this enzyme is located in
these granules, though we have not proven this. Our observation
that mature heterophils from adults show weaker histochemical
peroxidase activity than immature heterophils (Figure 1G-J) is also
consistent with that of Bielek,17 who found reduced peroxidase
activity in mature carp heterophils compared with immature carp
heterophils. Cyprinid heterophils are implicated in the processes of
acute inflammation and antibacterial defense. Adult carp heterophils showed respiratory burst and bactericidal activity to
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BLOOD, 15 NOVEMBER 2001 䡠 VOLUME 98, NUMBER 10
Aeromonas salmonicida.31 Peripheral blood granulocytosis occurs
in zebrafish experimentally infected with Listeria spp, though the
type of granulocyte is not described.32 Our studies in zebrafish
embryos demonstrate mobilization of peroxidase-expressing cells
to a site of acute inflammation within several hours of traumatization, and they confirm the presence of heterophil granulocytes in
tissues and circulatory areas proximal to the site of trauma. Hence,
we propose that the zebrafish heterophil indeed plays a role
analogous to that of the mammalian neutrophil in these respects.
The functional role of the zebrafish eosinophil is less certain.
Even observations made in other teleosts must be extrapolated with
caution in light of the considerable variation between species in cell
and granule morphology of granulocytes with eosinophilic cytoplasm. We have not yet identified a molecular marker for zebrafish
eosinophils, nor have we determined the point in development at
which production of eosinophil granulocytes is initiated. In due
course, it will be interesting to isolate zebrafish orthologues of
genes important for eosinophil functions in mammals,33 such as
major basic protein, eosinophil-derived neurotoxin, and eosinophil
cationic proteins, and to determine with which zebrafish granulocyte they are associated. Although mammalian eosinophils have
their own unique peroxidase, we have not identified a second
leukocyte peroxidase in zebrafish. Despite annotations in GenBank
suggesting that EST clones exist that may represent a zebrafish
myeloperoxidase and an eosinophil peroxidase, we have shown this
to be false. One clone (fj80f04) is the same as the cDNA we have called
mpx. The other (fj81h09) does not contain the peroxide sequence
AW419670, though it contains AW420468, leading us to think that
AW419670 has been mistakenly linked to fj81h09 in GenBank.
We isolated a zebrafish peroxidase gene expressed in myeloid
cells that we called myeloid-specific peroxidase, or mpx. Phylogenetic analyses of mpx gene on the basis of the catalytic and the
region 1 domains placed it at the base of Daiyasu and Toh24
subfamily 12. This led us to hypothesize that the gene duplication
and diversification in tetrapods that created myeloperoxidase,
eosinophil peroxidase, lactoperoxidase, and salivary peroxidase
postdate the last common ancestor of tetrapods and zebrafish.
Therefore, we suggest that mpx represents an ancestral subfamily
12 gene. Consistent with our hypothesis of an evolutionarily recent
duplication of this family in tetrapods, mammalian eosinophil
peroxidase, lactoperoxidase, and myeloperoxidase lie within 100
kb on human chromosome 17q23.1. In addition, within this region,
lactoperoxidase and myeloperoxidase lie head to head with minimal intervening sequence,34 an arrangement consistent with 2
recent intrachromosomal gene duplications. Similarly, in mice,
myeloperoxidase and eosinophil peroxidase both map to chromosome 11,35,36 (lactoperoxidase is not mapped in mice). Because we
have identified only one zebrafish peroxidase thus far, it is not
possible to say whether fish have undergone their own independent
process of duplication and diversification of this gene family,
though we hypothesize this to be the case. If this has occurred, there
is no reason to presume that the pattern of peroxidase gene
duplication in fish will resemble that in mammals. Indeed, it is
possible that the great diversity of morphology of granulocytes and
their granules within fish reflects this opportunity for divergent
evolution. Given the close location of these 3 peroxidases in the
mammalian genome, it will be interesting to search the genomic
sequence of linkage group 10 in the region of mpx for other
peroxidase genes as it becomes available from the zebrafish
genome project. It is also possible that, in the absence of extensive
ZEBRAFISH GRANULOCYTES AND MACROPHAGES
3095
gene duplication, the alternate splice forms we have described may
be deployed for different functions in zebrafish granulocytes.
Expression of mpx was first observed diffusely in the intermediate cell mass (ICM) at 18 to 19 hpf. This is the region undergoing
active erythroid commitment at this time, as indicated by the
expression of molecular markers of erythroid commitment. A few
hours later, discrete mpx-expressing cells are observed over the
yolk sac and in the axis of the embryo. The significance of the early
diffuse mpx expression in the ICM is unclear, nor is the mechanism
by which dispersed cells arise over the yolk sac with strong
expression of mpx, a marker of terminal myeloid–granulocytic
differentiation. We (G.J. Lieschke et al, manuscript submitted)
have characterized the expression pattern of zebrafish spi1, an
orthologue of mammalian PU.1 that has been shown to function as
a molecular antagonist of Gata-1 and can direct cells toward a
myeloid fate. Expressed in the caudal lateral plate mesoderm
before its convergence to form the axial ICM, spi1 was never
expressed in the axial structure itself. Interestingly, the earliest site
of spi1 expression is in the rostral lateral plate mesoderm anterior
to the heart field, a site from which the early macrophage
population, described by Herbomel et al,12 arises but not from
which granulocytes might be thought to arise, from the later
expression patterns of either c-ebp118 or mpx. However, our
fate-mapping studies confirmed that cells from this early anterior
location end up in the nascent circulation early on the second day of
life and look like the circulating granulocytes we visualized by
electron microscopy (Figure 3D). Whether a subset of these earliest
rostrally arising myeloid phagocytes are those that later express
mpx or whether these earliest mpx-expressing cells arise from a
separate site is uncertain. The expression of spi1 from 14 to 20 hpf
in the caudal lateral plate mesoderm and its later expression from
26 to 30 hpf in the posterior ICM, immediately caudal to the
posterior yolk extension, leave open the possibility that myeloid
commitment is directed at these sites from the moment these
posterior regions of spi1 expression are first observed.
In mammals, monocytes also contain myeloperoxidase granules,37 but this is not typical of quiescent tissue macrophages.38
Although mature macrophages and their immediate precursors are
myeloperoxidase-positive in the cyprinid Carassius auratus L.,39
we did not observe histochemical myeloperoxidase activity in
morphologically identified macrophages and their precursors in
cytospin preparations of adult zebrafish kidney leukocytes, nor did
we detect mpx expression in a pattern corresponding to the first
wave of zebrafish macrophage development.12 Hence, our observations collectively indicate that most cells with myeloperoxidase
activity in adult zebrafish are granulocytes and that there are
defined populations of adult and embryonic zebrafish macrophages
that do not express this enzyme. It remains possible, however, that
in zebrafish a minor population of the myeloperoxidase- or
mpx-expressing cells is monocytic rather than granulocytic, as in
goldfish and mammals. Clarification of the precise lineage specificities of myeloperoxidase activity and mpx expression awaits the
generation of additional independent markers of the several
zebrafish myeloid lineages.
The capacity to generate mutant zebrafish is large, and the tools
to translate mutants into identified genes, including the complete
sequence of the zebrafish genome, are now rapidly being collected.
Existing zebrafish mutants demonstrate the genetic dissociability
of myeloid and erythroid development in the early zebrafish
embryo. In mutants exemplified by spadetail, erythropoiesis fails
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BLOOD, 15 NOVEMBER 2001 䡠 VOLUME 98, NUMBER 10
LIESCHKE et al
(evidenced by a lack of gata1 expression), but myelopoiesis initiates,
because myeloid cells have been demonstrated morphologically
(G.J. Lieschke et al, manuscript submitted) and histochemically
(Figure 3M). A recent report alluded to the generation of a mutant
that initiated erythropoiesis but failed to initiate myelopoiesis on
the basis of a loss of L-plastin and c-ebp1 expression.18 In other
mutants typified by the mutant cloche, the first wave of erythropoiesis and myelopoiesis is absent, though even in the m39 deletion
mutant, erythroid cells and myeloid cells have now been identified
in the ventral vein region after the first day of life (our studies,
Rowley et al,10 and Liao et al26). With a basic understanding of
zebrafish myelopoiesis and an increasing repertoire of tools to
study zebrafish myelopoiesis specifically, it may be hoped that the
progressive generation and study of mutants with selective lineagespecific defects in hematopoiesis and myelopoiesis will contribute
significantly to a comprehensive genome-based understanding of
the genetic regulation of these developmental processes.
Acknowledgments
We thank Sony Varma for excellent technical assistance, Nadine
Watson for electron micrographs, Yi Zhou for radiation hybrid
mapping, Nathan Hall for bioinformatics advice, Ashley Dunn and
the Ludwig Institute Molecular Biology Laboratory for helpful
comments early in this project, Janna Stickland and Pierre Smith
for help with photography and figures, and Cuong Do and Bill
Robinson for comment and discussion. G.J.L. thanks Tony Burgess
for encouragement to apply zebrafish methodologies to the study of
myelopoiesis.
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2001 98: 3087-3096
doi:10.1182/blood.V98.10.3087
Morphologic and functional characterization of granulocytes and
macrophages in embryonic and adult zebrafish
Graham J. Lieschke, Andrew C. Oates, Meredith O. Crowhurst, Alister C. Ward and Judith E. Layton
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