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HEMATOPOIESIS
From the Department of Pediatric Oncology, Dana-Farber
Cancer Institute; Departments of Medicine and Pathology, Children's
Hospital; and Howard Hughes Medical Institute, Children's Hospital,
Boston, MA.
Genome-wide chemical mutagenesis screens in the zebrafish
(Danio rerio) have led to the identification of novel genes
affecting vertebrate erythropoiesis. In determining if this approach
could also be used to clarify the molecular genetics of myelopoiesis, it was found that the developmental hierarchy of myeloid precursors in
the zebrafish kidney is similar to that in human bone marrow. Zebrafish
neutrophils resembled human neutrophils, possessing segmented nuclei
and myeloperoxidase-positive cytoplasmic granules. The zebrafish
homologue of the human myeloperoxidase (MPO) gene, which is
specific to cells of the neutrophil lineage, was cloned and used to
synthesize antisense RNA probes for in situ hybridization analyses of
zebrafish embryos. Granulocytic cells expressing zebrafish mpo were first evident at 18 hours after fertilization
(hpf) in the posterior intermediate cell mass (ICM) and on the anterior yolk sac by 20 hpf. By 24 hpf, mpo-expressing cells were
observed along the ICM and within the developing vascular system. Thus, the mpo gene should provide a useful molecular probe for
identifying zebrafish mutants with defects in granulopoiesis. The
expression of zebrafish homologues was also examined in 2 other
mammalian hematopoietic genes, Pu.1, which appears to
initiate a commitment step in normal mammalian myeloid development, and
L-Plastin, a gene expressed by human monocytes and
macrophages. The results demonstrate a high level of conservation of
the spatio-temporal expression patterns of these genes between
zebrafish and mammals. The morphologic and molecular genetic evidence
presented here supports the zebrafish as an informative model system
for the study of normal and aberrant human myelopoiesis.
(Blood. 2001;98:643-651) During vertebrate hematopoietic development, common
myeloid progenitors give rise to separate precursors for cells of
the granulocyte/macrophage and erythrocyte/megakaryocyte
lineages.1 Studies of the genetic control of normal
myelopoiesis have focused on transcription factors and growth factors
and their receptors, largely through targeted disruption of these genes
in the mouse.2,3 Many of the genes known to regulate
erythropoiesis in mammals have been identified in another vertebrate
animal model, the zebrafish (Danio rerio).4
Homologues of known mammalian hematopoietic transcription factors, such
as gata-1, gata-2, c-myb, scl, fli-1, Imo2, and
cbfb are all appropriately expressed during zebrafish embryogenesis.5-9 Chemical mutagenesis screens in this
organism have resulted in the isolation of numerous zebrafish lines
containing mutations that affect both embryonic and definitive
erythropoiesis.5,10 Positional cloning of genes
altered in these mutant lines has identified genes that are homologous
to those linked to anemia,11,12 resulting in a new animal
model for human congenital sideroblastic anemia.11
Furthermore, the cloning of the reisling mutation, which
causes a defect in zebrafish erythrocytes analogous to that found in
human hereditary spherocytosis, led to the identification of zebrafish
The site of hematopoiesis in vertebrates changes during
embryogenesis.14 In mammals, primitive hematopoiesis is
largely erythropoietic and occurs outside the embryo in the blood
islands of the yolk sac. In later stages of development, hematopoiesis moves to the aorta-gonad-mesonephros region and the fetal
liver,15 whereas in adults definitive hematopoiesis occurs
in the bone marrow where all blood cell lineages are
produced.16,17 By contrast, primitive hematopoiesis in the
zebrafish occurs within the embryo in a region between the notochord
and endoderm of the trunk called the intermediate cell mass (ICM), as
well as anteriorly in the paraxial mesoderm over the yolk cell and
posteriorly in a small ventral cluster of cells in the developing tail
referred to as the posterior blood island.7,18,19 The
earliest site of zebrafish hematopoiesis is the ICM, a region analogous
to the blood islands of the yolk sac in mammals. Hematopoietic
progenitor cells from the posterior blood island enter the circulation
slightly later than those in the ICM and may represent the end stage of primitive hematopoiesis.7 In the 48-hour zebrafish embryo, cells expressing c-myb are found scattered along the ventral
wall of the dorsal aorta, an expression pattern analogous to the
observed expression of this gene in the progenitors of definitive
hematopoiesis in the aorta-gonad-mesonephros region of mammalian
species.7 This observation suggests that these cells may
represent the first progenitors of definitive hematopoiesis in
the zebrafish.4,7 During later stages of zebrafish
maturation and throughout adulthood the major site of definitive
hematopoiesis is the kidney, although some hematopoietic activity may
occur in the spleen.20
We propose that genome-wide mutagenesis screens in the zebrafish will
detect genes whose dominant or recessive mutation leads to deficiencies
or abnormal distributions of mature granulocytes and that a subset of
these genes will have human counterparts in which loss-of-function
contributes to myeloid cell neoplasias. The ultimate value of such
screens will depend greatly on the reliability of markers of
granulocyte development and the homology between zebrafish and
mammalian myelopoiesis.
The number of myeloid cell lineages and their distinct morphologies
vary among different fish species.21 For example, in Sparus auratus, heterophils (neutrophils) and eosinophils
are relatively numerous, whereas basophils are rare.22,23
In other fish species, only 1 or 2 granulocyte lineages have been
described,21,24,25 and the monocyte/macrophage lineage has
been identified in only a few species.26,27 These
considerations underscore the importance of defining normal myeloid
cell development in the zebrafish before embarking on studies to
dissect the genetic control of this process.
The morphologic, cytochemical, and genetic findings reported here
indicate a high level of conservation of the cell lineages and genes
involved in vertebrate granulopoiesis and support zebrafish mutagenesis
as a feasible approach for defining the molecular mechanisms of myeloid
cell development.
Fish maintenance and embryonic staging
For hematopoietic mutants, retsina and cloche
mutant embryos were generated from crosses between heterozygous males
and females. Both lines were obtained from the laboratory of Leonard
Zon (Howard Hughes Medical Institute, Children's Hospital, Boston, MA).
Blood collection and kidney harvesting
Cytochemical and histochemical stains Blood and kidney smears, as well as kidney touch preparations and cytospins, were stained for the identification of specific myeloid cell lineages, using Wright-Giemsa, leukocyte AP, periodic acid-Schiff, myeloperoxidase, alpha-naphthyl butyrate esterase, chloroacetate esterase, Sudan black, and toluidine blue (all from Sigma, St Louis, MO). Staining procedures followed the manufacturer's instructions.Electron microscopy Adult zebrafish kidney tissue was isolated and fixed in 2.5% formaldehyde and 0.03% picric acid in 100 mM cacodylate buffer (pH 7.4). Samples were washed in buffer and postfixed with 1% osmium tetroxide/1.5% potassium ferrocyanide and embedded in Epon-araldite. Semithin (1 µm) sections were cut and evaluated by light microscopy. Thin sections (60 nm) were then cut and stained with saturated uranyl acetate and lead citrate. These sections were examined and photographed with a JEOL 1200EX electron microscope (JEOL, Tokyo, Japan).Cloning of zebrafish myeloperoxidase Degenerate polymerase chain reaction was performed to amplify a 450-base pair (bp) fragment of myeloperoxidase mpo gene from adult zebrafish kidney complementary DNA (cDNA). This fragment was used to screen an adult zebrafish kidney cDNA library, and more than 50 positive clones were identified by overlapping sequence analysis. Twenty of these positive clones were confirmed to encode the zebrafish mpo gene. The 3486-bp cDNA sequence we obtained encodes a predicted 762-amino acid protein (GenBank Accession No. AF349034). The human and zebrafish proteins share 76.5% similarity and 49.6% identity. In the MPO catalytic domain, the 2 sequences share 82.8% similarity and 56.8% identity (Figure 1).
Whole-mount in situ hybridization Digoxigenin- and fluorescein-labeled RNA probes were transcribed from linear cDNA constructs according to the manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis, IN). Whole-mount in situ hybridization assays were performed according to the method of Schulte-Merker et al29 with minor modifications. Before in situ hybridization, fixed embryos (stored at 20°C in methanol) were rehydrated in PBST and incubated with
proteinase K solution. All incubations were done at room temperature
(RT); the proteinase K concentration and incubation times were
determined according to the embryonic stage of development. Embryos
younger than 24 hpf were not treated. Embryos collected at 24 to 30 hpf were incubated in proteinase K for 10 minutes (10 µg/µL), at 30 to
52 hpf for 20 minutes (20 µg/µL), and at 3 to 5 days past
fertilization (dpf) for 20 minutes (100 µg/µL). Embryos
were then fixed overnight in 4% paraformaldehyde at 4°C, washed in
PBST, transferred to microfuge tubes containing hyb
solution (50% formamide, 5 × sodium chloride/sodium citrate [SSC], 0.1% Tween-20, preheated to 68°C) and incubated for 15 minutes. Hyb solution was replaced with hyb+
(hyb with 5 mg/mL tortula yeast RNA type IV and 50 µg/mL heparin), and the embryos were incubated at 68°C for 1 to 4 hours with gentle agitation. Digoxigenin- or fluorescein-labeled
antisense RNA probe was added, and the embryos were hybridized
overnight at 68°C. After hybridization, they were washed at 68°C as
follows: twice with 2 × SSCT SSC plus 0.1% Tween/50%
formamide (30 minutes), once with 2 × SSCT (15 minutes), and twice
with 0.2 × SSCT (30 minutes), after which they were transferred into
12-well plates, and washed 2 times at RT with MABT (100 mM maleic
acid,150 mM NaCl, Tris pH 9.5 to bring the pH to 7.5, and 0.1%
Tween-20). Embryos were then transferred to blocking solution (MABT
with 10% heat-inactivated fetal calf serum and 2% blocking
reagent, Roche Molecular Biochemicals, Indianapolis, IN) for 1 hour,
followed by incubation with alkaline phosphatase (AP)-coupled
antidigoxigenin or antifluorescein Fab fragment antibody and incubated
overnight at 4°C. Embryos were washed once in blocking solution (30 minutes) and twice in MABT (30 minutes), followed by 3 washes (5 minutes) in staining buffer (50 mM MgCl2, 100 mM NaCl,
0.1% Tween-20, and 1 mM levamisol) containing 100 mM Tris at pH 9.5 for BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate, 4-nitroblue
tetrazolium chloride) staining or 100 mM Tris pH 8.2 for Fast Red
staining (Roche Molecular Biochemicals). The staining reaction,
with either BCIP/NBT or Fast Red, proceeded in the dark until
satisfactory staining of embryos was obtained. Reactions were stopped
by a brief rinse in PBST followed by storage in 4% paraformaldehyde at
4°C. cDNA clones for synthesis of RNA probes were provided by
Christine and Bernard Thisse (L-plastin),30
Graham Lieschke (pu.1, GenBank Accession No. AF321009;
manuscript in preparation), and Leonard Zon (embryonic alpha globin
[ E1]).11
For the hematopoietic mutants, at least 30 embryos from heterozygous incrosses were scored for each in situ hybridization assay, and the results were consistent with approximately 25% of the embryos exhibiting the homozygous recessive phenotype. Double in situ hybridization Double in situ hybridization assays were performed as described above employing simultaneous overnight incubations with digoxigenin- and fluorescein-labeled probes. Subsequent washes and blocking incubations followed the procedures outlined above, but antibody and staining reactions were done sequentially. Briefly, washed embryos were first incubated overnight with AP-coupled-antidigoxigenin Fab fragment, then washed, and reacted with BCIP/NBT. After detection, the embryos were washed twice (20 minutes) in MABT at RT, followed by an incubation (30 minutes) at 65°C in MABT with 10 mM EDTA to inactivate the antibody. The embryos were incubated in 100% methanol at RT to decrease background staining and were rehydrated through a series of 10-minute incubations (75%, 50%, and 25% methanol) back into MABT. The embryos were incubated in MABT for 1 hour, incubated with AP-coupled fluorescein overnight, and reacted with Fast Red, allowing detection of the expression of 2 different genes by the use of distinct chromagenic substrates.
Histology In the adult zebrafish, hematopoietic cells develop in the interstitium of the kidney (Figure 2A), which is positioned in the retroperitoneum along the dorsoposterior aspect of the body cavity. Hematopoietic tissue is situated between the renal tubules (Figure 2B), similar to the placement of developing mammalian blood cells among the fat and stromal elements of bone marrow. This compartment in the zebrafish contains a heterogeneous population of mature hematopoietic cells and their precursors (Figure 2C). Two granulocyte lineages were observed: neutrophils and a unique type of granulocyte, which exhibits characteristics of both basophils and eosinophils (referred to here as a basophil/eosinophil; Figure 2D). Like their human counterparts, zebrafish neutrophils have segmented nuclei; however, their nuclei are divided into 2 or 3 lobes instead of the 5 lobes typically found in human neutrophils (Figure 2F). Two distinct populations of granules were observed in the cytoplasm of zebrafish neutrophils. One population consisted of small, azurophilic granules discernible by Wright-Giemsa staining, and the other population comprised larger granules that failed to stain distinctly with Wright-Giemsa. By contrast, the mature basophil/eosinophil has a relatively smaller, eccentrically placed, nonsegmented nucleus (Figure 2F). Like that of the neutrophil, the basophil/eosinophil cytoplasm was highly granular, but the granules were relatively large and spherical and did not stain with Wright-Giemsa (Figure 2D,F).
The maturation of zebrafish granulocytes roughly parallels that seen in mammalian myelopoiesis. Blast cells were not discernible in the adult zebrafish kidney, but occasional granulocytic promyelocytes were observed (Figure 2D). These large cells contained round, eccentrically placed nuclei and moderate-to-abundant cytoplasm. The zebrafish neutrophilic promyelocyte lacks the coarse, azurophilic primary granules characteristic of human promyelocytes. As neutrophil maturation progressed, the cells became smaller in size, the chromatin condensed and divided into lobes, and the cytoplasm acquired the characteristic granules of mature neutrophils (Figure 2F). Although early basophil/eosinophil precursors were uncommon, occasional immature forms were observed. These larger cells exhibited diffuse nuclear chromatin and fewer, less-distinct cytoplasmic granules (Figure 2C). Rare macrophages were also observed, characterized by a high cytoplasmic-to-nuclear ratio and diffuse nuclear chromatin (Figure 2E). The macrophage cytoplasm was agranular but was often filled with vacuoles containing phagocytic material, particularly pigment, most likely derived from the pigmented connective tissue surrounding the kidney. Cytochemistry Cytochemical stains are useful in distinguishing cells of different lineages in human bone marrow and are routinely used to evaluate cell differentiation in patients with acute leukemia. We therefore applied such stains to normal zebrafish myeloid cells to further define their enzymatic properties.MPO is an abundant lysosomal enzyme present in the granules of mature human neutrophils and eosinophils. Monocytes are weakly positive for the enzyme, whereas basophils, erythrocytes, platelets, and lymphocytes are typically negative. In zebrafish, neutrophil granules were strongly MPO+, whereas the basophils/eosinophils were negative (Figure 2G). Acid phosphatase activity is present in many human hematopoietic cells, including lymphocytes, monocytes, and granulocytes. The granules of zebrafish neutrophils reacted strongly with acid phosphatase, whereas the lymphocytes and basophils/eosinophils were negative (Figure 2H). In human granulocytes, components of the cytoplasmic granules may react
with periodic acid-Schiff (PAS). Human neutrophils show a fine, diffuse
staining that is stronger in more mature cells, whereas human
eosinophils and basophils are usually PAS Other histochemical stains routinely used to identify the different cell lineages in human bone marrow, including Sudan Black, the monocytic esterases, chloroacetate esterase, toluidine blue, and AP, were also applied to the zebrafish kidney samples, but they failed to reveal distinctive staining patterns (results not shown). Ultrastructural analysis Electron microscopy was used to confirm the myeloid lineage assignments based on histochemical and cytochemical findings. At low magnification, hematopoietic cells were again observed among the renal tubules of the kidney (Figure 3A). The morphologic features of the hematopoietic cells were more clearly defined at higher magnification. The promyelocyte had a large, round nucleus with diffuse nuclear chromatin, and the cytoplasm contained a few small, round and dense granules (Figure 3B). As maturation progressed, neutrophil precursors at the myelocyte stage began to exhibit the 2 types of distinctive granules. The ultrastructure of one granule type was characterized by its round shape and homogeneous interior, whereas the second granule type showed needlelike paracrystalline inclusions (Figure 3C). Maturing neutrophils contained segmented nuclei and increasing numbers of both types of cytoplasmic granules (Figure 3B,C).
Although occasional immature cells were found, most of the basophils/eosinophils identified in the adult kidney were nearly fully mature, as in mammalian bone marrow (Figure 3D). The zebrafish basophil/eosinophil has a relatively small, eccentrically placed nucleus and abundant round cytoplasmic granules with homogeneous interiors that lack paracrystalline inclusions. Molecular genetic analysis pu.1 expression.
In mammals, the transcription factor Pu.1 is one of the earliest
hematopoietic genes expressed and is essential for normal hematopoiesis, specifically in the B-lymphoid and myeloid cell lineages.31 In the zebrafish, pu.1 expression
was observed by 16 hpf in the anterior yolk region of the developing
embryo, as well as in the posterior ICM (Figure
4A). The posterior expression was lost
between 22 and 24 hpf (Figure 4B), whereas anterior expression persisted and was observed in cells spreading anteriorly over the yolk
(Figure 4B,C). By 28 to 30 hpf, pu.1 expression was no longer observed (results not shown).
L-Plastin expression.
The actin-binding protein, L-Plastin, is present in human
peripheral blood leukocytes, predominately in monocytes/macrophages, but also in B lymphocytes, T lymphocytes, granulocytes, and natural killer cells.32,33 The zebrafish homologue of the
L-plastin gene has been cloned, and its expression was
observed in macrophages migrating along the yolk of early
embryos.30 In our studies, the expression of
L-plastin was first observed at 18 hpf and was limited to
cells in the anterior yolk region of the embryo (Figure 5A,B). By 28 hpf, L-plastin
expression was evident in cells in the posterior ICM and along the body
of the embryo (Figure 5C). By 5 dpf, expression of the gene was
drastically reduced, although a few cells continued to express it in
the area of the developing gill arches and thymus (results not
shown).
mpo expression.
In humans, MPO is present in the granules of neutrophils and
eosinophils from the promyelocyte stage through maturity. We cloned the
zebrafish homologue of mammalian Mpo (Figure 1) and used it
to create an antisense RNA probe for whole-mount in situ hybridization
to detect progenitors and differentiating cells of the granulocytic
lineage (Figure 6). The earliest
expression of zebrafish mpo was detected in cells at 18 hpf
and, in contrast to L-plastin, was first observed in the
posterior ICM. Within 1 to 2 hours, cells expressing mpo
were also observed anteriorly, spreading along the yolk sac (Figure
6A). At 3 dpf, the mpo-expressing granulocytic cells were
evident over the anterior yolk, in the posterior ICM, and in the
posterior blood island (Figure 6B); however, by 4 dpf they were
distributed throughout the embryo (Figure 6C).
mpo expression in adult kidney sections. The mpo RNA probe was also used for in situ hybridization assays on paraffin-embedded sections of normal adult zebrafish kidneys (Figure 6D). Expression of the gene was observed in a subset of cells with the morphologic features of neutrophils. Distinct segmented nuclei were observed in the mpo+ cells (Figure 6D, inset), confirming the specificity of mpo expression for granulocytes. Other hematopoietic cells, as well as those of neighboring renal tubules, do not express the gene. mpo expression in the hematopoietic mutants,
cloche and retsina.
The zebrafish cloche mutation affects both endothelial and
hematopoietic cell lineages, and these mutant embryos lack endocardium, head, and trunk vascular endothelium and nearly all blood
cells.34 The ICM of mutant embryos is severely depleted of
hematopoietic stem cells and transcription factors essential for early
hematopoiesis, such as scl, gata-1, and
gata-2, are not expressed except for a few cells in the
posterior ICM.8,35 The expression of mpo in
cloche mutant embryos was examined at several different
developmental stages and found to be absent throughout most of the
embryo, except for a very small number of cells observed in the
posterior ICM (Figure 7B,D,F). No cells
positive for mpo were seen along the anterior yolk or in the
trunk ICM as is seen in wild-type controls (Figure 7A,C,E).
The zebrafish retsina mutation (rettr265) causes a decrease in the red blood cell count after 3 days of development and is lethal during the first week of life.5 We performed double in situ hybridization experiments with retsina embryos using probes against mpo and E1, a member of the embryonic globin
gene family expressed in red blood cells.11 At 4 dpf, the
number of cells expressing E1 is markedly decreased in
the retsina mutant (Figure 7H) when compared with a
wild-type sibling (Figure 7G). By contrast, the number and distribution
of the mpo-expressing cells in the retsina mutant
is indistinguishable from its wild-type sibling.
Double in situ hybridization.
Assays to detect the simultaneous expression of 2 genes within
single cells demonstrated coexpression of mpo and
pu.1 at 22 to 24 hpf in some cells of the anterior yolk sac
and posterior ICM (Figure 8A,B,C).
Coexpression was observed during the brief period of time when
pu.1 expression was decreasing overall and mpo
was just beginning to be strongly expressed.
The double in situ hybridization studies with mpo and L-plastin confirmed our initial findings of independent expression of these 2 genes in distinct regions of the embryo, mpo in the posterior ICM and L-plastin along the anterior yolk sac of the embryo (Figure 9A). By 28 hpf, a mixture of both cell types was evident along the entire length of the embryo (Figure 9B), and occasional coexpression was observed (Figure 9C). However, the majority of cells appeared to express only one or the other of these genes, particularly at later stages of development (4-5 dpf).
Forward genetic screening in the zebrafish offers the opportunity to discover critical myelopoietic genes whose alteration leads to the differentiation arrest that characterizes congenital or acquired premalignant states or overt leukemia. One of the most promising strategies is to detect dominant or recessive mutations that cause a deficiency or abnormal distribution of mature granulocytes. The success of this approach will depend in large measure on the conservation of the genetic mechanisms regulating granulopoiesis in zebrafish versus mammals and the availability of reliable zebrafish markers of myeloid cell development. The findings reported here demonstrate that the zebrafish neutrophil, like its human counterpart, has a segmented nucleus and granular cytoplasm. The cytoplasmic granules appear to be of 2 types, one round and homogeneous and the other elongated with paracrystalline inclusions. Although not characteristic of human neutrophilic granules, these paracrystalline inclusions are common among the neutrophilic granules found in other teleost species.21 The maturation of zebrafish neutrophil precursors shares many of the features of granulocyte development in humans. Although relatively abundant in the kidney of juvenile zebrafish,36 myeloblasts were rare and difficult to identify in the adult kidney. Promyelocytes, large immature-appearing cells containing nonsegmented nuclei with diffuse chromatin, were also present in the zebrafish kidney. The primary granules of zebrafish promyelocytes were not evident by light microscopy, but rare granules were identified by high-magnification electron microscopy. As these granulocytic cells became more differentiated, they acquired their distinctive granularity and nuclear segmentation. One of the features that clearly defines zebrafish granulocytes as neutrophils is their positive staining for MPO, an enzyme found in human neutrophilic granules. A second granulocyte lineage identified in this study is more difficult to classify because these cells exhibit features of human basophils as well as eosinophils. Like human basophils and eosinophils, they possess highly granular cytoplasms, but, in contrast to the human cells, they lack segmented nuclei. The cytoplasmic granules are peroxidase negative, unlike those of human eosinophils, and the robust positive PAS reaction of the granules is uncharacteristic of either human cell type. The ultrastructural features of the granules most resemble those of human basophils and mast cells. In other teleost species, analogous cells have been identified as either basophils or eosinophils.21 Because of this ambiguity, we have tentatively classified these zebrafish cells as basophil/eosinophils. Conceivably, this unusual cell type may be the result of the divergence of mammals from teleosts and, therefore, may not be directly comparable to any single human myeloid cell type. Further functional analyses are needed for the definitive classification of this cell lineage. The results of in situ hybridization experiments using zebrafish homologues of 3 early mammalian hematopoietic genes, Scl, Lmo2, and Gata-2, indicate analogous expression patterns during zebrafish hematopoiesis.6,7 At 12 hpf, these genes are expressed in lateral plate mesoderm as 2 stripes that converge medially by 24 hpf to form the ICM.6,7 In the zebrafish, both hematopoietic and vascular tissues arise from the ICM, which serves as the counterpart of the yolk blood islands in the mammalian embryo.6,7,19 Similar to findings in mammals, the developmental expression of zebrafish gata-1 follows that of scl, lmo2, and gata-2 and is observed by 18 hpf in the ICM.19 By 48 hpf, lmo2 and gata-2 are no longer expressed in blood, and gata-1 expression is severely reduced. Taken together, these results indicate a high level of conservation of the genes and their spatio-temporal expression patterns in early vertebrate hematopoiesis. We cloned the zebrafish mpo gene and demonstrated its specificity for granulocytes by in situ hybridization of adult kidney sections in which the morphologic features of granulocytes could be clearly identified. Moreover, by whole-mount in situ hybridization we established that the expression of mpo persisted throughout early embryogenesis and was seen in regions of early myelopoiesis. These observations show that mpo expression is restricted to cells of the granulocyte lineage, as it is in humans. Furthermore, the profound depletion of mpo-expressing cells in the zebrafish bloodless mutant, cloche, confirms that nonhematopoietic cells do not express mpo. However, the mpo expression in the anemic mutant, retsina, was normal, emphasizing its distinct expression in nonerythroid blood cells. The robust and specific expression of mpo in normal zebrafish granulocytes demonstrates the suitability of this gene as a marker of maturing cells within the granulocyte lineage. We also analyzed the expression of the zebrafish homologues of 2 other known mammalian hematopoietic genes that are expressed in the pathway of myeloid differentiation, pu.1 and L-plastin. The expression of zebrafish pu.1, which is necessary for normal myeloid cell and B-lymphocyte development in the mouse,37 was observed in hematopoietic progenitors between 16 and 30 hpf but not in older embryos. Thus, cells expressing pu.1 appear later than those expressing scl, lmo2, and gata-2, consistent with findings in mammals showing that Pu.1 acts downstream of these transcription factors and may represent a commitment step in myeloid differentiation. In addition, the transient coexpression of pu.1 and mpo at a time when pu.1 expression was decreasing supports the idea that pu.1 expression in early zebrafish myeloid progenitors may regulate their subsequent development into mature mpo-expressing neutrophils. Zebrafish cells expressing mpo and L-plastin represent distinct hematopoietic cell lineages, which was particularly evident in the double in situ hybridization experiments demonstrating the general lack of coexpression of these 2 genes in zebrafish cells. These results correspond to findings in mammals, which show that Mpo expression is specific to granulocytes, whereas L-Plastin expression is specifically expressed in monocytes/macrophages and a subpopulation of lymphocytes.32,33 By injecting DNA constructs containing zebrafish promoter sequences fused to the green fluorescent protein (GFP) gene,38 it has been possible to generate transgenic zebrafish lines that exhibit tissue-specific expression of GFP. The potential utility of this approach was further demonstrated by the creation of transgenic lines capable of erythroid-specific expression of GFP from the gata-1 promoter and of T-cell-specific expression from the rag-1 promoter.38,39 We are presently developing a transgenic zebrafish line that expresses GFP under control of the mpo promoter to visualize GFP-expressing granulocytes. The ability to analyze the dynamic spatial and temporal changes in developing granulocytes of living embryos will enable us to better define the origin, migration, and lineage specification of these cells. Abnormalities of the genes controlling the myeloid differentiation
program can lead to myelodysplastic syndrome and acute myeloid
leukemia, a continuum of devastating diseases in children and adults in
which both stem and progenitor cells of the myeloid lineage are
affected. Heterozygous or homozygous inactivation of
still-to-be-discovered genes has been inferred as a cause of acquired
myelodysplastic syndrome in humans, based on the frequent finding of
nonrandom chromosomal deletions in clonally developing bone marrow
cells (eg, monosomy 7, 5q Genome-wide chemical mutagenesis screens in the zebrafish with the mpo probe described here should help to uncover genes in which heterozygous or homozygous inactivation leads to defective granulocyte development. Our morphologic, histochemical, and genetic analyses of myelopoiesis in the zebrafish support the use of this model to identify mutations that affect granulopoiesis. We postulate that the human homologues of many of the genes required for granulopoiesis in the zebrafish will prove to be the targets of inactivating mutations or deletions in congenital and acquired myelodysplastic syndromes and that forward genetic analysis in the zebrafish will prove to be a useful approach to gene discovery and the generation of reliable animal models for these diseases.
We thank John Gilbert for editorial review, Massimo Loda and his staff at the Dana-Farber In Situ Hybridization Core Facility for their assistance with tissue sectioning and processing, Bernard and Christine Thisse and Graham Lieschke for cDNA clones used for RNA probes, the HMS Electron Microscopy Core Facility, and members of the Look and Zon laboratory for their helpful comments and scientific discussion.
Submitted September 20, 2000; accepted March 4, 2001.
Supported in part by grant DK 02593 from the National Institutes of Health (B.H.P.). B.H.P. is supported in part by a Postdoctoral Fellowship for Physicians from the Howard Hughes Medical Institute.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: A. Thomas Look, Department of Pediatric Oncology, Mayer-630, Dana-Farber Cancer Institute, 44 Binney St, Boston, MA 02115; e-mail: thomas_look{at}dfci.harvard.edu.
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  M. E. Dodd, J. Hatzold, J. R. Mathias, K. B. Walters, D. A. Bennin, J. Rhodes, J. P. Kanki, A. T. Look, M. Hammerschmidt, and A. Huttenlocher The ENTH domain protein Clint1 is required for epidermal homeostasis in zebrafish Development, August 1, 2009; 136(15): 2591 - 2600. [Abstract] [Full Text] [PDF]
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D. Carradice and G. J. Lieschke Zebrafish in hematology: sushi or science? Blood, April 1, 2008; 111(7): 3331 - 3342. [Abstract] [Full Text] [PDF]
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Y. Liu, L. Du, M. Osato, E. H. Teo, F. Qian, H. Jin, F. Zhen, J. Xu, L. Guo, H. Huang, et al. The zebrafish udu gene encodes a novel nuclear factor and is essential for primitive erythroid cell development Blood, July 1, 2007; 110(1): 99 - 106. [Abstract] [Full Text] [PDF]
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L. J. Patterson, M. Gering, C. E. Eckfeldt, A. R. Green, C. M. Verfaillie, S. C. Ekker, and R. Patient The transcription factors Scl and Lmo2 act together during development of the hemangioblast in zebrafish Blood, March 15, 2007; 109(6): 2389 - 2398. [Abstract] [Full Text] [PDF]
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S. A. Renshaw, C. A. Loynes, D. M.I. Trushell, S. Elworthy, P. W. Ingham, and M. K.B. Whyte A transgenic zebrafish model of neutrophilic inflammation Blood, December 15, 2006; 108(13): 3976 - 3978. [Abstract] [Full Text] [PDF]
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J. R. Mathias, B. J. Perrin, T.-X. Liu, J. Kanki, A. T. Look, and A. Huttenlocher Resolution of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish J. Leukoc. Biol., December 1, 2006; 80(6): 1281 - 1288. [Abstract] [Full Text] [PDF]
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S. R. L. Young, C. Mumaw, J. A. Marrs, and D. G. Skalnik Antisense Targeting of CXXC Finger Protein 1 Inhibits Genomic Cytosine Methylation and Primitive Hematopoiesis in Zebrafish J. Biol. Chem., December 1, 2006; 281(48): 37034 - 37044. [Abstract] [Full Text] [PDF]
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H. E. Sabaawy, M. Azuma, L. J. Embree, H.-J. Tsai, M. F. Starost, and D. D. Hickstein TEL-AML1 transgenic zebrafish model of precursor B cell acute lymphoblastic leukemia PNAS, October 10, 2006; 103(41): 15166 - 15171. [Abstract] [Full Text] [PDF]
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M. Schorpp, M. Bialecki, D. Diekhoff, B. Walderich, J. Odenthal, H.-M. Maischein, A. G. Zapata, Tubingen 2000 Screen Consortium, Freiburg Screening Group, and T. Boehm Conserved Functions of Ikaros in Vertebrate Lymphocyte Development: Genetic Evidence for Distinct Larval and Adult Phases of T Cell Development and Two Lineages of B Cells in Zebrafish J. Immunol., August 15, 2006; 177(4): 2463 - 2476. [Abstract] [Full Text] [PDF]
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S. Gupta, H. Zhu, L. I. Zon, and T. Evans BMP signaling restricts hemato-vascular development from lateral mesoderm during somitogenesis Development, June 1, 2006; 133(11): 2177 - 2187. [Abstract] [Full Text] [PDF]
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M. A. Juarez, F. Su, S. Chun, M. J. Kiel, and S. E. Lyons Distinct Roles for SCL in Erythroid Specification and Maturation in Zebrafish J. Biol. Chem., December 16, 2005; 280(50): 41636 - 41644. [Abstract] [Full Text] [PDF]
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H.-F. Lin, D. Traver, H. Zhu, K. Dooley, B. H. Paw, L. I. Zon, and R. I. Handin Analysis of thrombocyte development in CD41-GFP transgenic zebrafish Blood, December 1, 2005; 106(12): 3803 - 3810. [Abstract] [Full Text] [PDF]
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G. J. Weber, S. E. Choe, K. A. Dooley, N. N. Paffett-Lugassy, Y. Zhou, and L. I. Zon Mutant-specific gene programs in the zebrafish Blood, July 15, 2005; 106(2): 521 - 530. [Abstract] [Full Text] [PDF]
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S. E. Craven, D. French, W. Ye, F. de Sauvage, and A. Rosenthal Loss of Hspa9b in zebrafish recapitulates the ineffective hematopoiesis of the myelodysplastic syndrome Blood, May 1, 2005; 105(9): 3528 - 3534. [Abstract] [Full Text] [PDF]
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D. M. Langenau, C. Jette, S. Berghmans, T. Palomero, J. P. Kanki, J. L. Kutok, and A. T. Look Suppression of apoptosis by bcl-2 overexpression in lymphoid cells of transgenic zebrafish Blood, April 15, 2005; 105(8): 3278 - 3285. [Abstract] [Full Text] [PDF]
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S. Berghmans, R. D. Murphey, E. Wienholds, D. Neuberg, J. L. Kutok, C. D. M. Fletcher, J. P. Morris, T. X. Liu, S. Schulte-Merker, J. P. Kanki, et al. tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors PNAS, January 11, 2005; 102(2): 407 - 412. [Abstract] [Full Text] [PDF]
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H.-D. Song, X.-J. Sun, M. Deng, G.-W. Zhang, Y. Zhou, X.-Y. Wu, Y. Sheng, Y. Chen, Z. Ruan, C.-L. Jiang, et al. Hematopoietic gene expression profile in zebrafish kidney marrow PNAS, November 16, 2004; 101(46): 16240 - 16245. [Abstract] [Full Text] [PDF]
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K. Hsu, D. Traver, J. L. Kutok, A. Hagen, T.-X. Liu, B. H. Paw, J. Rhodes, J. N. Berman, L. I. Zon, J. P. Kanki, et al. The pu.1 promoter drives myeloid gene expression in zebrafish Blood, September 1, 2004; 104(5): 1291 - 1297. [Abstract] [Full Text] [PDF]
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D. M. Langenau, A. A. Ferrando, D. Traver, J. L. Kutok, J.-P. D. Hezel, J. P. Kanki, L. I. Zon, A. T. Look, and N. S. Trede In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrafish PNAS, May 11, 2004; 101(19): 7369 - 7374. [Abstract] [Full Text] [PDF]
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A. C. Ward, D. O. McPhee, M. M. Condron, S. Varma, S. H. Cody, S. M. N. Onnebo, B. H. Paw, L. I. Zon, and G. J. Lieschke The zebrafish spi1 promoter drives myeloid-specific expression in stable transgenic fish Blood, November 1, 2003; 102(9): 3238 - 3240. [Abstract] [Full Text] [PDF]
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Z. Lian, Y. Kluger, D. S. Greenbaum, D. Tuck, M. Gerstein, N. Berliner, S. M. Weissman, and P. E. Newburger Genomic and proteomic analysis of the myeloid differentiation program: global analysis of gene expression during induced differentiation in the MPRO cell line Blood, October 16, 2002; 100(9): 3209 - 3220. [Abstract] [Full Text] [PDF]
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T. X. Liu, Y. Zhou, J. P. Kanki, M. Deng, J. Rhodes, H. W. Yang, X. M. Sheng, L. I. Zon, and A. T. Look Evolutionary conservation of zebrafish linkage group 14 with frequently deleted regions of human chromosome 5 in myeloid malignancies PNAS, April 30, 2002; 99(9): 6136 - 6141. [Abstract] [Full Text] [PDF]
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E. C. Liao, N. S. Trede, D. Ransom, A. Zapata, M. Kieran, and L. I. Zon Non-cell autonomous requirement for the bloodless gene in primitive hematopoiesis of zebrafish Development, January 2, 2002; 129(3): 649 - 659. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-spectrin.
