Hindawi Publishing Corporation
Advances in Hematology
Volume 2012, Article ID 830703, 13 pages
NovelInsights intothe GeneticControls of Primitiveand
RamanSood andPaul Liu
Oncogenesis and Development Section, National Human Genome Research Institute, National Institutes of Health, Bethesda,
MD 20892, USA
Correspondence should be addressed to Paul Liu, firstname.lastname@example.org
Received 28 March 2012; Revised 20 May 2012; Accepted 8 June 2012
Academic Editor: Elspeth Payne
Copyright © 2012 R. Sood and P. Liu. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Hematopoiesis is a dynamic process where initiation and maintenance of hematopoietic stem cells, as well as their differentiation
into erythroid, myeloid and lymphoid lineages, are tightly regulated by a network of transcription factors. Understanding the
genetic controls of hematopoiesis is crucial as perturbations in hematopoiesis lead to diseases such as anemia, thrombocytopenia,
or cancers, including leukemias and lymphomas. Animal models, particularly conventional and conditional knockout mice, have
played major roles in our understanding of the genetic controls of hematopoiesis. However, knockout mice for most of the
hematopoietic transcription factors are embryonic lethal, thus precluding the analysis of their roles during the transition from
embryonic to adult hematopoiesis. Zebrafish are an ideal model organism to determine the function of a gene during embryonic-
to-adult transition of hematopoiesis since bloodless zebrafish embryos can develop normally into early larval stage by obtaining
By providing specific examples of zebrafish morphants and mutants, we have highlighted the contributions of the zebrafish model
to our overall understanding of the roles of transcription factors in regulation of primitive and definitive hematopoiesis.
1.Zebrafishasa Model for Hematopoiesis
Recently, zebrafish have emerged as a powerful vertebrate
model system due to their external fertilization, optically
clear embryos, rapid development, availability of tools
for manipulations of gene expression during development,
and the ability to generate genetic mutants by random
(insertional and chemical) and targeted mutagenesis [1–3].
Microinjections of antisense morpholinos, which cause tran-
ysisof the effectsof loss and gain of function of specific genes
during development . Whole-mount in situ hybridization
(WISH) is a powerful technique to analyze the spatiotempo-
ral expression of genes, and placing genes in regulatory cas-
cades by analysis of genetic mutants and/or embryos injected
with morpholinos (commonly termed as morphants) [5, 6].
Specifically for hematopoiesis, zebrafish blood contains
cells of all hematopoietic lineages [7–11] and orthologs
of most transcription factors involved in mammalian
hematopoiesis have been identified indicating evolutionarily
conserved pathways of regulation [12–15]. Initial validation
of the use of zebrafish for hematopoiesis research came
from the forward genetic screens. In 1996, two large-scale
chemical mutagenesis screens were performed to identify
mutants with a variety of phenotypes [16, 17]. Of these,
characterization of 46 mutants with blood phenotypes by
allelic complementation suggested roles for at least 26 genes
in hematopoiesis [18, 19]. Subsequent efforts by several
groups identified the underlying genetic defects in many
of these mutants by positional cloning or candidate gene
approaches. In addition to identifying the genes previously
known to have a role in hematopoiesis (e.g., gata1, sptb, and,
alas2), these mutants also uncovered novel genes with roles
in hematopoiesis, (e.g., slc25a37, slc40a1, and glrx5) [20–
tional conserved pathways of regulation between zebrafish
and mammals [26–28].
This led to a surge of activity in zebrafish research
laboratories, developing a variety of tools for thorough
2 Advances in Hematology
analysis of hematopoiesis. Lineage-specific transgenic lines
genes driving fluorescent markers (reviewed in [29, 30]
and listed in Table 1), allowing for visual observations of
hematopoietic lineages in real-time during development.
Advances in imaging combined with the ability to perform
lineage tracing made it possible to follow the fate of specif-
ically marked cells during development in a live vertebrate
animal model [31, 32]. Sorting of hematopoietic cells by
fluorescence-activated cell sorting (FACS), in vitro culturing
using zebrafish-specific cytokines and kidney stromal cells,
and the ability to perform transplantation have facilitated
characterization of hematopoietic potential of different
While forward screens are biased by the phenotype being
reverse genetic approaches. This has been made possible in
zebrafish in the last decade by TILLING (Targeting-Induced
Local Lesions IN Genomes) [55, 60], and more recently by
targeted mutagenesis using zinc-finger and transcription-
activator-like-effector nucleases (i.e. ZFNs and TALENs)
[61–64]. Furthermore, effects of gene dosage can be analyzed
by injecting suboptimal doses of antisense morpholinos or
studying hypomorphic alleles generated by TILLING. In
this review, we discuss how the technical advances and
genomic tools discussed above went hand-in-hand with the
elucidation of genetic controls of hematopoiesis in zebrafish.
2.Ontogeny of VertebrateHematopoiesis
In mammals, hematopoiesis occurs in successive but over-
lapping waves that occur at distinct anatomical locations
. Overall, the hematopoietic process is distinguished
into primitive and definitive hematopoiesis based on the
type of blood cells generated. Primitive hematopoiesis is
transient in nature and produces unipotent blood cells that
arise directly from the mesoderm. Definitive hematopoiesis
produces multipotent blood cells that give rise to multiple
different lineages through cellular intermediates and support
blood cell development throughout the life of the organism.
hematopoiesis based on the studies using mouse models.
During embryogenesis, primitive hematopoiesis occurs
in two distinct waves in the extraembryonic yolk sac blood
islands, producing primitive macrophages and primitive
erythrocytes, respectively, thus providing the developing
embryos with oxygen and their first line of defense against
pathogens . There is some support for the presence
of additional lineages, particularly megakaryocytes, during
primitive hematopoiesis .
Definitive hematopoiesis also occurs in two distinct
waves. The first wave of definitive hematopoiesis produces
a transient population of cells, termed erythroid-myeloid
progenitors (EMPs) in the yolk sac and fetal liver [68,
69]. The second wave of definitive hematopoiesis pro-
duces hematopoietic stem cells (HSCs) from the hemogenic
endothelium of the embryo that includes the aorta-gonad-
mesonephros (AGM) region of the embryo, yolk sac, and
placenta [65, 70–72]. HSCs from these sites migrate through
circulation to fetal liver to support hematopoiesis during
embryogenesis [65, 70, 73]. Recently, Chen and colleagues
 demonstrated that EMPs and HSCs are derived from
two different hemogenic endothelial populations. Unlike
HSCs, EMPs lack the potential to give rise to lymphocytes.
The site of adult hematopoiesis, where HSCs undergo
differentiation to generate lineage-committed progenitors
that give rise to all the mature blood cell types and self-
renewal to maintain a constant supply of HSCs, is bone
marrow . The prevailing thinking, based on the current
data, is that HSCs emerging from the hemogenic endothelial
cells in the AGM region of the developing mouse embryo
give rise to most (if not all) bone marrow hematopoietic
cells [73, 76]. The shifting sites of hematopoiesis are thought
to provide specific microenvironment cues required for
the specification, and migration of precursors for lineage
commitment [77, 78].
Although the overall process of hematopoiesis is well
defined, we have just begun to elucidate the exact nature
of the molecular controls and lineage relationships using
in vitro colony assays and animal models, particularly
mice and zebrafish. The key questions revolved around
the generation, migration, and differentiation of HSCs into
lineage-committed progenitors and how these processes are
regulated to maintain a critical balance required for proper
functioning of the hematopoietic system.
2.1. Primitive Hematopoiesis in Zebrafish. In zebrafish, the
first blood cells can be observed in circulation at around
26 hours post fertilization (hpf). However, based on the
expression patterns of the genes involved in primitive
hematopoiesis, it is clear that the primitive hematopoiesis
starts at ∼11hpf in the lateral plate mesoderm (LPM) during
somitogenesis. The erythroid precursors are observed as
bilateral stripes in the posterior lateral mesoderm (PLM)
that fuse along the midline to form the intermediate cell
mass (ICM) located in the trunk dorsal to the yolk tube
extension by 24hpf [29, 75, 77, 79–81]. Primitive myeloid
progenitors initiate at the anterior lateral mesoderm (ALM)
and differentiate into macrophages in the rostral blood
island [80, 82]. Thus, primitive hematopoiesis in zebrafish
occurs in two waves, producing primitive macrophages and
primitive erythrocytes, respectively. In addition, neutrophils
and thrombocytes have also been detected during primitive
hematopoiesis in zebrafish. However, the origin of neu-
trophils during primitive hematopoiesis is not clear, as two
recent reports presented contradictory data on their origin
from either primitive macrophage lineage  or primitive
erythrocyte lineage  using fate-mapping techniques.
Thus, primitive blood cells in zebrafish appear to have
diverse lineages, similar to the mouse . However, further
studies are required to clearly define the lineage relationships
between these cell types during primitive hematopoiesis.
2.2. Definitive Hematopoiesis in Zebrafish. The hallmark
of definitive hematopoiesis is generation of multipotential
HSCs that can undergo self-renewal and differentiation to
Advances in Hematology3
Table 1: Lineage-specific mutant and transgenic lines for zebrafish hematopoiesis research.
Mutant linesTransgenic lines
Mutant designation and
References Line designationReferences
tal1/scl t21384, K183X
hkz3, truncation in
Developed by the
Zon lab, used in
[33, 34] cd41
m651 (vlad tepes), R339X
t26683, R797X 
produce cells of erythroid, myeloid, and lymphoid lineages.
In zebrafish HSCs can be identified by their expression of
runx1 and cmyb as early as 26hpf in the ventral wall of
the dorsal aorta and hence this region of the embryo is
referred to as the AGM [13, 29]. Two recent studies have
unequivocally demonstrated the origin of HSCs from the
hemogenic endothelium lining the ventral wall of the dorsal
aorta using time lapse imaging and lineage tracing in double
transgenic lines marking HSCs and endothelial cells with
different fluorescent markers [47, 85]. A novel process of
cell transition, termed endothelial hematopoietic transition
(EHT), appeared to be involved in the production of HSCs
from hemogenic endothelium . Similar to the mouse,
a transient multipotent progenitor population of EMPs
supports definitive hematopoiesis during embryogenesis and
these EMPs originate in the posterior blood island (PBI) of
The sites of adult hematopoiesis in zebrafish are kidney
marrow (analogous to the mammalian bone marrow) and
thymus (for T cells) [13, 29, 87]. Up until recently, a site
analogous to mammalian fetal liver was not recognized in
the zebrafish. Therefore, HSCs from AGM were presumed
to support embryonic definitive hematopoiesis and migrate
to thymus and kidney for adult definitive hematopoiesis.
However, two independent studies demonstrated the exis-
tence of an intermediate site of hematopoiesis posterior
to the yolk tube extension, termed caudal hematopoietic
tissue (CHT), using imaging and cell tracing techniques
[88, 89]. It was proposed that the function of CHT is
definitive hematopoiesis during embryogenesis. By tracing
the generation and migration of HSCs using cd41:GFPlow
cells, Kissa and colleagues  validated the migratory
route of HSCs as being AGM to CHT and then to thymus
and pronephros. Recently, Hess and Boehm  elegantly
imaged the process of thymopoiesis in real time in zebrafish
using triple transgenic lines and their data suggested that
AGM is a major source of thymus-settling lymphoid progen-
itors compared to CHT.
Thus, based on the current status of our understanding,
definitive hematopoiesis in zebrafish occurs in two waves:
first wave produces transient EMPs in the PBI region and
second wave produces HSCs in the AGM region that migrate
to CHT to support larval definitive hematopoiesis and to
thymus and kidney marrow to support adult definitive
hematopoiesis. It is not clear if the migration of HSCs from
AGM to kidney and thymus is via CHT only or also occurs
directly as was previously assumed.
3.Elucidationof GeneticControls of
Despite the spatial and temporal differences during
hematopoiesis between zebrafish and mammals as discussed
above, the overall process is highly conserved producing the
same effective repertoire of hematopoietic cells. It begins
from a cell, termed hemangioblast, that serves as a common
4 Advances in Hematology
precursor for hematopoiesis and vasculogenesis [92, 93].
A complex network of regulatory signals is involved in the
specification and lineage commitment of precursors during
primitive and definitive hematopoiesis in mammals. These
include homeobox, notch, vegf, and wnt signaling pathways
as well as specific transcription factors, such as Tal1 (Scl),
Lmo2, Gata1, Cmyb, Runx1, Spi1 (Pu.1), and Ikzf1 (Ikaros),
which are shown to function in a hierarchical manner
[5, 94–99]. The importance of proper functioning of these
transcription factors is evident from the preponderance of
mutations and genomic rearrangements disrupting their
activity detected in several blood disorders, particularly
leukemias and lymphomas [100–106].
Animal models, where level of gene activity can be
manipulated, have played a critical role in advancing our
understanding of the genetic controls of hematopoiesis.
However, knockout mice are embryonic lethal at mid-to-
late gestation for Tal1, Lmo2, Gata1, Sfpi1 (Pu.1), Myb,
and Runx1, thus precluding the examination of their roles
in later stages of hematopoiesis [107–112]. Conditional
knockout is a useful tool to determine the function of these
genes later in life; however, it has been difficult to use
this technology to study the initiating events of a lineage,
especially for the HSCs, since appropriate promoters to drive
Cre recombinase expression may not be available. Zebrafish
provide an advantage over mouse models due to their ability
to survive without blood for several days and are, therefore, a
function of genes that cause embryonic lethality in mice due
to the hematopoietic defects. Here, we discuss the contribu-
tions of zebrafish mutants, morphants, and transgenic lines
to our understanding of the regulatory cascade controlling
the hematopoiesis process (Table 1 lists the lineage-specific
transgenic lines and genetic mutants in transcription factors
involved in regulation of hematopoiesis). The common
theme in the studies reviewed below is utilization of the
unique features of zebrafish embryos and available tools for
analysis of the disruptions to the gene activity in an effort to
understand the overall process.
3.1. Genes Involved at the Hemangioblast Level: tal1 and
lmo2. Based on their expression in both hematopoietic and
endothelial cells, and the phenotypes of loss of function ani-
mal models, the T-cell acute lymphocytic leukemia 1 (TAL1)
and the LIM domain only 2 (LMO2) genes are both believed
to function at the hemangioblast level [12, 113]. Both genes
were identified from translocations occurring in T-cell acute
lymphoblastic leukemia, TAL1 from translocation t(1;14)
and LMO2 from translocation t(11;14) [102, 104]. TAL1 is a
bHLH domain is involved in DNA binding as part of a mul-
tiprotein complex that includes LMO2 as a bridging protein.
LMO2 belongs to the LMO family of zinc-finger proteins
that are characterized by 2 LIM domains, each composed
of 2 zinc fingers . Knockout mice for Tal1 and Lmo2
died in utero by embryonic days 9.5–10.5 (E9.5-10.5) due to
during definitive and adult hematopoiesis were investigated
by in vitro colony assays, chimeric mice, and/or conditional
knockout mice [114, 115]. Failure to produce any myeloid
colonies in vitro from Tal1−/−yolk sac cells indicated a
block at the EMP level . Using conditional knockout
mice, Hall and colleagues [114, 116] demonstrated that adult
hematopoiesis can occur independent of Tal1 function with
minor defects in erythropoiesis and megakaryopoiesis. On
the other hand, Lmo2 was shown to be absolutely necessary
for adult hematopoiesis based on the analysis of chimeric
mice derived from Lmo2−/−embryonic stem cells .
In zebrafish, tal1 is expressed in the ALM and PLM from
in primitive hematopoiesis [39, 117, 118]. First direct proof
for the exact site of HSC initiation between the dorsal aorta
and the posterior cardinal vein being analogous to AGM
in zebrafish came from the examination of Tg(tal1-PAC-
GFP) embryos by time lapse imaging . Loss-of-function
analyses for tal1 have been performed using morpholinos
and a genetic truncation mutation, K183X, which deletes the
(tal1K183X/K183X) exhibited lack of expression of markers of
both primitive and definitive lineages and also lacked visible
circulation at 26hpf . These studies not only confirmed
the role of Tal1 during primitive hematopoiesis, but also
provided direct evidence for the role of Tal1 in the initiation
of definitive hematopoiesis. However, mutant embryos died
due to pericardial edema and defects in heart morphogenesis
and could not be studied for the role of Tal1 in transition of
embryonic to adult stages of definitive hematopoiesis.
In zebrafish, lmo2 expression in the ALM and PLM is
detected about 20 minutes after the tal1 expression and
phenotype of lmo2 morphants is very similar to the tal1
morphants, supporting their roles aspartof the multiprotein
complex during hemangioblast development [41, 122]. To
date, no genetic mutants have been reported for lmo2. Over-
all, zebrafish studies have confirmed the strict requirements
for Tal1 and Lmo2 in initiation of both primitive and
3.2. Genes Involved at the HSC Level: runx1 and cmyb. The
onset of definitive hematopoiesis in the AGM is marked
by the specification of HSCs, which support hematopoiesis
used interchangeably as the earliest markers of definitive
hematopoiesis due to their expression in the AGM during
elucidate their precise roles in HSCs specification, migration
to the sites of larval and adult hematopoiesis, and differenti-
ation into erythroid, myeloid, and lymphoid lineages.
mals and 4 in zebrafish) that encode for the alpha subunits
of a heterodimeric complex that binds DNA through the
highly conserved runt domain. A single gene, CBFB, encodes
for the beta subunit, which does not bind to DNA by itself
but increases the affinity of alpha subunits to bind to DNA
after heterodimerization through their runt domains .
Promoters of many hematopoietic genes, for example, SPI1
and GATA1, contain RUNX1 DNA binding sites [125–127].
RUNX1 was first identified in the t(8;21) translocation
frequently observed in acute myeloid leukemias and its
Advances in Hematology5
dimerization partner, CBFB, is also frequently involved in
genomic rearrangements associated with leukemia [100, 128,
129]. Furthermore, mutations affecting the level of RUNX1
activity leading to loss of function, dominant negative gain
of function, and/or overexpression are associated with other
blood disorders such as familial platelet disorder with pre-
disposition to acute myeloid leukemia and myelodysplastic
syndrome, suggesting that the process of hematopoiesis is
very sensitive to the level of RUNX1 activity [130–132].
Runx1 is essential for the initiation of HSCs generation
during definitive hematopoiesis as the mutant mice failed to
develop fetal liver hematopoiesis and died in utero at E12.5
. Conditional knockout mice were able to develop all
lineages but showed defects in megakaryocyte maturation
and differentiation of B and T cells [133, 134]. Recent
elegant fate mapping experiments in mouse embryos by
Chen and colleagues demonstrated that Runx1 is required
. Taken together, these data suggest a strict requirement
of Runx1 in the generation of HSCs to initiate definitive
hematopoiesis and in further differentiation of certain
lineages but not for the maintenance of HSCs if they are
already produced (reviewed in ).
Zebrafish runx1 was identified based on its high simi-
larity to the human RUNX1 in the runt homology domain
[123, 136]. Since then, several studies have validated the
critical requirement of Runx1 in the initiation of definitive
hematopoiesis by morpholinos and characterization of a
variety of hematopoietic mutants [95, 97, 136, 137]. As
these studies were performed prior to the recognition of
CHT being the site of embryonic definitive hematopoiesis,
they did not address Runx1 requirements in specification
of EMPs and their transient nature precluded analysis
of Runx1 requirements in adult hematopoiesis. None of
the hematopoietic mutants from forward genetic screens
mapped to the runx1 locus.
Therefore, our group performed TILLING to identify a
truncation mutation, W84X, in the runt domain of runx1
[42, 43]. Homozygous mutant embryos displayed a complete
myeloid, and lymphoid lineages in the CHT and thymus
between 3–5dpf [42, 43]. However, utilizing Tg(cd41:GFP)
transgenic zebrafish, we were able to demonstrate that cd41+
cells were formed in the runx1W84X/W84Xfish in the AGM
and CHT regions and migrate to the pronephros, even
though they were negative for other HSC markers such as
cmyb. Based on the analysis of circulating blood cells, the
mutant fish displayed 3 distinct phases: first phase of normal
circulating blood cells until around 6–8dpf (presumably
from normal primitive hematopoiesis), second phase of
bloodless stage until around 20dpf leading to death in
most larvae (defective larval definitive hematopoiesis), and
astonishingly, ∼20% of the mutant larvae resumed blood
circulation and grew as phenotypically normal adult fish
with multilineage adult hematopoiesis . We do not
know exactly how these 20% runx1 mutant larvae were
rescued. One possibility is that the cd41+cells observed in
these embryos are hematopoiesis-committed or -primed
mesoderm cells, which could restart hematopoiesis in
permissive conditions, such as compensation by runx2a,
runx2b, and runx3 genes or other genetic and/or epigenetic
changes. Another scenario is that two waves of definitive
hematopoiesis exist, one for larval and the other adult,
while Runx1 is only required for the larval stage. For
both scenarios, most larvae died due to lack of circulating
blood cells resulting from defective larval hematopoiesis.
It is interesting to note that alternate runx1 promoters are
used during establishment of EMPs and HSCs (Table 1) as
demonstrated recently by Lam and colleagues .
Similarly, MYB, a cellular homolog of the V-MYB proto-
tive hematopoiesis. A number of mouse models, including
conventional and conditional knockouts as well as hypo-
morphic alleles, have been generated for functional analysis
of Myb requirements during hematopoiesis, as discussed
in a recent review by Greig and colleagues . These
studies have highlighted the key difference between Runx1
and Myb requirements during definitive hematopoiesis to
be the generation of HSCs. Myb knockout mice displayed
defects in erythroid and myeloid development and died in
utero at E15.5, which is much later than the stage when
HSCs are generated . Furthermore, Myb−/−ES cells
were able to produce T cell progenitors in Rag1−/−chimeric
mice . Thus, Myb deficiency causes a block in HSCs
differentiation and lineage commitment rather than HSCs
specification. Lieu and Reddy  demonstrated important
contributions of Myb to self-renewal and differentiation of
HSCs during adult hematopoiesis.
Recently, two groups reported characterization of loss
of function mutants for cmyb in zebrafish: (1) allele t25127
with a missense mutation, I181N, affecting DNA binding
domain and (2) allele hkz3, a splice site mutation leading
to truncation of the transactivation domain. These mutants
were identified from forward genetic screens for defects
in thymopoiesis and lack of lysozyme C (lyz) expression,
respectively [45, 46]. Homozygous embryos for either muta-
tion showed lack of definitive hematopoiesis but behaved
differently with respect to survival. cmybI181N/I181Nmutant
embryos displayed severe anemia and became bloodless by
20dpf. Although the mutants survived for 2-3 months with
stunted growth, there were no detectable hematopoietic cells
by FACS or histology . This is in contrast to our finding
with runx1W84X/W84Xmutants, thus suggesting differential
requirements for runx1 and cmyb activities during larval
and adult hematopoiesis. On the other hand, most of
the cmybhkz3mutants (splice site mutation affecting the
transactivation domain) died by 10dpf. The authors did
not explain the reason for this difference. We speculate that
the husbandry differences between laboratories might be the
reason for their differential survival in the absence of blood
cells. Using time-lapse imaging of cmybhkz3/Tg(cd41:GFP)
embryos and lineage tracing, Zhang and colleagues 
demonstrated an important role for cmyb in the migration
of HSCs from ventral wall of the dorsal aorta (VDA) to CHT,
thereby proposing that migratory defects of HSCs maybe the
cause of failure of definitive hematopoiesis in cmyb deficient
embryos. Thus, zebrafish models of cmyb deficiency have
6 Advances in Hematology
provided novel insights into its role in the migration of HSCs
from AGM to CHT during definitive hematopoiesis.
3.3. Genes Involved at the Level of Erythropoiesis, Myelopoiesis,
and Lymphopoiesis: gata1, spi1, and ikzf1. Differentiation
of HSCs during definitive hematopoiesis into lineage-
committed progenitors, which further differentiate into
mature blood cells, is mediated by lineage-specific tran-
progenitors lack the potential for self-renewal and thus
require a constant supply of HSCs for their production
[87, 140]. The first series of lineage-committed multi-potent
progenitors are termed common myeloid and common
lymphoid progenitors (CMPs and CLPs). In mammals,
CMPs further differentiate into megakaryocyte-erythroid
progenitors (MEPs) that produce mature erythrocytes and
platelets (erythropoiesis), and granulocyte/macrophage pro-
genitors (GMPs) for the generation of mature myeloid
cells (myelopoiesis). CLPs produce mature lymphoid lineage
cells (lymphopoiesis). However, intermediate multilineage
progenitors have not been identified in zebrafish yet, and
all lineage relationships are speculative. Here, we have sum-
marized the genetic controls of erythropoiesis, myelopoiesis,
and lymphopoiesis in zebrafish.
Erythropoiesis involves differentiation of erythroid-
myeloid progenitors into mature erythrocytes and throm-
bocytes. The master regulator of erythropoiesis is GATA1,
a transcription factor belonging to the GATA family (6
members) that contains a conserved DNA binding domain
consisting of two zinc fingers [140, 141]. Its consensus
DNA binding site, WGATAR, is found in regulatory regions
of most erythroid-specific genes . Human mutations
in GATA1 are associated with anemia, thrombocytopenia
and acute megakaryoblastic leukemia in Down Syndrome
patients . Gata1 knockout mouse embryos die by E10.5
due to severe defects in erythropoiesis during primitive
hematopoiesis, precluding assessment of its role in definitive
hematopoiesis without generating conditional knockout
mice [107, 144].
The zebrafish gata1 gene was identified by cross-
hybridization with the zinc-finger region of Xenopus Gata1
. Its expression is consistent with the sites of ery-
thropoiesis during primitive hematopoiesis starting at 5-
somite stage . Using positional cloning of one of the
bloodless mutants, termed vlad tepes or vltm651, identi-
fied in the 1996 large-scale forward screens, our group
identified a truncation mutation, R339X, distal to the C-
terminal zinc-finger domain in Gata1 . As expected,
homozygous mutant embryos displayed defects in primitive
onset of circulation. Evaluation of definitive hematopoiesis
by WISH revealed similar defects in erythropoiesis but
normal development of myeloid and lymphoid lineages,thus
demonstrating the specific role of Gata1 in generation of
erythroid progenitor cells not only during primitive but also
during definitive hematopoiesis [23, 48].
Myelopoiesis involves differentiation of erythroid-my-
eloid progenitors into differentiated macrophages/mono-
eosinophils [9, 80, 82]. The master regulator of myelopoiesis
is SPI1 (previously known as PU.1), an oncogene originally
identified as the site of genomic rearrangements by spleen
focus-forming proviral insertion in erythroblastic tumors
. SPI1 belongsto theETSfamilyof transcription factors
that bind DNA through a purine rich sequence, termed
the PU box . Sfpi1 knockout mice died around E18
due to multilineage defects, implicating additional roles of
Sfpi1 in erythropoiesis and lymphopoiesis . In vitro
studies have demonstrated the importance of a negative
cross-regulation of Gata1 and Sfpi1 during erythroid and
myeloid differentiation from CMPs . Unlike mammals,
the sites of erythropoiesis (PLM) and myelopoiesis (ALM)
are separate in zebrafish during embryogenesis [50, 51].
However, upregulation of myelopoiesis in gata1 morphants
and ectopic expression of gata1 in spi1 morphants proved
that similar cross-regulation of these two transcription
factors is critical for the proper commitments of erythroid
and myeloid lineages in zebrafish [147, 148].
Lymphopoiesis involves differentiation of lymphoid pro-
genitors into mature T and B cells that participate in a
functional immune system of the organism . Primary
lymphoid organs for T-cell maturation in zebrafish are
bilateral thymii, which are marked by expression of rag1,
ikzf1 and lck starting at ∼72hpf [56, 57, 59]. Pancreas has
been suggested as an intermediate site for the production of
B cells  between 4dpf to ∼3 weeks, at which point B
cells become evident in the kidney. However, this remains
to be verified, as no good transgenic markers of B cells
currently exist to follow their development in real time. The
master regulator of lymphopoiesis is the transcription factor
six zinc-fingers that are involved in DNA binding and
protein-protein interactions . By analysis of knockout
mice, Wang and colleagues  demonstrated differential
requirements of Ikzf1 for B- and T-cell differentiation during
fetal and adult hematopoiesis. Ikzf1 null mice displayed
complete blockage of differentiation of B cells during both
fetal and postnatal stages. On the other hand, they displayed
blockage of differentiation of T cells only during the fetal
stage. Postnatal T-cell development recovered, albeit with
deregulation of CD4 versus CD8 lineage commitment.
Overall, their data suggested that Ikzf1 is essential for lym-
phopoiesis (both B and T cells) during fetal hematopoiesis,
but it is dispensable for adult T cell development. Similar
to the knockout mice, zebrafish with a truncation mutation,
Q360X, in ikzf1 (ikzf1t24980), which removes the C-terminal
two zinc fingers essential for protein-protein interactions,
are adult viable . Mutant fish displayed complete lack of
lymphopoiesis during larval stage, and partial recovery after
14dpf. Although the mutant fish survived and lived up to
at least 17 months in nonsterile conditions, they displayed
abnormal and inefficient lymphoid development. However,
it is interesting to note that similar to our observations of
two phases of definitive hematopoiesis in runx1 mutants,
zebrafish lacking Ikzf1 activity potentially demonstrated two
phases of lymphoid development. In both cases, the larval
phase is gene activity dependent while the adult phase
develops to some extent despite the lack of gene activity.
Advances in Hematology7
erythrocytes and thrombocytes)
erythrocytes and thrombocytes)
Start of circulation
Kidney marrow (HSCs to myeloid cells, B cells,
Thymus (T cells)
CHT (HSCs to myeloid cells,
24 hpf 36 hpf3 dpf 5 dpf
Figure 1: A schematic of overall view of zebrafish hematopoiesis with shifting sites, types of cells produced at each site, and genes involved,
shown in 3 tiers as described below. Tier 1: lineage-specific transcription factors that control primitive and definitive hematopoiesis in
zebrafish. Tier 2: the sites of action during each stage of hematopoiesis and the types of cells produced at each of the sites. The site boxes
are color matched with waves of hematopoiesis and temporally placed according to the developmental stages in Tier 3. Tier 3: the time scale
depicting the stage of development in hpf (hours postfertilization) and dpf (days postfertilization) and different waves of hematopoiesis. The
abbreviations used are as follows: ALM: anterior lateral mesoderm, PLM: posterior lateral mesoderm, PBI: posterior blood island, AGM:
stem cells, TD: transient definitive wave.
Isoforms of the Same Transcription
Stages of Hematopoiesis
requirements of transcription factors in the hematopoietic
cascade as opposed to a simple on versus off situation [153–
156]. In zebrafish, it is relatively easy to manipulate gene
of hypomorphic alleles using TILLING. Therefore, differen-
tial requirements for some of the transcription factors either
in terms of level of activity or different isoforms have been
demonstrated recently in zebrafish, as discussed below.
4.1. Tal1. As discussed previously, Tal1 plays critical roles
during both primitive and definitive hematopoiesis. Using
different doses of morpholinos to completely or partially
abolish Tal1 activity, Juarez and colleagues  demon-
strated differential requirements of tal1 expression for
erythroid specification and maturation during primitive
hematopoiesis. Their work showed that lower activity of
Tal1 was sufficient for primitive erythroid specification but
not their maturation. Furthermore, by complementation
of Tal1, they demonstrated differential requirements for the
DNA-binding activity of Tal1 during erythroid specification
and maturation. Their data suggested different mechanisms
of target gene regulation during erythrocyte specification
and maturation by Tal1: direct binding to promoters of
the target genes involved in erythroid maturation and
indirect regulation through other protein complexes for
genes involved in erythroid specification.
Further complexity to Tal1 requirements during primi-
tive and definitive hematopoiesis became obvious from the
analysis of its two isoforms: the full-length form termed
Tal1-α and a shorter form lacking the first 146 amino
acids, termed Tal1-β. Using morpholinos to specifically
target the α and β forms, Qian and colleagues 
demonstrated that both forms act redundantly in initiation
of primitive hematopoiesis, while only the Tal1-β form is
required for the specification of HSCs in the AGM to initiate
definitive hematopoiesis. Renand colleaguesexamined
the requirements of Tal1-α and Tal1-β during angioblast
and HSC specification, also demonstrating the requirement
for Tal1-β in HSC specification. Thus, zebrafish research
has contributed significantly to our understanding of the
regulation of different stages of hematopoiesis by Tal1.
4.2. Gata1. Similar to Tal1, Gata1 activity is crucial
for erythropoiesis during both primitive and definitive
hematopoiesis. Recently, we described a hypomorphic allele
of Gata1 due to a missense mutation, T301K, in its C-
terminal zinc finger . This mutation reduces DNA
binding affinity and diminishes transactivation of target
gene expression by Gata1 . The gata1T301K/T301Kfish
had defective primitive erythropoiesis but normal definitive
hematopoiesis. By combining the T301K allele with the
8 Advances in Hematology
Gata1 null allele of vlad tepes, we were able to generate
an allelic series with different Gata1 activity levels, listed
in the descending order: gata1+/+, gata1+/T301K, gata1+/vlt,
gata1T301K/T301K, gata1T301K/vlt, gata1vlt/vlt. Analysis of fish
with these genotypes demonstrated that erythropoiesis dur-
ing primitive hematopoiesis requires higher activity level
of Gata1 than erythropoiesis and thrombopoiesis during
definitive hematopoiesis .
Depicted in Figure 1 is a schematic of the overall view
of zebrafish hematopoiesis emerging from these studies. It
is clear from the above-mentioned studies that zebrafish
has played a significant role in our understanding of the
genetic controls of hematopoiesis, particularly the dosage-
specific requirements during different stages. The viability
to adulthood with multi-lineage hematopoiesis in runx1
knockout zebrafish clearly demonstrated that Runx1 is
dispensable for adult hematopoiesis. Similarly, Ikzf1 was
found to be dispensable for adult lymphopoiesis. On the
other hand, Cmyb was found to be essential for adult
hematopoiesis, while dispensable for larval definitive stage.
Genetic mutants need to be generated for spi1 to elucidate
its exact role in maintaining proper balance between adult
erythropoiesis and myelopoiesis.
Proper functioning of the genetic controls regulating
hematopoiesis is crucial for normal development of all the
blood lineages. Mutations in critical genes at many of the
steps lead to leukemogenesis. Thus, adult viable mutant
zebrafish would allow us to understand the process of
leukemogenesis. Furthermore, the recent application of next
generation sequencing technologies to a variety of leukemia
samples have led to the identification of several new genes
mutated in leukemias [159, 160]. We anticipate that under-
standing their roles in normal hematopoiesis using the many
would aid in therapeutic advances in the coming years.
This study was supported by the Intramural Research
Program of the National Human Genome Research Institute,
National Institutes of Health.
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